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SOFTmax PRO for Lmax

1. Q 5 18 Long and Fast Kinetics s ceno eeiam t im otra o oet p m o 5 18 Custom Reduction Formulas 0 0 cece eee es 5 21 Recalculation Options quo drum aactor om erm 80 91 UR ADIAC LR RAUS uws 5 22 Displaying Data in Group Graph and Notes Sections 004 027 GTOUD SCCUONS 44eneteanaweaudgrereceaenenuageenaneaaeeanneaarens 5 22 DOLOS OBEBIOLIS usce nog e AUREUS S 5 28 Graph cit PR LE 5 28 Formulas in SOFTmax PRO for Lmax esseeeeeeeeeeeeee eee 5 46 Chapter 6 File Management and Printing 0 ccc cece cece ee eee e eee 6 1 File Creation and Management 0 eee ees 6 3 Bride e o 6 3 Locating Protocol and Data Files 0 0 eee eee eee 6 4 i is dui P X 6 4 Open gi M 6 6 Saving Files Manually 25 um hmm etm tact note m a 6 9 D D 6 9 Ix vio il 6 11 The galeis E 6 12 Customizing the Report 00 cece eee eee eens 6 12 The Print Dialog Box 33 ut urna ure mirata a ue i oa a a i gaan eas 6 14 Exporting Importing Information lesse 6 16 Exporting Data for Later Reimport 0 0 0 0 ce eee eee 6 16 Exporting Data General Information 000 0 c eee eee 6 16 Importing Data from Outside of SOFTmax PRO for
2. Figure 7 5 Instrument Settings Dialog Box Injection Wells Section Open with P Injector Enabled SOFTmax PRO User s Manual 7 9 Chapter 7 Tutorial You can click individual wells or click and drag across groups of contiguous wells to enable injection within them Note that the wells you select for the P injector will be shown with a red half circle if the M injector were enabled its wells would be shown with blue half circles on the other side of the wells Wells can be selected in any order if you select an incorrect well by mistake click it again to deselect it Select wells as shown in the following figure m Instrument Settings DO oo Endpoint Dual Read Fast Kinetic Injection Wells Figure 7 6 Instrument Settings Dialog Box Injection Wells Selected The wells you have selected are now also shown on the left side of the dialog box beneath the Injection Wells heading You do not need to change the default setting of 96 Well Standard for the Plate Type The settings are now complete Click to accept the settings as shown and close the dialog box 7 10 SOFTmax PRO User s Manual Chapter 7 Tutorial Step 3 Define the Template A Click the Template button in the Plate section tool bar This will open the Template Editor showing all wells empty Drag the mouse or shift click to select the first three columns of wells 1 through 3 These wells will be defined as Standards for the
3. 2 Chapter 8 Troubleshooting General Problems Insufficient Memory in a Macintosh If errors occur while using SOFTmax PRO for Lmax for the Macintosh it may be that the amount of memory allocated for the program is insufficient Two items need to be checked the amount of memory your Macintosh has avail able for use and the amount of memory allocated to SOFTmax PRO for Lmax To see the amount of memory available within the Macintosh first quit all open applications then go to the Finder and choose About This Macintosh from the Apple menu File Edit View Special About This Computer The information box that opens will list the amount of memory used by the System file and the amount of memory remaining under the heading Larg est unused block that can be assigned to applications such as SOFTmax PRO for Lmax The amount of memory available should be equal to or greater than minimum amount required by SOFTmax PRO for Lmax About This Computer Hasc 05 8 5 1 Mac 05 ROM 1 7 Built in Hemery 1 Virtual Hemory 200 HE vd on Avalon Largest Unwed Bleck 275 9 HB amp Apple Comguler Ime 1283 1998 Figure 8 5 About This Macintosh Dialog Box If you open many files simultaneously or if you open SOFTmax PRO for Lmax while other applications are open in the background memory errors can occur Also if you make a kinetic run with a large number of data points you may not have enough
4. e Text that appears in green with an underline contains a hypertext link to more detailed information about that item Figure 1 7 below shows some examples of hypertext links sOftmax Pro for Lmax Help Hg File Edit Bookmark Help Contents Search Back History s lt gt Softmax Pro for Lmax Window l Hypertext linked item clicking here will give more A Menu bar located at the top of the screen information about this item The window contains these major elements A Status bar located below the Menu bar The status bar is used to control the instrument and to display the current status af the instrumant One or mare sub windows containing collapsible sections in a repart format The window allows you to scroll around and examine sections in the report The report Is as wide as a piece of paper and shows you exactly what you will get when the report is printed Figure 1 7 Hypertext Links within SOFTmax PRO for Lmax for Windows Additional options are available depending upon where you are within the Help function More information about Windows Help can be found in the Microsoft Windows User s Guide that accompanies your Windows software Troubleshooting and Technical Support If you encounter a problem while using SOFTmax PRO for Lmax refer first to Chapter 8 Troubleshooting and try to locate a remedy for the problem For problems with SOFTmax PRO for Lmax that are not listed there or if you
5. Edit gt Cut Dispay Edit Paste a Well Edit gt New Section X Axis Y Axis Edit gt Text Style All Section Settings Control gt Instrument Setup Notes all items Plate all items Group all items Graph all items Only if data exists in the section Setting and Changing a Password Choosing Set Password from the File menu causes the Password dialog to appear If the active document is already password protected the menu com mand changes to Change Password Set Password Old Password Do New Password New Password Hetype New Password Retype New Password Cancer ox O Remove Password Cancel Figure 6 17 Set Password Dialog Box Left no password set previously right with previously set password This dialog appears differently depending upon whether or not you are set ting a password for the first time or if you are changing removing an existing password If you have not previously set a password the dialog box will appear like that on the left of Figure 6 17 To set a password type it in both 6 26 SOFTmax PRO for Lmax User s Manual Chapter 6 File Management and Printing boxes first to set it and then to confirm it After password protection is enabled a locked icon will appear on the right side of the status bar e When changing or removing a password the dialog box will appear as shown on the right of Figure 6 1
6. Figure 5 22 Graph Options Dialog Box Creating Multiple Plots 1 5 30 SOFTmax PRO for Lmax makes it possible to create multiple plots within a simple graph Each plot will represent data from a group that has been cre ated in the Template Editor or data from a summary or custom formula You may plot data from any group or from several groups on a graph even if the groups are in separate experiments It is possible for example to plot multi ple dilution series of an unknown to graph together a series of patient sam ples or to plot several controls that are run over time against one another Following is a summary of the steps required to create multiple plots within a single graph after which is an example that provides more detail Multiple Plots Step Summary e Create all required groups in the Template Editor e Read the microplate to obtain data Create a graph section e Add plots to the graph and choose the group and the X and Y values to be shown for each plot Example of Creating Multiple Plots This example shows how six groups all of which are Standards were plotted on the same graph Groups are created in the Template Editor For this example eight groups are created GA h GA native G5A h G5A native plec2 h plec2 native CHOMO6 h and CHOMO6 native SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data W Expenment 1 Plate tl Group CHOMOB h ww s
7. Written by Duane Borman Duff Caldewey Tae Kang and Tom Smith i 3 Testing coordinated by Emma Ratner Thanks Chris S JD SG LC and TM oA Copyright 2000 Molecular Devices Corp All rights reserved Figure 2 4 SOFTmax PRO for Lmax Title Screen After a few seconds the title screen will be replaced by an untitled SOFTmax PRO for Lmax window see Figure 2 5 SOFTmax PRO for Lmax User s Manual 2 9 Chapter 2 Installation imp SOFT mas PAD fos Lomas Fie Ed Yen Eipesmert Corin siae Buch Window Heb ale x Pa Untitled l x CB Endpoint Assay Default Protocol n E v E oen B6 6 a Dred aiit Pi otoc Tihai This protocol commis thes Introduction and ome experiment section H you are new to Sofiman PRO for Liner proceed to Chanter 7 and comtinie on this dui arid To Customire This Default Pretecot First delete redit ami sections you do mot wait Ged examgple this Introd amp gctie Secor make amy chamges you wish te the ineiriment settings by eking em Setup Third customize the Template to your miroplate by editirg the pre assigned Groups Last see thes dolau protocol with the name Dela Protocolipr in the same Tolder as the SOF Tmax PRO application For Tutus e use our will be asked m you wish to replace the esisiini default polecol choose Yes Experiment 1 Time Empat assay wilh Standards amd Unknown Gui and wrhour diliricn tacta Thee umnkrnewrnes are inberpedated fram a
8. Z Zoom box 5 15 SOFTmax PRO for Lmax User s Manual
9. eem Cic name will be included in the group EH of O for Unknown column formats 1 for other column formats descriptors Descriptor units Assigned in the Group Settings dialog box Supported strings are unit ml mg ml ug ml ng ml mg ng and ml If this column is left blank unit ml is assigned Sample Assigned in the d Settings dialog box Any text string can be entered in this descriptor field or it may be left blank Assigned in the Series dialog box Can be set to any number or left blank Exporting Graphs Graphs from Graph sections and Well Graphs can be exported in PICT format Macintosh and EMF format Enhanced MetaFile in Windows Graph sec tions can be exported by choosing Export Graph from the Graph menu Well Graphs can be exported by choosing Export Graph from the Plots menu that appears when a Well Graph is opened Copying and Pasting Parts of an experiment can be copied within a SOFTmax PRO for Lmax file to or from another SOFTmax PRO for Lmax file or to or from another program using the Copy and Paste commands Plate section data and text and summa ries in Notes sections can be copied and pasted to other sections within the same or a different experiment within SOFTmax PRO for Lmax or to other programs Virtually any ASCII formatted text can be copied and pasted into a Notes section A graph can be copied and the resulting picture pasted into another application Template information can be copied and paste
10. which is currently enabled Unchecking the box will allow you to view the name of the sum mary to the left of its text in the Standards section The formulas for MinStd and MaxStd are Min MeanValue Max MeanValue The MeanValue column is derived from the average of the well values found in the Values column the Standard replicates The minimum and maximum values from the MeanValue column in the Standards group are used to create the summaries and the summary formulas are used to determine our Outli rs Such a formula might be useful in finding points that might be better masked not used in the calculations for example Basic information regarding formulas and the information they can contain can be found in Chapter 5 Displaying and Analyzing Data For a complete discussion of writing formulas in SOFTmax PRO for Lmax as well as detailed SOFTmax PRO User s Manual 7 27 Chapter 7 Tutorial e E Standards Section Showing Replicates Default After Hiding Replicates 28 examples of formulas and customized protocols refer to the Formula Reference Guide Hide Replicates While the Standards section is active choose Hide Repli cates from the Group menu The display before and after hiding replicates is shown in Figure 7 35 standards Ha 24 Max value Maxi values Standards pg ul group blank 1 210 BEAU a EU O00 H1 6 900 7 000 0 100 7 100 He r000 ES T 100 ste eal
11. 22 e cece ences 6 12 Customizing the Report 0 sees 6 12 The Print Dialog Box eee ee eee eee ee 6 14 Exporting Importing Information 00 6 16 Exporting Data for Later Reimport 6 16 Exporting Data General Information 6 16 Importing Data from Outside of DOF Imax PRO for Lmax is ovau su tw te Swiss 6 20 Importing and Exporting Templates 6 20 Exporting Graphs 0 0 e eee eee eee 6 21 Copying and Pasting oer vb r opEP PEU te Se et ee 6 21 Password Protein ve ees ce Ve wae se wie a or Er oe EP 6 25 Setting and Changing a Password Ls 6 26 Chapter 6 File Management and Printing 6 2 SOFTmax PRO for Lmax User s Manual 8S Chapter 6 File Management and Printing File Creation and Management During routine use of SOFTmax PRO for Lmax you will create open and save numerous files You may wish to create file folders that will make it easy to locate particular files It is also important to understand the way in which SOFTmax PRO for Lmax finds and uses default protocol files to enable certain assay options automatically When you install SOFTmax PRO for Lmax certain files and folders directo ries are created on your computer s hard disk One of these files is known as the Default Protocol so named on the Macintosh called DEFAULT LPR in Windows The Default Protocol file
12. 4 20 A ANNONA Changing the column format setting will cause the columns in the group to revert to their original defaults as shown in Table 4 2 on page 4 18 If you have added columns or changed column information within the existing section for the group you are editing these changes will be removed The Button Allows you to deselect or clear wells in the map of the microplate Highlight the wells you wish to clear and then click the Clear button Data for cleared wells will no longer be shown in the data display The Sample Area The Sample area contains the Sample drop down list and sample descriptor values active if defined in the Group Settings dialog box or if assigned auto matically by SOFTmax PRO for Lmax Sample 5ta01 we Sample Name or ID Concentration Sample Descriptor Figure 4 17 Modifying Sample Names and Descriptor Values in the Template Editor NIS When selecting wells to change sample names or descrip tors be sure all wells are replicates of the same sample and group or that you want them to become replicates of the same sample and group SOFTmax PRO for Lmax does not prohibit selecting individ ual wells from different samples and or groups for example and changing their descriptions and they will change to become the same Sample Drop Down List The sample drop down list consists of two areas divided by a horizontal gray line Before other sample names have been defined a single pre defin
13. Button Equivalent in the Group section SOFTmax PRO for Lmax User s Manual 3 31 Chapter 3 SOFTmax PRO for Lmax Overview BO nwu nuunuu BEEEERE BBEBEEE LA 5 1548 gs a Yay Window Tile Cascade Arrange Icons w T Untitled anwuwuwuuwu g TL T 1459 55 Help Contents Indes Using Help About SOFT max PAO for Lmas 3 32 Autosize Choosing this command expands or shrinks the width of selected columns to accommodate the largest piece of text in the column Mouse Equivalent Double clicking the line to the right of the column heading performs the same function Hide Hides the selected column from view This command is dimmed if a column has not been selected Mouse Equivalent Dragging the line to the right of the column head ing until it is on top of the line to the left of the column performs the same function Hide Replicates Hides data for all replicates if any from view Show All Shows all columns or replicates that were hidden previously Show Formulas Causes a new row to appear beneath the headings of each column showing the formulas used to determine the data in the col umns After choosing this command the menu name changes to Hide Formulas Group Settings Opens the Group Settings dialog box allowing you to re name a group and or change the sample or group descriptors used as well as to modify the units used with each descriptor Mouse Equivalent doubl
14. Testing coordinated by Emma Ratner Thanks Chris S JOW SG LC and TH Copyright 2000 Molecular Devices Corp a All rights reserved Figure 3 1 SOF Tmax PRO for Lmax Title Screen SOFTmax PRO for Lmax User s Manual 3 3 Chapter 3 SOFTmax PRO for Lmax Overview BO After a few seconds the title screen will be replaced by an untitled SOFTmax PRO for Lmax window Figure 3 2 below shows an example default protocol screen for the PC The SOFTmax PRO for Lmax screen contains these major elements e A menu and status bar located at the top of the screen e An untitled window showing an experiment containing various sections depending upon the configuration of the default protocol used and the type of instrument chosen in the Preferences or connected to the computer The menu and status bars are always visible while you are using SOFTmax PRO for Lmax no matter where you are within the program Title Bar Menu Bar Minimize Button Maximize Button Status Bar lose BF SOFT max PAO for Lae Box File Edi View Experiment Contel Assays Window Help fre Coot 1 a Experiment Bi Untitied Scroll Section Be r amp experimenuitt Arrow Tool Bar v E nates 24 sf Notes T UA fa Dishes cmd Setup Teplaba 5 Fiii i Dong kel zx Plates Open 1 2 4 4 5 amp i T a Tool Close A Bar Triangle indicator Instrument Settings Pisis Las
15. The status bar shows the following items from left to right Instrument icon An icon such as that shown at the left indicates that the Lmax is communicating properly with the computer If an X appears on top of the icon the computer is not making proper contact with the Lmax NOJ Double clicking the Lmax icon will open the Preferences dialog box Temperature display A small window after the Lmax icon shows the current temperature within the microplate chamber in degrees Celsius The Read button If a single Plate section has been created in the experiment window clicking the Read button starts the reading This reading is based on the settings chosen in the Instrument Settings dialog box refer to Instru ment Setup for the plate If more than one Plate section has been created in the experiment window and no Plate section is currently active clicking the Read button opens a dialog box asking you to select which plate to read If a Plate section is active when you click the Read button that section is read automatically The Incubator button Clicking this button causes the Incubator dialog box to appear allowing you to set and regulate the temperature of the microplate chamber The temperature setting can be left at the default or can be specified by typing a different value in the box The incubator setting is independent of the protocol being run Run ning an assay does not automatically set the temper
16. gm SOFT max PRU for C TEMP Swncows Z Drives Cancel Select Assays c COMPUTER Cancel Figure 6 2 Set Folder Dialog Box Macintosh and PC Select the folder you wish to use and then click the large button at the bottom Mac or right Windows of the dialog box to set this folder as the default Kesey Molecular Devices recommends that you have only one default protocol Opening Other Protocol Files The bottom half of the Assays menu provides a shortcut for opening other protocol files contained in the set folder These usually consist of protocols you or others create for repeated use To choose a different protocol file click the protocol file you wish to use An untitled copy of that protocol file will open in a new window Locating Protocol and Data Files You may find it useful to create separate folders subdirectories within which to store the data files that SOFTmax PRO for Lmax creates In addition you may want to create and save various protocol files for use as templates for later readings In a multi user environment it might be helpful to create a folder subdirectory for each SOFTmax PRO for Lmax user or to create sepa rate folders subdirectories for different types of experiments We recommend that you consider the way in which you will organize your files before creat ing too many of them otherwise you may find that locating a particular file among the many that fill your hard disk
17. mle 5 300 6 000 0 100 17 6 100 6 000 6 100 SIE 125 000 5i 4900 5 Dt 0 100 F100 5 000 5 100 3 900 4 000 0 100 25 4 100 4 00 410 aJ 3 000 Bi 33 3 100 3 00 3 10 1 90 SAUL 0 100 40 2 100 n stl 7000 d zh 1 000 0 100 100 1 100 1 000 1 100 imalest standard salue T Largest standard vale 7 000 m Standards Standards pg group blank 1 210 malest standacd yale 1 000 Largest standard value 7 000 Figure 7 35 Standard Section Before and After Hide Replicates More information about modifying group sections can be found in Chapter 5 Displaying and Analyzing Data SOFTmax PRO User s Manual 8S Chapter 7 Tutorial Step 8 Data Analysis Standard Curve The Default Protocol which is the same as the protocol file created in this tutorial example contains the predefined groups Standards Unknowns and Unk w dilution In addition it contains a graph section which has been cre ated to display a standard curve If you have not already done so open the Default Protocol to create a new Unknown file and with the simulator still running click the Read button to gather simulated data Scroll down within the SOFTmax PRO for Lmax window until you see the section tool bar for Std graph This section is closed click the indicator on the left side of the tool bar to open it This graph contains a single plot as shown in Figure 7 36 v Z stdoraph i Ft 0 100 200 300 400 500 600 700 0
18. plicate and New on page 6 24 Text Style 381 CTRL T Allows you to change the attributes of selected text This command is dimmed if no text has been selected Preferences double click the instrument icon Through the Preferences dialog box you can choose settings for the following options e Serial port connection modem port or printer port for the Macin tosh COMI or COM2 for the PC that will be used to connect to the Lmax A third option Neither should be used when no instru ment is connected to the computer Export format Time versus Plate and whether or not to include labels e Whether or not files will be saved automatically and the location where saved files will be stored Autosave e Whether or not reports should be printed automatically Auto print Suspend Recalculation Stops SOFTmax PRO for Lmax from performing cal culations based on changes you make to the settings or other program information This command is used most often when you want to make a number of changes to the settings without having to wait while the results of each change are recalculated Recalculate Now 8 CTRL 2 Causes SOFTmax PRO for Lmax to recalcu late data based on the current settings This command is used most of ten when you have suspended automatic recalculation SOFTmax PRO for Lmax User s Manual 3 25 Chapter 3 SOFTmax PRO for Lmax Overview BO View Menu View Minimize
19. to de scribe how the series will fill the wells and a number of replicates Various op tions are diagrammed in Figure 4 23 using a 4 x4 block of wells for more information about replicates see Replicate Samples and Samples in a Series on page 4 21 The maximum number of replicates depends on the selection of wells if you are filling down from the top for example and you have chosen a block of wells that is 4 wells wide by 8 wells high the maximum number of replicates will be four with the same block of wells selected filling from the left would allow eight replicates If the number of replicates you choose does not divide evenly into the number of rows or columns you select depending on the filling direction the remaining wells that cannot contain replicates will be labeled as additional individual wells in the series For example if you fill a block of 4 x 4 wells from the top and choose three replicates then the first three columns will contain the replicates and the last column will contain the additional wells in the series see the last diagram in Figure 4 23 This repli cate feature allows you to quickly define the number of replicates and in cases where the series has more samples than a single row or column to name them simultaneously To create a series with all 96 samples highlight all the wells in the plate select the fill direction and set the replicates to 1 Fill from Top one replicate Fill from Bottom one r
20. 1 10 Glossary OF TCs isan oe hy DEAE a A 1 11 Chapter 1 Welcome to SOFTmax PRO for Lmax 1 2 SOFTmax PRO for Lmax User s Manual 2 Chapter 1 Welcome to SOFTmax PRO for Lmax Introduction The Lmax Microplate Luminometer uses SOFTmax PRO for Lmax soft ware This software allows you to create files containing all of the parameters required to acquire and analyze data from the Lmax SOFTmax PRO for Lmax version 1 0L is available in either Macintosh or Windows format The Macintosh version of the software is a fat binary application that includes native code for both 68K and Power Macintosh computers The Windows ver sion is a 32 bit application that is Windows 95 98 2000 and Windows NT 4 0 compliant SOFTmax PRO for Lmax is powerful yet easy to use It performs three major functions e Instrument Control You can set up and run a complete assay Instrument settings can be saved as a protocol file and used repeatedly for reading dif ferent microplates Data Collection SOFTmax PRO for Lmax collects and stores all raw data received from the Lmax Data is displayed on the computer screen in a grid format that corresponds to the wells in a microplate How the data is shown depends on other settings made using the software e Data Reduction and Plotting You can reduce the data using built in for mulas or you can define custom reduction formulas More than one reduc tion can be shown and results from different m
21. Button Equivalent 4 in the Notes section Show Formulas Section Mame New Window Edit Summary lIf you have created a summary and need to change it you can do so by first highlighting the summary you wish to edit and then choosing Edit Summary This command is dimmed if you have not yet created a summary or if the summary has not been selected The dialog box then appears showing the name and formula Mouse Equivalent Double clicking the summary performs the same function Button Equivalent in the Notes section Show Formulas Choosing this command shows the formulas associated with the summaries you have created in the Notes section After choosing Show Formulas the menu name changes to Hide Formulas This com mand is dimmed if you have not created any summaries Section Name Allows you to change the name of the Notes section Mouse Equivalent double click the name of the Notes section introduction to the right of the Notes section icon New Window Opens the active section in a separate window Mouse Equivalent double click the icon in the Notes section Graph Menu Graph This menu item changes to show Plate Notes Graph or Group depending E p on the type of section that is currently active If no section is active this menu item does not appear 2S MIS Y Axis Graph Options You can select from three types of graphs Scatter with uem Esel symbols plotted and or the poin
22. Displaying and Analyzing Data BO Data Reduction 5 16 1 2 3 To remove masking from one or more wells highlight the masked well s and click the Mask button in the Plate section tool bar toggles masking on and off or choose Mask from the Plate menu again The diagonal lines covering those wells and masking function will be removed Raw data received from the Lmax can be displayed in an analyzed or reduced form The reduction process within SOFTmax PRO for Lmax is based on formulas that reduce the raw data to show a single number for each well Further analysis of this reduced number then takes place in the Group and Graph sections SOFTmax PRO for Lmax performs calculations hierarchically when reducing the luminescence information collected from the instrument Calculations in the Plate section are performed in the order shown below If an option has either not been selected in the Instrument Settings dialog box or has not been defined in the template and or Reduction dialog boxes SOFTmax PRO for Lmax starts continues with the next listed calculation Plate blank subtraction Endpoint or kinetic reduction Group blank subtraction You can access the Reduction dialog box in several ways by clicking the Reduction button in the tool bar of the active Plate section by choosing Reduction from the Plate menu or by clicking the Reduction but ton in the Well Graph display The appearance of the Reduction dialog box
23. Plate 1 the topmost print command in the File menu would be Print Plate 1 If more than one section is select ed the menu will show Print Selection You can select multiple sections to be printed by holding down the Shift key and clicking on the section tool bars to make them active 5 Enlarged displays well graphs of kinetic data can be printed by click ing the Print button in the Well Graph window The size of the print out is dependent on the size of the well graph If you enlarge the well graph to greater than 12 inches the printout will be truncated Regardless of which print option you select make sure the printer is con nected properly to the computer and that it is turned on prior to sending the print request If Autoprint is enabled make sure the printer is connected and that it is turned on prior to starting a reading If you are using a Macintosh you can check the Chooser for the currently selected connected printer If you are using Windows you can select a printer by choosing Print Setup from the File menu In the Print Setup dialog box choose the default printer or select from any other printers have been configured for use with Windows See your Windows documentation for further information SOFTmax PRO for Lmax User s Manual 6 11 Chapter 6 File Management and Printing BO The Printed Report You can create customized reports by including or excluding experiments and individual se
24. Plate with Template Graph Section Plots Created with Information from Standards Group Table Initial Data from Lmax Unknowns Group Table Reduced Number from Plate Reported Here Interpolated Values Results Reported from Graph Section Figure 5 1 Relationship of Sections This chapter discusses the initial display and reduction of data in the Plate section first followed by display and data analysis in the Group Graph and Notes sections SOFTmax PRO for Lmax User s Manual 5 3 Chapter 5 Displaying and Analyzing Data BO Initial Data Display and Reduction The manner in which data is displayed in a Plate section depends on the type of reading that was performed endpoint dual read long kinetics or fast kinetics but can be customized by choices made in the data display and reduction settings Depending on the type of reading performed display options include e Number or plot displays for RLU relative luminescence units with or without reduced number The number format use of commas or periods in numeric notation chosen for the computer operating system in general is reflected in the way that SOFTmax PRO for Lmax displays data e Number Plot Threshold Ranged or Grayscale with or without reduced number e If you have chosen 384 well plates from the Instrument Settings you can display the wells in different ways Normal Vertical Rotated Large or Interleaved Default displays are s
25. Printer Modem b Figure 8 2 8 Pin Din Connection to Macintosh Computer If you are connecting the Macintosh to an Lmax the other end of the cable should also be an 8 pin DIN connector Figure 8 3 8 Pin DIN to 8 Pin DIN Serial Cable You should use a double shielded straight through serial cable If you are unsure of the quality of the cable you are using Molecular Devices suggests that you purchase a cable of known quality and try the connection again 8 8 SOFTmax PRO for Lmax User s Manual Chapter 8 Troubleshooting F PC Cable Connections The connector required to attach to your PC will depend upon the type of serial port s available on your computer Typically at least one 9 pin or 25 pin serial port is available on a PC most have two which are known as COM1 and COM2 respectively The connector for your computer may be a 9pin or 25 pin connector or you can use an adapter that converts from 9 pins to 25 pins see Figure 8 4 4soad 9 pin to 25 pin adapter 25 pin Figure 8 4 25 Pin DIN to 25 Pin or 9 Pin Serial Cable The cable itself should be a double shielded straight through serial cable If you are unsure of the quality of the cable you are using Molecular Devices suggests that you purchase a cable of known quality and try the connection again SOFTmax PRO for Lmax User s Manual 6 9 Chapter 8 Troubleshooting Display Problems A large Group section table You are attempting
26. This manual does not represent a commitment on the part of Molecular Devices Corporation The software described in this document is furnished under a license agreement or nondisclosure agreement The software may be used or copied only in accordance with the terms of the agreement It is against the law to copy the software on any medium except as specifically allowed in the license or nondisclosure agreement ii SOFTmax PRO for Lmax User s Manual e SOFTmax PRO for Lmax Software License Agreement and Warranty Important The right to use this Molecular Devices Corporation MDC software is sold only on the condition that the user agrees to the terms of this license agreement If you do not agree to the terms of this license you may return the unopened package to MDC or its authorized agent for a full refund However opening the package indicates your acceptance of these terms and conditions License Agreement In consideration of payment of the license fee which is part of the purchase price you n for this product Molecular Devices Corporation MDC the licensor enr grants a non exclusive license to you the Lic ensee User to use SOFTmax PRO for Lmax and its utilities Software included with SOFTmax PRO for Lmax upon license The user is licensed to copy the software for a backup or archival copy as necessary Simultaneous use of the software with more than one MAXline Microplate Reader per license is eod prohibited Sim
27. This mechanism is discussed in detail later in the Appendix SOFTmax PRO for Lmax User s Manual A 3 Appendix A SOFTmax PRO for Lmax Remote Command Language BO SOFTmax PRO for Lmax Commands SOFTmax PRO for Lmax commands are single words If parameters are required with the command a colon is used between the command and the parameters Close Close the current document NISNUNGNB If the data has not been saved the document is closed any way with no user warning Copy This is equivalent to pressing CTRL C or selecting Copy from the Edit menu Notes e If a plate section has just been read or is the only selected section the plate data will be copied to the clipboard e The data copied will follow the display settings for the current plate nor mally Raw Data Other sections such as Groups can also be copied The SelectAll command should be used first ExportAs XXX Tell SOFTmax PRO for Lmax to export data in the standard SOFTmax PRO for Lmax format Importlemplate XXX Have SOFTmax PRO for Lmax import a template file The file should comply with SOFTmax PRO for Lmax s template file format A plate section should be selected prior to importing a template NameCurSection This re names the current selected section If no section is selected nothing happens Notes e This command is used typically to identify different plates in an automated environment e The data copied will follow the displ
28. Use either the default name Group X where X is an integer that is initially set to 1 and increments i e 1 2 3 etc as other groups are created or enter a custom name up to 32 characters in length In the Sample Descriptor area of the Group Settings dialog box you can enable the application of the sample descriptor if appropriate by clicking the checkbox You may also choose the units used for the sample descriptor by selecting an option from the Units drop down list or by typing the units you wish to use into the list box The Sample Descriptor is automatically enabled when either the Standards or Unknowns Dilution column format see below is selected The descriptor defaults to Concentration with the Standards column format and to Dilution with the Unknowns Dilution column format The Sample Descriptor may also be used for time or fraction number information No sample descriptor is assigned automatically for the Basic or Unknowns column format You may assign a sample descriptor to these column formats manually but if you choose to do this the information will not appear automatically in the Group section To see it you will need to create a new column containing the formula ISampledescriptor For more information about creating columns see Adding Columns on page 5 26 Column Format defines the default column types that will be created for a new group or shows the original column format setting that was us
29. if the data shown in the Values column of the Unknowns section is greater than or equal to the calculation of MinStd found in the Standards group section and less than or equal to the calculation of MaxStd found in the Standards group section then show nothing signi fied by two quotation marks with nothing between them otherwise show the word Outlier Looking at the Standards section you will not immediately see the MinStd or MaxStd calculations used by this If statement They are there but their names SOFTmax PRO User s Manual Chapter 7 Tutorial are hidden Look at the bottom of the Standards section and you will see two summaries as shown in the following figure F3 0 stid ba 5l E 3300 4 000 0 100 25 Ez 4 000 ES 4 100 5th 31290 D1 2 300 3 000 0 100 33 De 3 000 D3 3 100 ot 15 525 C1 1 900 e DL 0 100 5 0 Ce SAEI C3 SUL Sun FOO B1 0 900 1 000 0 100 10 0 Be 1 000 LH dB3 1 100 malest standard wahe 1 000 Largest standard vale 7 000 Figure 7 33 Summaries in the Standards Section Double click the first summary Smallest standard value A Calculation dia log box will appear W Calculation Description Smallest standard value Formula Mint Mean alue Decimal Places oce Cancel Figure 7 34 Calculation Dialog Box from Summary in Standards Section This dialog box contains a checkbox for Hide Name
30. need additional assistance in the U S call Molecular Devices Technical Ser vices group at 800 635 5577 or 408 747 1700 elsewhere call your local repre sentative 1 10 SOFTmax PRO for Lmax User s Manual 8S Chapter 1 Welcome to SOFTmax PRO for Lmax Glossary of Terms Active The window or icon that you are currently using or that is currently selected The active section is the section in which you are currently working The title bar for the active section or window will be highlighted gray inactive sec tions are not highlighted white Only one section or window can be active at a time To make a section or window active click anywhere within it If you scroll past the bottom or top of the active section the tool bar for that section sticks to the top of the window you can always tell which section is active in this way and can always access the items in its tool bar ANSI Character Set The American National Standards Institute 8 bit character set It contains 256 characters Area Under Curve Reduction formula for a kinetic reading that determines the area under the kinetic plot or under a plot in a Graph section First the area under the curve is divided into a series of trapezoids then the area defined by each trapezoid is computed and all areas are summed ASCII Character Set The American Standard Code for Information Interchange 7 bit character set It consists of the first 128 0 127 characters o
31. same sample name and description within a group to multiple wells to create replicates For example you might want to read standards in replicate to ensure that anomalies can be excluded prior to generating a standard curve Replicates can be created using the Assign button or with the series function SOFTmax PRO for Lmax User s Manual 4 21 Chapter 4 Reading Microplates BQ Creating a series is faster than assigning replicates individually The series option can be used as long as the replicate values conform to a pattern that can be described by a mathematical operator multiply by 2 or add 5 for example A series is defined in one direction starting from left right top or bottom and therefore samples must be arranged sequentially in either ascending or descending order To create a series do the following 1 Inthe Template Editor highlight the wells to be part of the series The wells must be in the shape of a rectangle EXDEIMITIEN Ecko EC rate oup onra F E meer m Concentration Series Group Name Button Pull Down MASS Sample Name Box and Selected Wells Units Figure 4 21 Template with Wells Selected for a Series 2 Click the button to open the Series dialog box Series Start From First Sample Sta 8 i Top px Sample Descriptor E onu eet ane I en b C Left Starting value Replicates 3 Figure 4 22 Series Dialog Box 3 Specify the name of the fi
32. sei uu mem oe a a a a eae 0 001 f Serre erry es 0 001 0 01 0 1 1 10 100 1000 Conc Tmo well a PhD ATF 405 dels Concentration vi Meanalus Figure 5 29 Graph with No Fit You can fit any plot to one of nine curve fitting algorithms Linear Semi Log Log Log Quadratic 4 Parameter logistic Log Logit Point to Point Expo nential and Cubic Spline These selections are shown in the Fits pop up menu in the Graph section toolbar click on the box next to Fit Typically a standard curve refers to the curve fitted to the plot of concentra tion versus mean value for the Standard group Once you have selected a Fit type SOFTmax PRO for Lmax determines the parameter values that best fit the data The function with these parameters is then drawn on the graph Ideally the type of fit used should be determined by the underlying chemistry of the assay and could be set before data is read A good discussion of curve fitting appears in Data Analysis and Quality Control of Assays A Practical Primer by R P Channing Rogers in Practical Immuno Assay edited by Wilfrid R Butt published by Marcel Dekker Inc New York 1984 When a fit is chosen the Graph section is updated to show the coefficients describing the fit when applicable These values are displayed in the legend at the bottom of the graph Figures 5 30 through 5 37 show the same graph with the different fit algorithms applied Note that the legend information chang
33. will be different depending on the type of read mode you have chosen and the options you have chosen in the Instrument Settings dialog box Figure 5 11 shows the Reduction dialog box for endpoint dual read and kinetic modes along with the default reductions for each of those modes SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data Reduction Dialog Boxes Default Reductions m Reduction x Endpoint Custom w Male kaiaa Wavelength Combination AAA i MW use pre read plate Switch normal and pre read Cancel m3 Reduction Dual Read Custom TON w Wale Dee Wavelength Combinatiorr Du D E IPV alue IMValue l PV alue AIN AONE TET AEE TAE A SE EES OUT MME MH E RES IPalue MM Value IIS alue PY alue IP alue IM Value Cancel my Reduction xi Long Kinetics TET and v IMValue us Wavelength CambiliStiB Pr mmm nnnnnnnnnnnnnnnnnnnn nnne ene ennnnnnnnnnnnnnnnnnnnnnanitttstsestesettntnnnnnnnnnsnananaanius Fast Kinetics Vmax B c IU C ea a ce EE E UNAM IA UM ME SUM E Vina units per sec i Time to Vira Ymax units per sec Ww v hax Points 20 Onset Time ha aximum j Minune Min RLU Lag Time bo IV Absolute Values Time at Maximum Max RLL End Time 2 Time at Minimum Time at 142 Maximum Slope Area Under Curve Custom Figure 5 11 Reduction Dialog Boxes for All Read Modes SOFTmax PRO for Lmax User s Manu
34. 3 27 4 29 entire plate 4 12 microplate 4 29 mode 4 9 Dual Read 1 3 Endpoint 1 3 4 9 Fast kinetics 1 4 Long kinetics 1 4 partial plate 4 12 strips 4 12 Recalculate Now command 3 25 5 22 Recalculation options 5 22 Reduced Number display 5 9 Reduction 5 4 5 16 command 3 28 custom formula 5 21 defaults 5 18 5 19 kinetic 5 19 options 5 4 parameters 5 3 Registration dialog box 2 8 Replicates 4 21 Report 6 12 customizing 6 12 default setting 7 31 printing 6 11 6 12 7 31 including excluding sections 6 12 RLU values absolute 5 19 S Sample descriptor 4 17 Save command 3 22 6 5 6 9 7 18 Save As command 3 22 6 5 6 9 Saving files manually 6 9 SOFTmax PRO for Lmax User s Manual e Scroll 1 20 Scroll bar 3 9 Section 3 8 active 1 11 3 8 3 9 3 20 3 23 3 24 3 26 3 30 changing order 6 13 creating 3 9 deleting 3 7 3 9 Graph 3 8 3 17 5 28 5 35 curve fit 5 34 5 35 options 5 44 plots 5 35 Group 3 8 3 15 5 22 7 21 columns 7 22 customizing 3 8 text format 6 13 inactive 3 8 Notes 3 8 3 12 5 28 text format 6 13 opening closing 3 10 order 6 13 Plate 3 8 3 13 printing order 6 12 relationship 5 3 Standard Curve 7 29 tool bar 3 9 Select All command 3 24 Semi Log curve fit 1 20 5 35 5 36 Serial port 1 20 2 11 8 5 Series 4 21 4 23 replicates 4 21 Show All command 3 32 5 23 Show Formulas command 3 30 3 32 5 24 5 27 5 28 Size box 5 15 Slope 1 20 5 21 Specifications Ma
35. 7 14 Unknowns Changed to aSeries eller 7 15 Group Settings Dialog Box for Unknowns with Dilution 7 15 Templie CONIDISIO La ed secu rau qut uro Rum RCUREULRURER EUR RRCRA RUE RURRUERE RR ER ORE 7 16 The Plate Section Showing Template Group Boundaries 7 16 Display Dialog Box Endpoint 0 cece eens 7 17 Display Dialog Box after Choosing Reduced and Clicking the Display Choices Drop Down Menu 0s eee cnet eens 7 17 Save As Dialog Box Macintosh 0 eee eens 7 18 Save As Dialog Box Windows 0 ce eee eee 7 18 SOFTmax PRO for Lmax User s Manual xi Figure 7 22 Figure 7 23 Figure 7 24 Figure 7 25 Figure 7 26 Figure 7 27 Figure 7 28 Figure 7 29 Figure 7 30 Figure 7 31 Figure 7 32 Figure 7 33 Figure 7 34 Figure 7 35 Figure 7 36 Figure 7 37 Figure 7 38 Figure 7 39 Figure 7 40 Figure 8 1 Figure 8 2 Figure 8 3 Figure 8 4 Figure 8 5 Figure 8 6 Tables xii Table 3 1 Table 3 2 Table 3 3 Table 3 4 Table 4 1 Table 4 2 Table 5 1 Table 6 1 Table 6 2 Table 6 3 Table 6 4 Table 6 5 Simulator Choice in the Preferences Dialog Box 0 00 eee eee ee 7 19 Plate Section with Simulated Data 0 cece eee eee eee 7 20 Display Dialog Box Updated with Low and High Limit Settings 7 20 Plate Display with Data Displayed as Gray Scale 0 2 eee eee 7 21 The Group sec
36. Contiguous column formulas may be copied and pasted together Summaries must be copied and pasted individually To copy a formula or summary highlight it and then select Copy 38C CTRL C from the Edit menu Activate the area or section in which you would like to paste it if necessary with a formula access the particular dialog box into which it will be pasted After placing the cursor in the correct location and highlighting any existing information to overwrite it select Paste 3 V CTRL V from the Edit menu The pasted formula or summary will appear in the new location Formulas and summaries can be deleted by highlighting them and pressing the Delete key or choosing Cut from the Edit menu Duplicate and New All sections and even complete experiments can be duplicated by making the section or experiment active and then selecting Duplicate from the Edit menu All of the information in the section or experiment including data is copied to the newly created section or experiment The name of the new section or experi ment is the same as that of the original with the addition of the word Copy e g OldName Copy Duplicated sections are created at the end of the exper iment Duplicated experiments are created beneath the other experiments Group section tables can also be duplicated When a Group table is dupli cated its name is added to the Template Editor s list of group names but the group is not assigned to any wells Gra
37. Copy Template Paste Template Export Template Import Template Section Mame New Window Figure 4 27 Copy Template and Paste Template Commands in the Plate Menu The group names and sample names of the pasted template are identical to those of the source template In effect the destination template is considered an extension of the original template and all wells on the destination plate are considered replicates of the wells on the source plate same group and sample names Any changes made to the template on the destination plate will also be made on the source plate Data from the two Plate sectionss will be analyzed together Copying and Pasting between Different Experiment Sections To copy a template make the section containing that template active and choose Copy Template from the Plate menu the menu name changes to reflect the type of active section Then activate the destination Plate section and choose Paste Template from the Plate menu The group names and sample names of the pasted template will be identical to those of the source template but since they are in different experiments the full name will be different for example group experiment 2 instead of group experiment 1 Any changes made to the template of the destination Plate section will not affect the source section Data from the two Plate sections will not be analyzed together Copying and Pasting Template Regions Contiguous regions of templ
38. Data mode Luminescence Data type Raw or Reduced Pre read included TRUE or FALSE Kinetic points Reduced plates and endpoint plates 1 Kinetic plates number of reads Kinetic read time Kinetic read time in seconds otherwise blank field Kinetic interval Kinetic read interval in seconds otherwise blank field Blank field Blank field Blank field Number of wavelengths read 1 for endpoint and kinetics 2 for dual read Wavelengths used Always All First column read First column read 1 to max Number of columns Last column read 1 to max Number of wells Number of wells in a plate 96 or 384 The information for the first plate section is provided in the lines following the descriptors The number of lines of data depend on the export format and the type of test that is run A minimum of one line of data is exported when the Time format is used with an endpoint read NOH The temperature at the time of the reading will appear before the first data point in the reading The second line of a file for a Group table section includes fields that corre spond to the number and types of columns found in that section they are labeled as the table is labeled and look very much like the table itself Following the data a blank line is included and then a line with a single ter mination field End Each subsequent data set in the file includes the lines described above a blank line and a line with the termination field SOFTmax P
39. Dialog Box for the Group Named Patient 1 4 19 Groups Settings Dialog Box Showing Original Column Format 4 19 Modifying Sample Names and Descriptor Values in the Template Editor 4 20 The Sample Drop Down List When this list first appears the only selection shown below the dividing line is BL the active sample name that is ready to be assigned appears above the line 4 20 Sample Drop Down List 5ample Names Defined and Wells Selected 4 2 Assign Area of the Template Editor Tool Bar 0 cee ee eee eee 4 21 Template with Wells Selected foraSeries cece eee eee ee eee 4 22 De tles DO BOX CC E E E E E E E EEA 4 22 Fill Options and Replicate Settings 0 cece eee eee 4 23 Pre Read Plate Blanking Section of the Instrument Settings Dialog Box Endpoint Mode Only 00 eect cece e 4 24 Graphic Representation of Plate Blank Subtraction 00 ee eee 4 26 Template Editor Showing Plate Blank and Group Associated Blank Wells 4 27 Copy Template and Paste Template Commands in the Plate Menu 4 28 belect5echon Dialog BOX a2 aue tt ont trt drm RUSO REUS ERE een 4 29 Alert Regarding Replacing Data for Plate Section 0 0 00 4 29 Status Bar during a Long Kinetics Reading 0 0 0 cece eee ee eee 4 30 Relationship of Sections 0 ee ec cence e 5 3 Endpoint Dual Read and Long and Fast Kinetics Displa
40. Graph command This command is dimmed if the instrument settings are for an endpoint reading or if no well in the Plate display has been selected Button Equivalent in the Plate section tool bar Mask Allows you to hide certain wells so that they will not be taken into account when performing analyses This command is dimmed if no data has been acquired or if no wells in the plate have been selected Button Equivalent in the Plate section tool bar Copy Template While a Plate section is open and active choosing this com mand allows you to copy the template from this Plate section to the Clipboard allowing you to paste it into a different Plate section in the same or in a different experiment or into a Plate section in another SOFTmax PRO for Lmax file Paste Template If you have copied a template from a Plate section you can then create or activate open another Plate section and choose Paste Template In this way you can use the same template for more than one Plate section or you can modify the pasted template as desired If you are pasting a template into a new experiment and groups from the source template do not exist in the destination experiment pasting the template will create them Existing groups of the same name will not be changed by the pasting process For example if the template be ing copied contains the groups Standards and Unknown and the group Standards already exists in the destination exp
41. It will contain the SOFTmax PRO program If you want SOFTmax PRO for Lmax to be able to communicate with your Lmax be sure to turn the Lmax on prior to starting the SOFTmax PRO for Lmax program After the instrument Ist on and has completed its start up sequence double click the Ipf icon to start SOFTmax PRO for Lmax NOH In Windows a shortcut for the program will be available in the Start menu under Programs SOFTmax PRO for Lmax pf SOFTmax PRO for Lmax User s Manual 2 7 Chapter 2 Installation a What Is Installed A folder called SOFTmax PRO for Lmax will be installed in the area you specified on your computer s hard drive Within this folder are a number of different files and subfolders folder and filenames are the same for Windows and Macintosh except that Windows files have the extension lpr appended to the file name An uninstall program is available for Windows computers Should you need to remove an older version of SOFTmax PRO for Lmax from your hard drive we recommend using the uninstall program since it also removes information regarding SOFTmax PRO for Lmax from the Windows Registry To remove any version of SOFTmax PRO for Lmax from a Macintosh computer simply drag the SOFTmax PRO for Lmax folder and any other SOFTmax PRO for Lmax files you may have on your hard drive to the trash Enter Registration Information Turn on the Lmax and allow it to complete its start up sequence
42. Lmax 6 20 Importing and Exporting Templates 0 0 0 0 cece eee eee 6 20 Exporting Graphs 02 cece cece eee eee ee ete te tenons 6 21 Copying and Vast e eus a np dr np ACUAEUREURUS A Gas 6 21 Password Proteco un ma i daret RUE 176 8794 81997081 4890 EAA 6 25 Setting and Changing a Password 0 c eee eee cent eens 6 26 v1 SOFTmax PRO for Lmax User s Manual e Chapter 7 Tutotial 222a xacpeE RR Rua days ss Ebr Rossa Rc Ra dob ed P ee doe 7 1 lau ole a e EFE TETTTITIC TOT TITOTIC TIT OTIC C10 0111 1 T1 T1 T7107 7 3 Hard and Soft Parameters ees 7 3 Description of the Assay Example 0 cece eee eee ee eee 7 4 Define the Protocol 0 ccc ccc cee ec eee eee cree ee eens eee seeeseveees 7 4 Start SOFTmax PRO for Lmax and Open the Tutorial File 7 5 Define the Instrument 5ettinps cre cesecebeReReRI RO RI DEPRECOR REO ve PERO 7 6 Denne the T5mpldl8 e acceded acuta cute ce beefed be ce EErEE 7 11 Set the Display Parameters ded t cde be ce ie cuba ce ba VET EET ERE EETE TRETEN 7 17 Save the Protocol 1 2 duds dedo8sdedsdedudedededetedwiwiededeivdudeds 7 18 Read thePlate cc RII RII RIA 7 19 Data Analysis Group Sections esee eee 7 21 Data Analysis Standard Curve 0 0 cece een 7 29 PML AINCCOll oes ibunt REA ETUR RA ERA RETE RAE TA WAR WUER RETIA WE dS 7 31 Chapter 8 Troubleshooun 2 22 euo sx ERE Ra
43. Plot 1 1s integration pg ml ws Mean saluel 0 092 0 686 0 995 Figure 5 32 Graph with Log Log Fit simulated data Quadratic The quadratic function fits the best parabola to the data The parabola is a curved line based on the equation y A B x C x which is shown on the screen as y A B x C x 2 where A is the intercept B is the slope of the curve at the intercept and C is the measure of the curvature of the parabola The quadratic fit is most appropriate when the standard curve has a tendency to curve up or down An example of a quadratic fit is shown in Figure 5 33 5 38 SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data we 7 Graph Fit _Quadratic Y braph 1i T a m JT T ka 15 Concentration y Bx s C o STD Standards Concentration ws lean alue f z 18e 4 Figure 5 33 Graph with Quadratic Fit simulated data 4 Parameter Logistic and Log Logit If the standard curve has a sigmoidal shape when plotted on the semi log axes it may be appropriate to use either the log logit or 4 parameter fit Both fits are based on the equation y A D D e ThE where D is the Y value corresponding to the asymptote i e the flat part of the curve at high values of the X axis and A is the Y value corresponding to the asymptote at low values of the X axis The coefficient C is the X value corre sponding to the midpoint between A and D The coefficient B d
44. Printing the Well Graph You can print the Well Graph window if desired by clicking the Print button at the bottom left corner of the dialog box NOF If you enlarge the Well Graph to greater than 12 inches Molecular Devices does not recommend printing it since the graph will not fit on an 8 5 by 11 inch page and the printout will be truncated Masking Wells If you find data in certain wells outliers that should be excluded from the calculations to be performed by data reduction you can mask these wells Select the well s to be masked and then click the Mask button in the Plate section tool bar or choose Mask from the Plate menu Masked wells are shown with an overlay of diagonal lines and are marked with the word Masked The masking function can be used as a what if tool For example you could suppress or enable certain functional groups within the experiment Suppose you have included a group blank in the template and want to see the data with and without the blank Masking the group blank wells would sup press the blanking function unmasking them would enable it again S EE i Ymax units per sec Platez1Copy Fast Kinetic Integrate 1 sec 20 P Injection Off M Injection Ott Lag Time 0 00 O C lene RLU Max 50 mas Pte 20 20 mem Combination Mi alue Masked Plate Last Read Wells 10 03 AM 6 21 00 Figure 5 10 Masked Wells SOFTmax PRO for Lmax User s Manual 5 15 Chapter 5
45. Raw or reduced data is displayed proportionally in a plate format using seven shades of gray ranging from light shading at the low end to dark shad ing at the high end based on user defined high and low limits Group Wells can be assigned to group types using the Template Editor Depending on the default protocol used certain group types such as Standard or Unknown may be created automatically you can create others as required Highlighted Indicates that an object or piece of text is selected and will be affected by your next action Highlighted text appears in reverse video or in color Highlighted objects may change color or be surrounded by a selection cursor Icon A graphical representation of an element of the computer or software such as a disk drive application or file Import Bringing information or data from another application into SOFTmax PRO for Lmax Information can be brought into SOFTmax PRO for Lmax in two ways by copying tab delimited text to the clipboard and then pasting this information into the Plate section within SOFTmax PRO for Lmax or by using the Import command under the File menu to import ASCII text files Incubator Choosing Incubator from the Control menu or clicking the incubator button opens a dialog box allowing you to start or stop temperature regulation and to select an elevated temperature for the microplate chamber on instruments that have temperature control capability Injection The pr
46. Then dou ble click the program icon inside the SOFTmax PRO for Lmax folder Mac or on the desktop Windows or choose SOFTmax PRO for Lmax from the Windows Start menu to begin the software program The first time you open SOFTmax PRO for Lmax a registration dialog box will appear asking that you enter your name company and the serial num bers of the software program and Lmax The software serial number can be found on the original SOFTmax PRO for Lmax CD on the inside cover of this manual and on the software Warranty Registration Card please remember to fill out this card and return it to Molecular Devices Corporation to validate your warranty The instrument serial number S N is located on a label affixed to the back of the Lmax Type the information into the appropriate boxes and then click OK Note the option to click will not be available until all information has been entered If you have difficulty entering the reg istration information refer to Chapter 8 Troubleshooting for assistance 2 8 SOFTmax PRO for Lmax User s Manual Chapter 2 Installation Molecular Devices Please register this copy of SOF Tmax PRO for Lmax by providing the follayving information Software Serial Number fF Instrument Serial Number SM o Figure 2 3 Registration Dialog Box After you click OK a title screen will appear see Figure 2 4 showing the registration information you entered Version 1 0L
47. V Unk w Dilution Hinai Unas iin 3 Hn 4 inns Hnat Unde Und Un 4 Unies Lindt Unde in s Unda Unas omes OK Figure 7 16 Template Complete The template is now complete Click the button to accept the entries and close the Template Editor The outlines of the groups you have defined in the Template Editor will now be shown in the Plate section a E Plate 1 E REM MEN EM UNE 10 11 12 Endpoint Integrate 1 sec fF d bL P Injection f Volume 100 uL Delay 3 Sec f RIeettons oti I m om ncm F Wavelength Combination IM yalue Figure 7 17 The Plate Section Showing Template Group Boundaries SOFTmax PRO User s Manual Chapter 7 Tutorial Step 4 Set the Display Parameters Click the button to open the Display dialog box m Display Figure 7 18 Display Dialog Box Endpoint The default display for Endpoint readings is raw RLU This tutorial example displays reduced data as a number To do this click the Reduced button at the top of the dialog box and then choose Number from the choices that appear wm Display Figure 7 19 Display Dialog Box after Choosing Reduced and Clicking the Display Choices Drop Down Menu Click to close the dialog box SOFTmax PRO User s Manual 7 17 Chapter 7 Tutorial BO 18 Step 5 Save the Protocol Choose Save from the File menu Because the file has not been saved before and
48. a new experiment directly following the active experiment the new experiment then becomes the active experiment All sections shown in the experiment that was active when this command is cho sen are duplicated in the new experiment New Notes Creates a new Notes section below the active section New Plate Creates a new Plate section below the active section The new Plate section will be a copy of the active Plate section or if no Plate sec tion currently exists the new Plate section will use SOFTmax PRO for Lmax default settings New Graph Creates a new Graph section below the active section and brings up the Plots dialog box allowing you to select the graph options SOFTmax PRO for Lmax User s Manual 2 Chapter 3 SOFTmax PRO for Lmax Overview Control Menu Control Head Incubabx Mash Inecines Prime inea Fass Inpechots Move to Senke Fosi Instrument Setup This command is available only if a Plate section is ac tive Choosing Instrument Setup opens the Instrument Settings dialog box which allows you to enter the mode for the run endpoint dual read or kinetic and the settings for pre read blanking strips timing for kinetic runs and plate type Button Equivalent Setup in the Plate section tool bar Read In a single plate experiment the Read command reads the plate imme diately If no Plate section is active in an experiment containing multi ple Plate sections the Rea
49. a set of data for log X plot ted against Y The resulting curve displayed will be a straight line with the X axis drawn in logarithmic scale The equation for a semi log fit is Y A B log49 X and is shown on the screen as y A B log x where A is the y intercept of the line and B is the slope note log in this equa tion is the common or base 10 logarithm 5 36 SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data lt 7 Z Standard Curve Fit Standard Curve T m T1 T E Concentration y A 6 Logix o Std Standards Concentration vs lean alue Figure 5 31 Graph with Semi Log Fit simulated data Log Log The log log function fits the best straight line to the set of data which consists of the logarithm of the readings on the Y axis the response and the logarithm of the dose on the X axis The resulting display will be a straight line with both axes drawn in logarithmic scale The equation for the log log fit is log4o Y A B gt log49 X and is shown on the screen as log y A B log x where A is the log Y intercept of the line for log49 X 0 and B is the slope note log in this equation is the common or base 10 logarithm SOFTmax PRO for Lmax User s Manual 5 37 Chapter 5 Displaying and Analyzing Data 10000 4 100 4 l lean value 0 1 0 07 1 1 1 1 100 1000 10000 Concentration Logty amp 8 Loaxr A B RAZ
50. all individual well values obtained during the reading of micro plates Plate Blank subtraction may be turned off in the Reduction dialog box so that the data may be seen with or without plate blank subtraction Plate blanks are only subtracted from wells on the same plate to which they are assigned The checkbox for Plate Blank subtraction only appears in the Reduction dialog box after data has been col lected For kinetic data the blank value is averaged and subtracted on a point by point basis The example below shows a graphic representation of original and average plate blank data followed by the resulting data with average subtracted SOFTmax PRO for Lmax User s Manual 4 25 Chapter 4 Reading Microplates Time Time Time Blank Subtracted Sample Well Sample Well Average of Blank Wells Figure 4 25 Graphic Representation of Plate Blank Subtraction Group Associated Blanks Blank wells can be assigned within any group other than the Blank group in the Template Editor The average reduced value of the blank wells within that group will be subtracted from individual reduced values within that group only This blanking function uses the reduced number example Lm1 Lm2 Vmax etc only it does not subtract on a point by point basis When a group blank has been assigned it is automatically subtracted To see the reduced number without the group blank subtracted mask the group blank wells Group blanks apply to
51. all wells in a group and thus may be subtracted from wells in more than one Plate section Blanks in Combination It is possible to use the blanking functions in combination For example you could perform plate blanking and also include blank wells within certain groups The effects of the different blanking functions are cumulative so it is important to understand the function of each before using both types of blanking in a single assay Plate blanking is applied to the raw data acquired from the instrument whereas group associated blanks are applied to the reduced number Group associated blank subtraction is only visible in the Plate section when the reduced number is displayed Figure 4 26 shows plate blanks and group associated blanks in the Template Editor 4 26 SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microplates Experiment 1 Plate 1 Edit Group TR Samples bull Clear Sampe vli Blank Group Plate Blank Group Associated Blank Wells Figure 4 26 Template Editor Showing Plate Blank and Group Associated Blank Wells In the figure above note that the plate blank in the upper left appears as a separate group named Blank and individual samples within this group are named BL The group associated blank wells shown in the upper right are not a separate group the samples also named BL are contained within the boundaries of the large group called Unknowns and are the sam
52. also manually save the file using the Save as command Append to File Text only If you are saving data to an ASCII text file you may choose to save all data to a single file for all plates by choosing Append to File This option is dimmed not available if you are saving to a SOFTmax PRO for Lmax file File Prefix The information entered into the File Prefix field is automatically given to new files that are saved automatically The default prefix is Data Accepting this default saves files starting with the name Data followed by a sequential number beginning with 1 For example the first microplate read would be saved under the file name Data 1 File Prefix File Prefix Field Append Date Figure 2 9 File Prefix Field Windows You can change this default prefix to any other prefix provided the entire file name will not exceed 31 characters To change the prefix click within the File Prefix field highlight the existing information and type over the word to enter a new prefix If the File Prefix field is left blank the date of the run will be used as the file prefix 2 14 SOFTmax PRO for Lmax User s Manual 2 Chapter 2 Installation Append Date Autoprint Assays Folder Enabling the checkbox for Append Date will cause the date to be included in the automatic file name for the saved data file For example using the file pre fix Data the first file saved on January 31 2000 w
53. and save the resulting data file under a different name Congratulations You have just created your first SOFTmax PRO for Lmax protocol file Now would be the time to read the plate and then save the data file In this tutorial example however you will not perform an actual plate reading but will use a simulator program built into SOFTmax PRO for Lmax to generate data that you will manipulate as if it had been obtained from an actual reading Note that the Default Protocol which ships with SOFTmax PRO for Lmax con tains a template and display parameters that are equivalent to the protocol file you just created in this tutorial example Step 6 Read the Plate If you were reading an actual microplate at this time you would prepare the microplate as it is defined in the Template Editor Before an actual reading you might find it helpful to print a copy of the template to assist in properly filling the plate from the Template Editor click the Print button in the lower left corner You would then place the filled microplate in the drawer of the MAXline instrument Since we are using a simulator you don t need to load anything in the instrument To enable the simulator open the Prefer ences choose Edit Preferences and then click on the instrument type shown in the lower left corner of the dialog box A menu will appear offering a choice of instrument or simulator Choose Simulator see the figure below m Preference
54. assay Click the Group pull down menu Because no groups have been created yet Blank is all that is available you will have to create a new one Choose New W Expenment 1 Plate Group Pull down Menu sample v Geres I n m c Oo F Cancel DK Figure 7 7 Template with Three Columns of Wells Selected and New Group Being Created B The Group Settings dialog box will appear allowing you to name the new group and select the appropriate information for it Name the new group Standards and choose the Standards Column Format Doing this auto matically enables the Sample Descriptor and enters the default units ug mL The tutorial assay uses pg uL however so you will need to change this Clicking on the arrow next to units shows all possible options but pg uL is not among them You must enter it manually by overtyping the value in the box When you are done entering information the Group Settings dialog box should appear like the one on the right below mw Group Settings Ea wm Group Settings E Sample Descriptor vere d Due OR OURO T rv Sample Descriptor ringed IET i e a age Encre Jute Tm Column Format RRRRRRRRRRRSSSERRRRRRERREERRERERRRSRERERERRRRRERREEEREREESRERRRRRREERSRRRERERE Column Format n S SCSS Cancel Cancel Figure 7 8 Instrument Settings Dialog Box Left shows initial settings right shows settings after entering info
55. by clicking the button in the Graph Options dialog box After clicking New a dialog labeled Plot 2 is opened Choose GA h for the group and change the X and Y axes to show concentration and values respectively for GA h W Graph Options xc Height roo onts sv fe Scatter Tithe Sample Text Mh C Cluster bar lil Stack bar Legend sample Text Connect points wee Sample Text w Plot symbols Pots ae x Concentration Ach Platz Y Values Ath M Errors Mo Error Y Errars Mo Error Cancel Figure 5 26 Graph Options Dialog Box with Second Plot Added for GA h This process is repeated for a the rest of the Group plots in this order Plot 3 G5A native Concentration vs Values Plot 4 G5A h Concentration vs Values Plot 5 plec2 native Concentration vs Values Plot 6 plec2 h Concentration vs Values Plot 7 CHOMO6 native Concentration vs Values Plot 8 CHOMO6 h Concentration vs Values SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data 6 Now that all plots information is complete clicking in the Graph Option dialog box will close it and the resulting graph will show the eight plots created v 2 Graphe Ft 5 Gr apti 4000 3000 RLU Ane under Curve hi c UTF Conc uel D Polis Anaseesr Concentdion vz veles LJ Pici AT Concendralion vs v alusg Pai CS names Concentrsion ve Yakaz C Pio ioar Concen
56. data for log X plot ted against Y The resulting curve displayed will be a straight line with the X axis drawn in logarithmic scale Serial Port Windows Series Slope A connection on a computer usually COMI where you plug in the cable for a serial device such as a plate reader a printer or a modem Serial asynchro nous and RS232 interfaces are all the same type by which the computer sends single bits of information to the connected device one after the other Win dows supports COM1 through COM4 The Lmax requires COM1 or COM2 Lets you assign a set of wells within groups in the Template so that the stan dard value or dilution factor increases or decreases in specified steps and the name increments as well A reduction option for a kinetic run that determines the slope of the com bined plot i e the slope of the line using linear regression This reduction uses all timepoints visible in the reduction window Standard Curve A mathematically idealized representation of the relationship between the concentration of a standard calibrator X value and the optical density Y value The standard curve may be set to a linear semi log log log qua dratic 4 parameter logistic log logit 2 parameter logistic exponential cubic spline or point to point curve fits SOFTmax PRO for Lmax User s Manual Chapter 1 Welcome to SOFTmax PRO for Lmax Status Bar Shown above the SOFTmax PRO for Lmax window the Sta
57. enlarged window Select the well in the data display hold the Shift key if you are selecting non contiguous wells and then click the Graph button or dou ble click in one of the selected wells A AECL ape LIS TEHE UT ai Tiri i3 vet Pind s wc Tua n pm n F HI2 Vena Pas Gann AE Ji 3d Aim E HEN on par Coo e Jpn Figure 5 9 Well Graph for Three Fast Kinetic Wells of a Microplate Raw Data The choices you make for viewing the data in the Plate section also affect the way the Well Graph appears Figures 5 8 and 5 9 show Well Graphs for raw data displays If you display one of the reduced options reduced number Threshold Ranged or Gray Scale the Well Graph will show the plot with an indicator of the reduced value and will include the word Reduced in the window title SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data Zoom Box You can enlarge the Well Graph even further by clicking the zoom box in the upper right hand corner of the window the center box of three in the upper right corner in Windows Clicking the zoom box once will cause the window to fill the total display on your monitor clicking the zoom box again will return the window to its previous size Size Box Clicking and dragging the size box located in the lower right corner Macintosh or clicking and dragging the lower right corner of the window Windows allows you to make the Well Graph any size desired
58. for well A1 is writ ten into the first line of the file information for well A2 is written to the sec ond line of the file and so on until a line has been created in the export file for each well in a plate If no template information exists for a well in a plate that line is left blank The ASCII file terminates with the string End The order for importing template information must match the export order Similarly blank lines in the file are assigned to a well providing a well with no template setting Table 6 3 describes the data that is included in each column of the ASCII tem plate file 6 20 SOFTmax PRO for Lmax User s Manual 8S Chapter 6 File Management and Printing Table 6 3 ASCII Template File Columns and Descriptions 1 Groupname name Assigned in Assigned in the Group Settings dialog box If this field is missing the rest of the Group Settings dialog box If this field is missing the rest of the line is ignored Any text string can be entered in this field Column format Assigned in the Group Settings dialog box Four text strings are supported in SOFTmax PRO for Lmax Basic Standards Unknowns and Unknowns Dilution Blank groups have a Basic column format If this field is empty the Basic column format is assigned o qmm name The sample name is set in the Sample field of the Template Editor dialog box If this field is left blank no sample name will be assigned to the well although the
59. housekeeping code Private Declare Function FindWindow Lib user32 Alias _ FindWindowA ByVal IpClaesName As String _ ByVal IpWindowName Ae String As Long Private Declare Function SendMessage Lib user32 Alias _ SendMessageA ByVal hWnd As Long ByVal wMsg As Long _ ByVal wParam Ae Long _ ByVal IFaram As String As Long Private Declare Function RegisterWindowMessage Lib user32 Alias _ RegieterWindowMeeeage ByVal mgsName As String As Long Const WM_SETTEXT 12 define a windows message value Function SendMsgToPRO ByVal msgStr As String e Long hWnd FindWindow SOFTMaxPROMainWna SOFTmax PRO for Lmax IF hWnd O Then one beep means Fro ie not running Deep Elee Fro ie running eo use the SendMessage AFI softmaxMsg RegieterWindowMeeeage SOFTMaxFROMeq If softmaxMsg O Then This version of PRO does not understand external msgs Beep Beep Else SendMessage hWnd WM_SETTEXT softmaxMsg msgStr End If End If End Function Ree RR RR RR RR RR RR RR RR RR RRR RR RRR RRR RR RR RR RRR RRR RR RR RM RR RRR RRR RRR RRR RR RR RR Ree eee ee This ie an example of how to use the above routine SOFTmax PRO for Lmax User s Manual A 7 Appendix A SOFTmax PRO for Lmax Remote Command Language a Sub SendNewMsgToPRO SendMsgToPRO New End Sub MFC C Interface to SOFTmax PRO for Lmax Remote Com mands that Return Values Some SOFTmax PRO for Lmax IPC commands return values B
60. is automatically used as a template for new files you create You can however create one or more different tem plate protocol files Default Protocol When first installed SOFTmax PRO for Lmax looks for the Default Protocol file in the same folder or subdirectory as the SOFTmax PRO for Lmax pro gram file You can instruct SOFTmax PRO for Lmax to look in a different loca tion however through the Assays menu Suppose for example that you create a folder subdirectory called MYASSAYS and that you create a new protocol file called Default Protocol or DEFAULT LPR within this folder The Assays menu shows two sections at the top a command entitled Set Folder which allows you to choose the folder within which SOFTmax PRO for Lmax will look to find the Default Protocol file when creating a new experiment At the bottom of this menu beneath the dividing line is a list of all protocol files contained in the currently chosen folder Assays Set Folder Cellular Chemiluminescent Default Protocol Diowetane Glow DNA Probe Figure 6 1 Example Assays Menu To have SOFTmax PRO for Lmax look in a different folder for the Default Pro tocol file choose Set Folder A dialog box will open allowing you to locate and set a different default folder SOFTmax PRO for Lmax User s Manual 6 3 Chapter 6 File Management and Printing 6 4 Select Directory Directory C Eject
61. is not named the Save As dialog box will open 53h SOFT maxe PRO for Lmax 1 0 iL Assays Save this document as Untitled i amp Data File 39 Protocol File Figure 7 20 Save As Dialog Box Macintosh Save this document as El x Save in C3 SOFTmax PRO forLmas 1 0 m E fad _ Assays aa GHL E 23 lda chemiluminescence lda ix D ual Luciferase 8 lda e elisa glow Ida Save as type Pro for Lmax Data Files 1da Cancel Figure 7 21 Save As Dialog Box Windows On the Macintosh click the option button next to Protocol File In Win dows click in the box under Save File as Type and choose Pro for Lmax Protocols lpr Type Tutorial the name for the file in the text box high lighted over Untitled Then click Save to place a copy of the protocol file to the hard disk The title bar of the window will still show Untitled rather than Tutorial after saving This does not mean the protocol has not been saved When you create a protocol file SOFTmax PRO for Lmax saves a copy of the current file under that file name but does not display the name on the screen Protocol files are saved as template files on the Macintosh these are known as stationery files When you open a pro SOFTmax PRO User s Manual Chapter 7 Tutorial tocol file an Untitled window appears containing all the protocol information saved previously so that you can collect data
62. kaa ex 8 mi uobsded 1 C pi ud onvali vx Lin Pate 0 n35uwgovasgeaesm vas ka mi ees Aulae a mntdcPmsr Ce 3 uM EV alee ws ES8 ml eR E 10 uM rv sir aed vs Pale eee 3 uM ov asini vem Hos rd tae 100 0M eile vie IH md Epod abel Figure 5 36 Graph with Point to Point Fit SOFTmax PRO for Lmax User s Manual 5 4 Chapter 5 Displaying and Analyzing Data BO Cubic Spline This curve fit generates a fit to a cubic equation between each pair of data points The general form of a cubic equation is y A Bx Cx Dx The equations are computed with the requirement that the first and second derivatives of the equations are continuous throughout the range of the data As multiple curve fits are performed with this routine fit parameters are not shown with the graph gt 2 Grepnaz FEL ciis T dag Graph C D ul aaa ve alaa LJ 01 3 uid omi mp ed ve den pee Figure 5 37 Graph with Cubic Spline Fit Exponential The exponential function used to generate this curve fit is y A B 1 exp x C ad it lal Graphs5 ovos e Fe Pea reed ve Carcesk retina y Ae Bd ee A B c E e PE rou conceniralien ws Means nue 0 128 0375 4B 304 D 553 Figure 5 38 Graph with Exponential Fit simulated data 5 42 SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data SOFTmax PRO for Lmax does not plot samples with desc
63. memory with a typical allocation SOFTmax PRO for Lmax requires a minimum of 7223K over 7 megabytes of memory to run but this is not optimal The preferred size is 8223K over 8 megabytes or larger Try increasing the amount of memory allocated to the program To do this quit SOFTmax PRO for Lmax if it is running Then highlight the SOFT max PRO for Lmax program icon click it once and then choose Get Info 1 from the File menu The dialog box that opens lists suggested and minimum memory allocations see Figure 8 6 SOFTmax PRO for Lmax User s Manual 8 11 Chapter 8 Troubleshooting SS SPF for Lmax 1 0 Info SS Ej SOFT Max PRO forLmax 2000 Molecular Devices Corp Kind application program Memory Requirements Suggested Size 10000 K Minimum Size 8000 k Preferred Size 10000 k Hote Memory requirements will increase by 2 152 K if virtual memory is turned off Locked Figure 8 6 SOFTmax PRO for Lmax Info Dialog Box Enter a larger amount of memory in the box next to Suggested size the amount you enter may be limited by the total amount of memory available The next time you start SOFTmax PRO for Lmax it will use the larger mem ory size you have allocated You may also enter a different number into either the minimum or preferred size boxes It is suggested that you do not reduce the number in the Minimum size box you may increase it however The Preferred size can be as large as you
64. not warrant that the functions contained in the Software will meet the User s requirements nor that the operation of the pro ram will be uninterrupted or error free Some states do not allow the exclusion of implied warranties and therefore the above exclusion may not apply to you Limitation of Remedies MDC s entire liability and the User s only remedy with respect to the Limited Warranty set forth above is 1 The replacement of any discs not meeting the Limited Warranty set forth above provided such discs are returned to MDC with evidence of date of purchase or 2 If MDC is unable to delivery ar replacement disc which is free of defects in materials and workmanship the user dvi terminate this license agreement upon return of all materials including manual and disc s to MDC for a full refund of purchase price In no event will MDC be liable to the User for any damages including any lost profits lost savings or other incidental or consequential damages arising out of the use or inability to use the software even if MDC has been advised of the possibility of such damages or for any claim by another party Some states do not allow the limitation or exclusion of liability for incidental or consequential damages so the above limitations or exclusions may not apply to you Duration and Termination The License is effective from the date of the first use of the Software by the User until terminated or until the expiration of fifty years w
65. read the entire plate To configure the settings for a partial plate read choose the Wells to Read set ting Wells To Read XX I Q nm s um r C C Ct Figure 4 1 Selected Partial Plate Read Wells to Read Dialog Box with wells B2 through F9 Highlight the wells to be read Wells must be contiguous but do not need to start in any particular place If you enable a partial plate reading only the 4 12 SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microplates Template Editor Template Group Groupi Y Editor Tool Bar Group Drop Down List Sample Descriptor Field Microplate Map wells to be read will be visible in the data display indicating that no data will be collected for the other wells all wells will be present in the Template Edi tor however The Template Editor is used to describe the location of samples in the wells of the microplate providing the link between the raw data and the analysis groups The Template Editor is used to create a template which provides SOFTmax PRO for Lmax with a map of what is in the microplate When you first open the Template Editor for a new Plate section the grid or display is empty The number of wells shown depends upon the type of plate selected in the Instrument Settings After selecting wells you can assign groups and indicate the types of samples they will contain Depending upon the default protocol you
66. section is a table which shows all of the information for a particular group A group is a set of associ ated samples which have been created in the Template Depending on the default assay chosen certain group types such as Standard or Unknown may have been created you can create others as required Group sections may also be created by duplicating existing Group sections The Group section is divided into three areas e The tool bar e The body of the Group table e The summaries The Group menu contains the following items shown with their tool bar or mouse equivalents These menu items and buttons are active only when a Group section is active Table 3 3 Group Section Menu Commands and Tool Bar Buttons Menu Command uU New Window in the Opens the Group section in Group menu a new window Name of Group sec Opens the Group settings Group tion dialog box double click double click Group Settings in the Opens the Group Settings Group menu dialog box Opens the Calculation dia log box allowing you to create a summary Create Summary in the Group menu Opens the Calculation dia log box allowing you to create a column Create Column in the Group menu SOFTmax PRO for Lmax User s Manual 3 15 Chapter 3 SOFTmax PRO for Lmax Overview e 3 16 Menu Command Mouse Equivalent Edit Summary in the Group menu or dou ble click the summary pointer changes to double headed arrow Edit Colum
67. simulated data 0 0 cee eee 5 40 Graph with Point to Paint Fit ger cebwo vor owOpOIParCrPq Cre ER Paene 5 41 Giaph wih Cubic Spline EI ceseque eee eu oor rrie rrr Era 5 42 Graph with Exponential Fit simulated data 0 cece eee eee eee 5 42 Graph ODHODS Dialog BOK as tc pcde ceded ednee teens cope rrei rnr erar 5 44 Scatter Graph with Points Connected Linear Fit Applied 5 45 Bar Graphs Cluster and Stack eese 5 45 As xus Dido DO Socesevee ee ee er er as ee ee ee doing 5 46 xomple Aissay s IGI S 1o dures eiua corres uiri VI EOeUIieivem ED 6 3 Set Folder Dialog Box Macintosh and PC 0 eee eee 6 4 Save as Dialog Box Macintosh 0 0 eee cece eens 6 5 Save as Dialog Box Windows 0 cece cece eee ee erence cence scene 6 5 PC Exchange Dialog Box 0 2 eee ence ere ee ere eee eene 6 7 Add Mapping Dialog Box 0 0 cee eens 6 8 Preferences Dialog DOX o isdem im so t RR RR URN RURAL UR 6 10 I ut DidlOe BOM ad eseus se uus ouetqo ER ERELERERPERATAREREREPRTSERERPANES 6 12 Format Text Dialog Box Macintosh sssseeeeeeeeeee eee ees 6 13 Print Dialog Box for the Macintosh 00 ccc eee cece eee eee 6 14 Print Dialog Box for Windows Epson Stylus 800 Shown 6 15 Choosing an Export Format from the Preferences Dialog Box 6 16 Display Didl0e DON x ide sordo dirt RR UH UR ERUDITUS EHE URS 6 17 EXPOSE M
68. standard curve This protocol can be used dor dsDNA Quantitation by LMAX DL Pei eH Setup ES Tap E eksa Grp L xe olj I C m c oc m e Tiaan Conmiinaion fco b M Sanana a Be s b L7 Std graph Ft b ffi UniT ATi LA me d b ffi Link v Caution z infra E If pb E Figure 2 5 SOFTmax PRO for Lmax Default Protocol Untitled Window Communicating with the Lmax After installation of SOFTmax PRO for Lmax you will note that the instru ment icon in the upper left hand corner of the window appears with an X and the words No port selected through it You must access the Preferences settings from the Edit menu choose the Preferences command to select the proper communications port modem or printer for the Macintosh COMI or COM2 for the PC An additional setting which is the factory default is Nei ther used when you do not have an instrument connected to the computer and wish to use SOFTmax PRO for Lmax or if the port connection must be disabled for other reasons Choose the appropriate setting for your com puter s physical serial port connection and click OK 2 10 SOFTmax PRO for Lmax User s Manual Chapter 2 Installation W Preferences Serial Fort COM1 lt Set serial port here Export Format ee LE Ive UU MVE AR hile Eimer emere Br Time aos ee Roe e SOF Tmax PROG for Lmax 1 D zw Iv Include Labels Append Date f SOF Tmax fo
69. that opens the Control menu for the window It is located at the left of the SOFTmax PRO for Lmax title bar The Control menu can be activated by clicking in this box with the mouse or by pressing ALT SFACEBAR Copy A function common to most software applications that duplicates the selected text or item and places this duplicate on the clipboard The copy function is enabled by pressing 3C on the Macintosh and CTRL C in Windows Copy and Paste A method of copying selected text or graphics from one location and pasting them to a different location in the same file or into another file which could be in an application other than SOFTmax PRO for Lmax Cubic Spline Curve Fit This curve fitting option generates a fit to a cubic equation between each pair of data points The cubic equations are computed such that these equations and their first two derivatives are continuous everywhere More information regarding curve fits can be found in Chapter 5 Displaying and Analyzing Data 1 12 SOFTmax PRO for Lmax User s Manual Chapter 1 Welcome to SOFTmax PRO for Lmax Curve Fit Specifies the type of curve linear logarithmic quadratic and so on that is applied to the data points in a plot The curve is a graphic representation of the mathematical relationship between the data points in the plot More infor mation regarding curve fits can be found in Chapter 5 Displaying and Ana lyzing Data Cut A function common to mo
70. the cable from the instru ment is connected to the computer If the port setting is correct the instrument is on and communication is still not functioning properly quit SOFTmax PRO for Lmax turn off power to both the computer and the Lmax and check that the cable connections between the instrument and computer SOFTmax PRO for Lmax User s Manual 6 5 Chapter 8 Troubleshooting e Communication 2 8 6 are secure Turn both machines on again and restart SOFTmax PRO for Lmax If an X still appears over the icon it may be that you are using the wrong type of cable Macintosh Computers Having a Single Serial Port Some Macintosh computers have only a single 8 pin DIN serial port which serves as both the printer and modem port Please check to see if your model is listed as compatible hardware on the SOFTmax PRO for Macintosh Version System Requirements and Compatible Hardware sheet If your computer is not listed the performance of SOFTmax PRO for Lmax version 3 0 for Macintosh cannot be guaranteed when using this model When running SOFTmax PRO for Lmax the single serial port Macintosh can either communicate with the Lmax or print through the single serial port but both printer and instrument cannot remain connected to the serial port at the same time To avoid uncabling the instrument in order to attach the printer you must either print across a network using the ethernet port of the com puter or the instrument
71. the left side Injection and Delay The Instrument Settings window should look like the following PERTIESCITUT aT ES Oot Dual Read Fast Kinetic Injection and Delay Injection and Delay Figure 7 3 Instrument Settings Dialog Box Injection and Delay Section Open SOFTmax PRO User s Manual 7 7 Chapter 7 Tutorial BQ Click in the checkbox next to P Injection to enable it Drag over the default volume 25 uL to highlight this value and overtype it with the new volume of 100 uL Highlight the default of 1 9 seconds for the delay and enter the new value of 3 seconds Leave M injection disabled The Instrument Settings dialog box should now look like this we Instrument Settings Endpoint Dual Read Fast Kinetic Injection and De lay m Injection and Delay Figure 7 4 Instrument Settings Dialog Box Injection and Delay Section Revised 7 8 SOFTmax PRO User s Manual Chapter 7 Tutorial Since the entire plate will be read and that is the default setting you do not need to do anything to the Wells To Read section You do need to define the wells that will be injected by the P injector how ever Click on the Injection Wells heading on the left side of the dialog box to open that section on the right The default shows no wells selected and the P injector enabled as shown below ws Instrument Settings Endpoint Dual Read Fast Kinetic Injection Wells Injection Wells
72. to click on a button choose a com mand from one of the drop down menus in the menu bar or press a key stroke combination to activate a command or function The Plate section is divided into four areas e Tool bar Data display shown in a microplate grid format e Instrument settings shown to the right of or below the data display depending on the plate type and printer settings e Reduction settings shown to the right of or below the data display depend ing on the plate type and printer settings When the grid in the Plate section shows colors this means that a template has been defined for this Plate section Each group defined in the template has a different color the icon of the corresponding group table will have the same color If your experiment requires multiple plates you can create as many Plate sec tions as needed The Plate menu contains the items shown in Table 3 2 shown with their tool bar or mouse equivalents These menu items and buttons are active only when a Plate section is active SOFTmax PRO for Lmax User s Manual 3 13 Chapter 3 SOFTmax PRO for Lmax Overview Table 3 2 Plate Section Menu Commands and Tool Bar Buttons Menu Command Deseriotion Tool Bar Mouse Equivalent P Button Plate section icon Opens the Plate section in a new window l double click Name of Plate section Opens the Section dialog box Hd E 5 double click Instrument Setup in the Control Opens the Instrument Setting
73. to display more than 2500 data points in a group becomes gray or disappears 1 From the View menu choose Minimize Delete the large Group table then create a new smaller table 2 If this does not work from the View menu select the large group then choose Delete groupname from the Edit menu Save the file close the file reopen the file Checkbox for Plate blank This option is not visible until after data has been collected subtraction in the reduction dialog box is not visible Y axis units on a graph either This can occur when the range on the Y axis is very small Increase the Well Graph or Graph section height of the Y axis are repeated Text appears too large for In the Windows Control Panel check the settings for Display Ensure wells in plate on Windows that you are using small fonts platform General display problems in Change the video display settings to 16 bit and high color or better Windows Printing Problems Text in the header field of the The text is still there but is not visible It will print If you click in the print dialog box disappears box and drag the mouse to the left it will reappear if you press the return or enter key The Notes section is longer Insert a linefeed into the Notes section at the appropriate place than one page The bottom line of the first page and the top line of the second page are partially printed 8 10 SOFTmax PRO for Lmax User s Manual
74. to the top of the window SOFTmax PRO for Lmax User s Manual 3 9 Chapter 3 SOFTmax PRO for Lmax Overview Active Tool Bar Active Open Section Scrolling Upward Wavelength Combination Male Plate Blank Used Lint 1 6 Figure 3 5 Example of a Tool Bar Sticking to the Top of the Window Opening and Closing Sections Newly created experiments and sections are initially shown in their full size open form You can close them however to show just their tool bars if desired Sections that are created automatically by SOFTmax PRO for Lmax may be shown initially either open or closed depending on the default proto col file in use To open or close a single section within SOFTmax PRO for Lmax click its indicator located in the top left corner of the tool bar When an experiment or section is closed the indicator points to the right when open the indica tor points down xz Figure 3 6 shows an open experiment containing three sections the first two are closed the last is open File Edit View Expenment Control Assays Plate Window Help Gres n I Untitled v Ge Experiment ZN Closed be Ie Notes 1 24 b Ea Platez1 eg Setup B Template Reduction Display k E hs J Pieter Open Platez B T Figure 3 6 Closed and Open Sections The Notes 1 and Plate 1 sections are closed the Plate 2 section is open To open or close multiple sections within an experiment or multipl
75. to well variability The pre read values will be stored in the data file and the individual well values collected during the pre reading of the plate will be subtracted from the individual well values obtained during the actual reading of samples Pre read blanking may be turned off in the Reduction dialog box click on the Reduction button so the data may be seen with and without pre read blanking subtracted This option is only available within the Reduction dialog box if pre read blanking was chosen in the Instrument Settings for endpoint mode Molecular Devices recommends that you fill the wells of the pre read plate with appropriate buffer Other Types of Blanks In addition to the pre read plate blanking selection found in the Instrument Settings dialog box other types of blanking are available in SOFTmax PRO for Lmax and these others can be used with other read modes Understanding the differences between the types of blanking functions will enable you to choose the type of blanking that is appropriate for your reading Note that blanks can be used in combination and have cumulative effects The other types of blanking besides pre read plate blanks that are defined in SOFTmax PRO for Lmax are e Plate blanks e Group associated blanks Plate Blanks Wells can be assigned to a Blank group in the Template Editor Unlike pre read plate blanks the average value of all the wells in a plate blank group is sub tracted from
76. under Preferences in the Edit menu You may choose to export the data in plate format 8 x 12 array of numbers for a 96 well plate for example or in time format a single line or row You may also choose for the exported file to be created with or without labels Set Password Allows you to enable password protection for the file to pro tect it from unauthorized or unintentional changes Page Setup Print Setup You should access this command after you select a different printer in the Chooser Macintosh if you choose a different printer using Windows or if you want to use a different paper size or printing option Choosing Page Setup Print Setup opens a dialog box with various options for printing More about this function can be found in the documentation that accompanies your computer and or printer ese The Page Setup Print Setup command also controls how SOFTmax PRO for Lmax sections are displayed on your monitor the width of the display corresponds to the width of the page size Print Section name Selection Allows you to print just the active section s Print 67 CTRLF With the Macintosh choose this command and then click either Print all or Print report Print all sends all sections to the printer in the order they appear in the window Print report sends those sections selected to be in the report to the printer in the order they appear in the window From Windows choose Print and then specify a page range
77. when you are in the Finder Macintosh or in the File Manager Windows Note that you can open SOFTmax PRO for Lmax files using either platform regardless of whether they were created using Macin tosh or Windows see Cross Platform Files below for more information Opening SOFTmax SOFTmax PRO for Lmax Files Earlier Versions SOFTmax PRO for Lmax allows you to open files from previous versions of SOFTmax PRO for Lmax as well as from SOFTmax From within SOFTmax PRO for Lmax select the Open 38 O CTRL O command from the File menu The file will open and will function in the same way as a SOFTmax PRO for Lmax file If you wish you can save the file in SOFTmax PRO for Lmax for mat by selecting Save As from the File menu If you do not change the name of the file and save it to the same location a message box will appear asking whether or not you want to replace the existing SOFTmax file with the new SOFTmax PRO for Lmax version If you do not you can either choose a differ ent location or rename the file While SOFTmax files can be opened using SOFTmax PRO for Lmax two items do not translate completely across the programs sample blanks and auto range limits in graph sections The data for sample blanks obtained using SOFTmax will be available when these files are opened in SOFTmax PRO for Lmax but no calculations are per formed automatically using this data Auto range limits are not avail able when SOFTmax files are ope
78. your computer Exporting Data General Information 1 Export Format e Time Selection The Export command a sub menu of Import Export located in the File menu creates a file of data from one or more Plate sections and or Group table sections within a SOFTmax PRO for Lmax file The type of data that is exported depends upon three things Selections made in the Preferences dialog box regarding the export format Choose Preferences from the Edit menu Within the Preferences dialog box that appears select the format for data export that you prefer for Plate sec tions you can choose either time based which produces a linear format for the data or plate based which exports data in a matrix format corresponding to a microplate grid You also must choose whether or not to include labels with the exported data Click OK to close the dialog box Export Form at ETE kf Include Labels NOF The preference settings chosen above also affect the copying and pasting of data and Autosaving to ASCII text l Including labels will affect all data that is exported but doing so is most noticeable with Group section data since the column headings in the Group table will be shown as will any other calculated data in sum maries etc Labels included with Plate sections show 1 12 headers for 96 well Plate section data repre sented as an 8 x 12 matrix 384 well plate data will show 1 24 headers SOFTmax PRO for Lma
79. 0 900 1000 dsDNA pgiwell y a Bx A Bg Ree amp Plot 1 Standards paAwell vs Mean s aluel 2 5 0 005 0 771 Figure 7 36 Standard Curve Graph Section Linear Fit Applied Note that this sim ulated data does not work well with this linear curve fit This graph is set to show pg well on the X axis and mean value RLU on the Y axis A linear fit has been applied to this data but because the simulated data is not accurate the fit is not good If you were to try to find a good fit for this data you could apply other curve fits by clicking in the box next to Fit in the Graph section toolbar and making a different fit selection Try some of them as you wish The figure below shows the result of applying a 4 parameter curve to this simulated data for a much closer fit SOFTmax PRO User s Manual 7 29 Chapter 7 Tutorial 30 DNA Quantitation in the Lmax 6 5 M cM c OUR ee N zz 4 3 M ee Tec e LE D P 2 10 100 dsDNA pg well y ttA Dyu xo o A B M D R amp Plot 1 Standards palvvell vs Mean value 196 5565 0 013 125 204 5568 1 Figure 7 37 Standard Curve Graph Section with 4 Parameter Fit Applied You can also display the data in a bar graph or another format by clicking the graph options button which opens a dialog box like that shown in the fol lowing figure Graph Options Standard Curve Height w onts ae e scatter Title sample Text Set Ob C2 Clust
80. 1000 10000 100000 1 le Conc ipe D Pati Aeddi igheel es Mean ne O Pte Frei fof ve Meer ele Figure 5 28 Plot Showing Error Bars Choices for displaying error bars relative to a particular axis are the same as those available for displaying data on that axis any column from that group that contains numerical information not columns with text To display error bars click and hold the box next to Error Bars in the Plot X dialog and drag to select one of the choices in the menu To disable error bars choose No Error for the X and or Y axis Error bars show both positive and negative error Depending on the range set tings for the graph portions of the error bars may not be visible Curve Fitting When you first create a graph of the data it does not have a fit associated with it The graph might appear something like this 5 34 SOFTmax PRO for Lmax User s Manual 2 Chapter 5 Displaying and Analyzing Data No Fit selected mia j ATP Standard Curve in the Lmax b m pam mpm Re p neds hed ed ale old in uleg de ho aja philo ne a iet a mm mi a ig Li a a uu L im m pm Li a mh 1 b mu jew 3 d34E pipa ge i 24th gam 1 qr Hii mp ian i r1 5EN i m erat m rs x ais mney snl L Tr F a Tht pap ale fe MT pe z oy esate Bera eer eee Mer poe abi Ln nar mag U 1 TeeeENIDOEEUUONICE ERECTO RO SY SE te ee 0 00 eerste
81. 3 9 Figure 3 10 Figure 3 11 Figure 3 12 Figure 3 13 Figure 3 14 Figure 4 1 Figure 4 2 Figure 4 3 Figure 4 4 Figure 4 5 Figure 4 6 Figure 4 7 Figure 4 8 Figure 4 9 Figure 4 10 Figure 4 11 Figure 4 12 Figure 4 13 Figure 4 14 Help Menu for SOFTmax PRO for Lmax for the Macintosh 1 7 POR aC VIN ro PRO 1 7 Help Menu for SOFTmax PRO for Lmax for Windows 45 1 8 Help Contents for SOFTmax PRO for Lmax for Windows 1 8 Using the Search Function within Help in SOFTmax PRO for Lina ford VIDdOWS 220042440464 dad EINA E sede Ded ve Pepe dee edunt 1 9 Choose a Topic within Help in SOFTmax PRO for Lmax for Windows 1 9 Hypertext Links within SOFTmax PRO for Lmax for Windows 1 10 8 Pin DIN to DB 25 Macintosh Straight through Serial Cable 2 3 25 Pin DIN to 25 Pin or 9 Pin Serial Cable 0 0 00 cee eee eee 2 4 Registration Dialog BOX sque uus wr SORS UN RUINA ECOUTER EE UR ERTOIHE RUD EROR UR o 2 9 SOFTmax PRO for Lmax Title Screen 1 0 eee eee 2 9 SOFTmax PRO for Lmax Default Protocol Untitled Window 2 10 Preferences Dialog Box Windows 0 c eee e ee eee ene een eens 2 11 Autosave Information Windows 0 00 cc cee cee eee RR I eee 2 13 Autosave Location Dialog Box Windows ce eee eee eee eee 2 13 File Prefix Field Windows 0 0 c cc cee ccc cece eee eee RIRs 2 14 S
82. 4 8 Instrument Settings for a Fast Kinetics Reading Plate Type This setting determines the display of wells in the microplate it should be set to match the type and number of wells in the actual microplate that will be read SOFTmax PRO for Lmax User s Manual 4 11 Chapter 4 Reading Microplates Select a plate type 96 well plates 96 Well Standard 384 well plates 384 Well Standard Figure 4 9 Plate Type Setting Choose either 96 well or 384 well standard plates Changing the Plate Type within a Plate section causes well assign ments stored in the previous template to be discarded The groups that were created previously remain however so you can select new wells and simply apply existing groups to these wells Switching between 96 and 384 well plates requires a hardware reconfiguration on the Lmax as well as the designation of Plate Type in the software Choosing a different plate type will cause a message to appear on the screen reminding you that the physical setup must be altered and that you must also choose an additional setting in the Control menu to ensure that the proper hardware for that plate type has been configured Wells to Read Selection You may choose to read only certain wells in the microplate or to read the entire plate Partial plate reading is available for all read modes the time required for readings may be significantly reduced using this setting if the instrument does not have to
83. 7 Before you can change or remove the password you are required to enter the old password in the top text box After entering the old password checking the box next to Remove Password and typing nothing in the rest of the boxes will clear the password from the file when you click OK As with all password protection be sure to choose a password that you will be able to remember since the file will be locked to changes of nearly any kind should you forget it If you try to perform an unauthorized action on a protected file a dialog box will appear asking you to enter the password Password Required O suspend Password Cx Figure 6 18 Password Required Dialog Box When this dialog box appears you must enter the password in order to con tinue with the action you have chosen If you do not remove password protection prior to trying to change the file every attempt to change the file will result in this dialog box appearing This repeat prompting can be stopped by enabling the Suspend Password option click the box to check it NISSEEOOSE SOFTmax PRO for Lmax provides no hidden commands that will override password protection If you are concerned that you might forget your password write it down and store it in a safe place for future reference SOFTmax PRO for Lmax User s Manual 6 27 Chapter 6 File Management and Printing 6 28 SOFTmax PRO for Lmax User s Manual Chapter 7 Tutorial Bn od Ue BOR o
84. 7 5 or later you will need to make sure that File Exchange is available in the Control Panels 1 If you have not yet enabled File Exchange on your Macintosh select Control Panels from the Apple menu From within the Control Panels choose the Extensions Manager and turn File Exchange on by placing a checkmark next to it Restart your computer File Exchange will be enabled 2 Choose File Exchange from the Control Panels A dialog box like the one below will appear File Exchange File Translation a Map PC extensipns to kar 05 file types an PC disks of Open unmapped files on any disk using mappings helow Charges 3 iese mapa ras esa affect Enfer net Preferences ce Samglefext Plage Pra B SoundA pa dB soundapa d HE S Shall zu Fui eP Laser Add Remove Change PCSCSEDIsES f Mount at startup Mount Now Figure 6 5 PC Exchange Dialog Box 3 In this dialog box click Add This will cause a second dialog box to appear SOFTmax PRO for Lmax User s Manual 6 7 Chapter 6 File Management and Printing 6 8 4 5 6 Add Mapping Extension Map to amp KPT QuickShowLT 1 2 2 E Remote Access Status ar Acrobat Catalog 3 0 Be Acrobat Distiller 3 01 p Acrobat Reader 3 01 Adaptec Toast 3 5 j Adobe Gamma retype TL 15 Cra Figure 6 6 Add Mapping Dialog Box In order to allow the Macintosh to automatically recognize protocol and d
85. 8 1 Macintosh G4 PowerMac 7600 120 with OS 8 1 PowerMac 7100 80AV with OS 7 5 5 Printer tested Apple Laserwriter Select 360 SOFTmax PRO for Lmax User s Manual 2 5 Chapter 2 Installation Windows 95 98 Windows NT 2000 Recommended System Configuration Recommended System Configuration e Pentium III 450 MHz computer e Microsoft Windows 95 or 98 e 17 inch color SVGA monitor e Video adapter and software driver capable of displaying 800 x 600 minimum resolution set to 16 bit high color and small fonts or better e 64 MB RAM or greater e 6 GB hard disk drive e 1 44 MB HD high density 3 5 inch floppy disk drive e CD ROM drive e Bus mouse e Serial port COMI or COM2 e Pentium III 450 MHz computer e Microsoft Windows NT 4 0 or 2000 e 17 inch color SVGA monitor e Video adapter and software driver capable of displaying 800 x 600 minimum resolution set to 16 bit high color and small fonts or better e 128 MB RAM or greater e 6 GB hard disk drive e 1 44 MB HD high density 3 5 inch floppy disk drive e CD ROM drive e Bus mouse e Serial port COM1 or COM2 Windows 95 98 Windows NT 2000 Minimum System Configuration Minimum System Configuration e 486 33 MHz computer e Microsoft Windows 95 or 98 e Color monitor e SVGA graphics adapter set to 256 colors and small fonts or better e 24 MB RAM or greater e 1 GB hard disk drive e 1 44 MB HD high density 3 5 inch floppy disk drive e Access to a CD RO
86. 84 well plates End Time Reduction parameter used to omit the end of a kinetic run from data reduc tion Export Data from SOFTmax PRO for Lmax is copied to a new file that can be opened by another program such as a spreadsheet or word processing application Data from some or all Plate or Group sections may be exported as tab delim ited ASCII files Exponential Curve Fit The exponential function used to generate this curve fit is y A B 1 exp x C More information regarding curve fits can be found in Chapter 5 Displaying and Analyzing Data Fast Kinetics During fast kinetics readings data is collected over time with multiple read ings made at regular intervals up to a specified number of points The values calculated based on raw kinetic data are Vmax Vmax per Sec Time to Vmax and Onset Time Fluorophore A material which absorbs light energy of a characteristic wavelength under goes an electronic state change and instantaneously emits light of a longer wavelength SOFTmax PRO for Lmax User s Manual Chapter 1 Welcome to SOFTmax PRO for Lmax Four Parameter Logistic Curve Fit The equation used to generate this curve fit is y A D 1 CP D More information regarding curve fits can be found in Chapter 5 Displaying and Analyzing Data Gain The amount of increase in signal power expressed as the ratio of output to input for a photomultiplier tube Gray Scale Data Display
87. Bar Data Display Reports Figure 1 4 Help Contents for SOFTmax PRO for Lmax for Windows 1 8 SOFTmax PRO for Lmax User s Manual 8S Chapter 1 Welcome to SOFTmax PRO for Lmax Help Options from the Contents Window e You can search for a particular item or subject by clicking the Search but ton located in the tool bar at the top of the Contents window Type a word or select one from the list Then choose Show Topics Enter the subject you are searching for in this box Select a topic then choose Go To Figure 1 5 Using the Search Function within Help in SOFTmax PRO for Lmax for Windows After entering a search subject clicking the Show Topics button will cause a list of related topics to appear in the box below me Search Type a word or select one from the list Then choose Show Topics Formulas Formulas Fract Function Getting Started Graph Menu Graph Section Select a topic then choose Go To Edit Column Edit Summary Choose a Formula Help topic from Group Menu PLA list Hecalculate Now Heduction Show Formulas Figure 1 6 Choose a Topic within Help in SOFTmax PRO for Lmax for Windows e You can return to the window viewed previously by clicking the Back button SOFTmax PRO for Lmax User s Manual 1 9 Chapter 1 Welcome to SOFTmax PRO for Lmax BO e Clicking the History button will provide a list of all areas you have viewed within the Help function
88. Connect the Hardware 00 cece cece ee eee eee eee e eet cnet nne 2 3 Computer System Requirements a au dti a qos d SORRUE RUPRUERA UE RUPEE nosiri sirati 2 5 Macintosh Computer 0 0 0 eee cee e 2 5 Windows Compatible Computer 02 0 0 0 cece cece eee ee 2 6 Install SOFTmax PRO for Lmax 0 cc cee eens 2 7 What Is Ins Calli c uu uaa arem io tor mim ip RR i i ium arn eran BUE RR UR 2 8 Enter Registration Information 0000 eee ees 2 8 Communicating with the Lmax 00 cece cece eee ee 2 10 dui M C 2 11 vogue D D M 2 11 E O FO esmonmensm emt EN EPETEERESPEATATAEASAEATAEREERE ERES 2 12 ur P I 2 12 PUODMM DN ETETETT ETETETT 2 15 PSSA Ss VOIGCl C M 2 15 Chapter 3 SOFTmax PRO for Lmax Overview eeeeeeeeee cece eee 3 1 IDOL OCIUC BOITE dot n5 6 9us o pecu E RUIN S RUE QUPD S HOUR 0 B0 MUR d 3 3 The SOFTmax PRO for Lmax Window eeeeeeeee eee 3 3 VDO DIZBG onu mas ds vh ER E S hod a iOS PRA SUB EU GbR 2 dI BUS BUS di 3 5 PIAS 3 6 Experitiients and SCCHONS cccsccctacausaageeae een eeaaeenaereanaea esa RE 3 7 LXDSLIBIEH E E EEEE EE TETTETETT 3 7 vieni EE E E E E T EEE 3 8 Noles DECHON eee nr etenune nenn TERETE TEREE ARENE E E ES 3 12 Plate eE Ee E E EE E ETE E EEEE 3 13 Eole en o EREE EEEE 3 15 E e e a EEEE EE RS 3 17 Hast RU
89. Data describes how data is dis played during and after a reading and how this display can be customized Additionally it describes data reduction and the different means for analyz ing the raw data received from the Lmax Chapter 6 File Management and Printing explains the printing options available within SOFTmax PRO for Lmax along with how to customize reports Other topics include managing files and importing exporting 1 4 SOFTmax PRO for Lmax User s Manual Chapter 1 Welcome to SOFTmax PRO for Lmax copying pasting and duplicating data both within SOFTmax PRO for Lmax as well as to and from other external programs e If you are new to SOFTmax PRO for Lmax Chapter 7 Tutorial provides a written series of steps that will help you learn the basics of working with the software Chapter 8 Troubleshooting describes alert messages that may appear while using SOFTmax PRO for Lmax and what to do if they occur Other information in this chapter pertains to possible communication problems with the Lmax and other computer related issues This manual assumes that you are familiar with the basic operating environ ment of your computer We recommend that you become familiar with your computer s operating environment prior to installing and using SOFTmax PRO Conventions Used in This Manual This manual uses visual cues and standard text formats as follows Visual Cues The following typographic conventions
90. Dialog Box Injection Post En Number Integration J Injector Ah Wells Wells Run Plate P and or M Injection of Reads Time Sp Volume P amp M toRead Time Type Injection Delay per Well Injector Endpoint e gt gt gt DualRead 9 T A A a Patke p d uu jd ce 1 7 e pL 7 ce c9 n e o Long Kinetic ee NOH Incubator control is an instrument setup parameter that is not found in the Instrument Settings dialog box 4 4 SOFTmax PRO for Lmax User s Manual BO Chapter 4 Reading Microplates Endpoint Mode Figure 4 1 Instrument Settings Dialog Boxes for an Endpoint Reading SOFTmax PRO for Lmax User s Manual 4 5 Chapter 4 Reading Microplates Dual Read Mode Figure 4 2 Instrument Settings Dialog Boxes for a Dual Read 4 6 SOFTmax PRO for Lmax User s Manual BO Chapter 4 Reading Microplates Long Kinetics Mode Figure 4 3 Instrument Settings Dialog Boxes for a Long KineticsReading SOFTmax PRO for Lmax User s Manual 4 7 Chapter 4 Reading Microplates Fast Kinetics Mode Figure 4 4 Instrument Settings Dialog Boxes for a Fast Kinetics Reading 4 8 SOFTmax PRO for Lmax User s Manual BO Chapter 4 Reading Microplates Instrument Settings Options Read Mode Choices for the read mode are endpoint dual read long kinetics or fast kinet ics To select a read mode click th
91. Endpoint When you finish your entry in the Calculation dialog box and click OK the formula you have entered then appears to the right of the word Custom and becomes the default selection for the Custom option You may subse quently edit this formula or enter a new one by clicking the Formula button again For a full discussion of custom formulas see the Formula Reference Guide SOFTmax PRO for Lmax User s Manual 5 21 Chapter 5 Displaying and Analyzing Data BO Recalculation Options By default SOFTmax PRO for Lmax performs continuous recalculation of the data when you read plates create or change formulas and so on At certain times it would be useful to disable the recalculation feature so that you can make alterations to the experiment without waiting for individual recalcula tions to be completed Two commands are available in the Edit menu that allow you to modify the way in which SOFTmax PRO for Lmax performs recalculation Suspend Recalculation and Recalculate Now 3 CTRL Choosing Suspend Recalculation disables the automatic recalculation feature of the program and places a checkmark to the left of the command in the menu to indicate that it has been selected While this function is enabled no recalculation will occur regardless of what you may change add or delete from the experiment This is especially useful when creating or changing col umn formulas within Group sections for example as it can speed up
92. File menu Printer Name Epson Stylus 800 ESC P 2 Properties Status Ready Type Epson Stylus 800 ESC P 2 Where LPT1 Comment Print to file Print range Copies r All 3333 pages Number of copies fi v Pages from te 12 2 Jel mu Collate Options v Experiment Title v Date and Time v File Name v Page Numbers C Print All Print Report Header T ext OK Cancel Figure 6 8 Print Dialog Box 6 12 SOFTmax PRO for Lmax User s Manual Chapter 6 File Management and Printing Other options regarding the appearance of the printed report can be chosen in this dialog box you can add text to be included in the header on every page of the report maximum of 100 characters in a single row and you can include or exclude the experiment title the name of the experiment in the file as well as the name s of sections and you can include the date and time of printing as well as add page numbers to the report which are printed at the bottom right corner of each page These are all enabled by default until you disable them by clicking in the box with the checkmark next to each option which will deselect that option If a Notes section is longer than can be printed on one page a line may be cut off not printed at the bottom of the first page To fix this problem insert a carriage return in the appropriate place above the page break in the Notes
93. If the width of a section extends beyond the page width boundary the portion that is not visible will not be printed To size two or more columns to the same width highlight those columns and then drag one of the dividing lines to the desired width The width of all high lighted columns will be sized the same as the column to the left of the divid ing line you are moving Columns can also be resized to the minimum amount of space required by choosing Autosize from the Group menu By highlighting the entire table or individual columns and then choosing Autosize the amount of space each column requires will be reduced to the minimum size required by the largest piece of text in that column Double clicking the line dividing two columns causes the single column to the left of the divider to be sized according to the largest amount of text in that column Showing and Editing Existing Column Formulas 0 24 When group sections are first created the formulas used to calculate the data are not displayed These formulas can be shown until you choose to hide them again by mak ing a Group section active and then choosing Show Formulas from the Group menu Note that the command in the menu changes to Hide Formulas after they are shown SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data Group Create Column Create Summary Edit Column Edit summan Autasize Hide Chrl H Hide Replicat
94. If you have not yet installed SOFTmax PRO for Lmax refer to Chap ter 2 Installation and follow the steps to install the software before continuing with this tutorial Hard and Soft Parameters The only settings that must be defined before reading a microplate are the instrument settings found in the Instrument Setup dialog box These set tings tell the instrument what conditions should be in place in order to collect data They include the read mode endpoint dual read long kinetics and fast kinetics the wells to read any injections required and so on These settings called hard parameters define the conditions under which the data is obtained Once a microplate has been read the instrument settings cannot be changed without deleting the data Soft parameters include the template definition data reduction and data display settings These parameters can be defined prior to reading a micro plate but they are applied only after the plate is read and can be changed at any time In this tutorial you will build a complete protocol that will include both hard and soft parameters You will save the completed protocol file prior to collect ing data NOJ This tutorial has been written specifically for use with the Lmax Microplate Luminometer A synopsis of the steps you will take during this tutorial is given below Create a new file Instrument setup Template setup Set reduction parameters Set disp
95. K If you are creating a protocol file from a file containing data first save the file as a data file if you wish to save the data and then per form a second save as a protocol file Data will not be saved with a protocol file Protocol files are stationery pad files when you choose to open a protocol file you are actually presented with a copy of the file which appears as an untitled file Choose the Ci SOFTmaxa PRO for Lmax 1 0 Hard Drive ication tortbe Cp Assays Hec new file here New Enter a name for the new Protocol file here Save this document as Untitled File type Data File 3 Protocol File option buttons Figure 6 3 Save as Dialog Box Macintosh Save this document as TE i sium Choose the Save in SOFT mas PRO for Lmax 1 0 PET iy CF WES location for the J Assays a GHL 5 23 lda new file here xn aequarin Ida in Chem amp 25 lda Enlera cue ix chemiluminescence lda Dual Luciferase 8 lda for the new elisa alow lda Protocol file here File name File type choices Save as type Pro for Lmas Data Files Ida R Cancel Figure 6 4 Save as Dialog Box Windows SOFTmax PRO for Lmax User s Manual 6 5 Chapter 6 File Management and Printing BO Opening Files You can open a SOFTmax PRO for Lmax file by selecting Open 380 CTRL 0 from the File menu within SOFTmax PRO for Lmax or by double clicking on the file name or icon
96. LT ESC means to press and hold down the ALT key and press the ESC key Then release both keys H Keyl Key2 A comma between key names means to press and release the keys one after the other For example press ALT F means to press and release the ALT key and then press and release the F key The names of keys found on different Windows compatible PC computer keyboards may vary somewhat from machine to machine The key names shown in this manual reflect typically used key names Yours should be similar but may not be exactly like those shown For example the print screen key may be shown on some keyboards as Prt Scrn other keyboards may spell out the complete key name soa soa Messages The following types of messages may appear in this manual Ose Calls attention to information that should be followed during instal lation use and or servicing of the instrument or software KAAU ON Indicates a potentially hazardous situation which if not avoided might result in minor or moderate injury loss of data or mechanical damage It is also used to alert against unsafe practices BAER Used whenever an action or condition might potentially cause serious injury to yourself or to others who might use the instru ment or computer 1 6 SOFTmax PRO for Lmax User s Manual Chapter 1 Welcome to SOFTmax PRO for Lmax Getting Help Macintosh On Line Help On line help is available while working with SOFTmax
97. Ltrl M Expand Eti tE Hide Status Ex perimentit The View menu provides three commands Minimize 3 M CTRL M Expand 3 E CTRL E and Hide Status In addition to these choices the names of the experiments appear at the bottom of the menu sections within experiments are shown as submenus allowing you to go to a specific experiment or sec tion within an experiment by choosing the item If the section was closed selecting it from the View menu will open and activate it The currently active section is shown in the menu with a checkmark next to it Minimize 3 M CTRL M If you select Minimize while a section within an ex periment is active the sections within that experiment are minimized If you choose Minimize while an experiment tool bar is active all ex periments will be minimized sections within are hidden and only the experiment tool bars are visible Expand 3 E CTRL E If you select Expand while a section within an experi ment is active the sections within that experiment are expanded If you choose Expand while an experiment tool bar is active all sections within all experiments become visible the sections may be minimized or expanded Hide Status Show Status Hides or displays the status bar Experiment Menu Experiment New Experiment New Notes New Plate Ctrl F Mew Graph 3 26 The Experiment menu provides five commands New New Notes New Plate and New Graph New Creates
98. M drive e Bus mouse e Serial port COMI or COM2 e 486 33 MHz computer e Microsoft Windows NT 4 0 or 2000 e Color monitor e SVGA graphics adapter set to 256 colors and small fonts or better e 32 MB RAM or greater e 1 GB hard disk drive e 1 44 MB HD high density 3 5 inch floppy disk drive e Access to a CD ROM drive e Dus mouse e Serial port COMI or COM2 2 PC hardware tested for the SOFTmax PRO for Lmax release Dell Dimension XPS R350 with Windows 98 Dell Dimension XPS H266 with Windows 2000 HP Vectra XU 3 200 with Windows NT Compaq Presario 9234 with Windows 95 Printers tested HP DeskJet 812C and HP LaserJet 4000N 2 6 SOFTmax PRO for Lmax User s Manual 2 Chapter 2 Installation Install SOFTmax PRO for Lmax 1 2 3 4 5 6 7 8 Follow these instructions to install SOFTmax PRO for Lmax from the CD to your computer s hard disk Insert the SOFTmax PRO for Lmax CD into the CD ROM drive Macintosh Double click the CD ROM drive icon to open it Windows Open My Computer and then double click the CD ROM drive icon to open it Double click the Installer icon Macintosh or Setup icon Windows and follow the screen prompts The Welcome screen suggests that you close all other programs before begin ning installation of SOFTmax PRO for Lmax software It is also recommended that you disable any virus protection software you may have running before installing SOFTmax PR
99. MEN OCCU Co oie RU ER ROPSUT TERME rnern EA 3 19 Menus and COMMANCS ccinrsenseecka aa RHEIN RURAL ORC LR CREARI 3 19 Interprocess MOSSA IND au dans nena mra EESE REC E E GU OR UR AE RUR CR 3 33 SOFTmax PRO for Lmax User s Manual U Chapter 4 Reading Microplales 22x RREEERESOEREERENRERREEFSTEN ERES 4 1 WOON EE cers een aes eed cae gees ee ee oe es ees 4 3 Metue nt SeN eoe coe ooetereedro ve vbt OASIS RESP IST ed PET ee eee 4 4 Insttumen 5er unes ODUOLIS rinni i tate OE RIPE 4 9 Ten ae BdilgE ad e tu esancte E E A E 4 13 Selecting Wells in the Template Editor 0 000022 ee 4 14 The Template Editor Tool Bar 2 cece eee eee eee eens 4 15 iru dg P PEE m 4 24 Copying and Pasting Templates 0 0 0 eee 4 27 Reading a Microplate 2 2 coe be iadcdeiedetodateculudeiadetaantaastecatada 4 29 Chapter 5 Displaying and Analyzing Data cece ccc ee eee ee ene 5 1 IMU OCUCHONT EL A 5 3 Initial Data Display and Reduction 0 00 5 4 The Data CO 12220 secesacegacucucucneueeeaqeneeeeeuaseauzeceaueesacd4 5 4 The Display Dialog DOX 5e aucma ure mon epe dup um e RR 5 5 Well Graphs of Kinetic Wells 0 0 0 cee eee eee eee 5 12 Masking Wells Gelagewecacayatagatauaenustasataeeteustaeaeagauas 5 15 DAA Red cUON 2 4 ecatecese eens PoRCR RR EPRE HACER D ETERNE EERE EEEE EA 5 16 ENG OOING P 5 18 Dig
100. Manual 2 Chapter 6 File Management and Printing Saving Files Manually SOFTmax PRO for Lmax provides several ways to save files manually If the current file has not been saved is Untitled it can be saved under any name If you have opened and modified an existing file you can save it under the same name overwriting the old information or under a different name You can also save the file in the same or in a different location on your hard disk in a different folder or subdirectory Saving Files under the Same Name If you have made changes to a file and want to save these changes thereby updating the same file select Save 35 CTRL S or Save As from the File menu If you select Save 5 CTRL S the current version of the file will replace the old one without notification If you select Save As an alert box will appear asking if you want to replace the previous file by that name If you are intentionally replacing the file click Replace or Yes The new information will be written over the old information under the same file name Saving Files under a Different Name or in a Different Folder Autosave If you have made changes to a file and want to save these changes as a new file or if you wish to save the file to a different location select Save As from the File menu A dialog box will open allowing you to enter a new name and or a new location for the file To choose a different locatio
101. O for Lmax Click Cancel to exit the installer and close any programs and or disable virus protection and then start the installer again from step 3 above To continue with installation click Next The screen named Choose Destination Location asks you to select the drive where the software will be installed A default location will be shown on the screen If you have more than one hard drive in your computer you may change the destination to the disk drive where you wish SOFTmax PRO for Lmax to reside or accept the default location The Setup Type screen asks you to choose between Complete the default or Basic Basic installation installs all but the tutorial files If you wish to omit the tutorials choose Basic Otherwise just click Next to continue NOH The Back button is available on all but the first screen allowing you to go back to previous screens and review or change information that you have entered Click Next to start the actual installation of SOFTmax PRO for Lmax files When installation is complete the Finished screen will appear Click Close to end the installer setup program On the Macintosh remove the CD by dragging its icon to the Trash On the PC close the window for the CD and My Computer and then press the CD eject button on the CD ROM drive Replace the CD in its holder and store it in a safe place An open folder named SOFTmax PRO for Lmax will be visible on your screen
102. O for Lmax affect the way in which data is displayed the type of reduction specific time settings and more These settings are discussed in Chapter 5 Displaying and Analyzing Data SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data ItttOQ UC HOEEos A cor ys sgh prr ppp daba oap one etl nap dob ay ge 5 3 Initial Data Display and Reduction 004 5 4 The Data Display 1 ee buerPPPUPPPERPVEPPHPASERE TP 5 4 The Display Dialog Box seeeeeeeeeeess 5 5 Well Graphs of Kinetic Wells 0 5 12 Maskine Wells Guopxbtrabutv aep bp IU UE qid 5 15 Data Reducir sere ears PD APEAT APUD pP pU 5 16 PC DOME v eppppE pares aoe A DU Eb E See 5 18 Daal Read 4 5 secs P EEPPDAVEAP ERO DAVERP MPO esa ate 5 18 Long and Fast Kinetics uos ao poPAPPEPPAPEPPMeD EA 5 18 Custom Reduction Formulas 004 5 21 Recalculation OpHODS s cass ore Pp pP RIP Pd 5 22 Displaying Data in Group Graph and Notes Sections 5 22 SOUP eC MONS Seema age vot eU Pop bbs 5 22 INOS eC NON care ID ARAM ee ae te ee ae 5 28 Graph SeCtoB evi epPRUDUUPEPPVUPRPUPIHPEPIIP ES 5 28 Formulas in SOFTmax PRO for Lmax Lees 5 46 Chapter 5 Displaying and Analyzing Data 5 2 SOFTmax PRO for Lmax User s Manual 2 Chapter 5 Displaying and Analyzing Data Introduction Initial display and reduction of the data received from the Lmax is performed in the Pl
103. OFTmax PRO for Lmax Title Screen 1 0 ec eee eens 3 3 SOFTmax PRO for Lmax Screen and Window for the PC Windows 3 4 vcn P 3 6 Tool Bar from an Active Graph Section 0 cee eee lees 3 9 Example of a Tool Bar Sticking to the Top of the Window 3 10 Closed and Opel SECORS acces aua uud nva dioc tatam aen E E 3 10 Result of Minimizing an Experiment Tool Bars Hidden 3 11 Deparale Section WindOW a a aco dod ucc dud qi t ania dp id ia no Uu 06 13 310 06 14 0 1 0 3 11 Example Notes Section Named Results llle 3 12 PIAS DOCHIOEe oed ados E qv ara MUR doo ori E EE cds don erue esennsenasens 3 13 Group Section example shown prior to collecting data 3 15 Graph Section example shown before data collection lusus 3 17 Menu Bar Macintosh menu top Windows menu bottom 3 19 SOFTmax PRO for Lmax Menus Windows version esses 3 21 Instrument Settings Dialog Boxes for an Endpoint Reading 4 5 Instrument Settings Dialog Boxes for a Dual Read 0 045 4 6 Instrument Settings Dialog Boxes for a Long KineticsReading 4 7 Instrument Settings Dialog Boxes for a Fast Kinetics Reading 4 8 Instrument Settings for an Endpoint Reading 00 00 esses 4 9 Instrument Set
104. PRO for Lmax for the Macintosh by clicking the Help menu at the far right of the menu bar i Click here for the Help Menu t About Balloon Help Show Balloons SOFT max Pro for Lmax Help SOF Tmax PRO for Lmax Formula Help Help Options Figure 1 1 Help Menu for SOFTmax PRO for Lmax for the Macintosh Choosing either of the two help options at the bottom of this menu SOFTmax PRO for Lmax Help or Formula Help causes a Help window to appear This window provides a list of topics on the left and information about the selected topic on the right Figure 1 2 shows the window for Formula Help In this figure the topic Math Functions has been selected on the left and the corresponding information about that topic appears on the right Eu Formula Help Formula Help Topics Math Functions Comparison Operators a NOTE All math functions are calculated in radians 180 T e Conditionals radians e Direct Well Accessors Errors Abs number e Group accessors Returns the absolute value of a number ar list of numbers Interpolation Example Abst 103210 Abst 103210 Kinetic and Spectrum Accessors Acos number Math Functions EN Returns the arccasine of a number ar list of numbers Math Operators Example cosi 0 25321 82248 Other e PathCheck Accessars AntiLog number e Plate Data Accessors Returns the AntiLog t base e of a number or list of numbers This e Plate Informa
105. Plate section data the format of the copied information must match the Instrument Settings of the target Plate section and the settings chosen in the Export Format in the Preferences Pre read data cannot be pasted into a Plate section If there is a mismatch between the data to be pasted and the target sec SOFTmax PRO for Lmax User s Manual Chapter 6 File Management and Printing tion SOFTmax PRO for Lmax will attempt to paste the data but if it cannot do so an error message will result an example of such an error message for a Plate section is shown in Figure 6 16 After data is pasted into a Plate section the information to the right of the display will show imported data 5 Pasted Tent doesn t match plate settings Figure 6 16 Error Message Generated When Pasting Mismatched Data If you have created a template for a Plate section prior to pasting data numbers will be shown only in the group s you have created in the template Data in other wells will not be reported but will be available if you alter the template Clearing the template or assign ing groups to all wells in the plate will show the complete data set Copying to Plate section Group table section and Notes section data can be copied from Other Programs SOFTmax PRO for Lmax and pasted into another program that will accept the data such as a spreadsheet or word processing program No copy or paste features are available for Graph sections or Well G
106. RO for Lmax User s Manual 6 19 Chapter 6 File Management and Printing BO Importing Data from Outside of SOFTmax PRO for Lmax SOFTmax PRO for Lmax imports data in tab delimited ASCII text format allowing you to import data collected by a non Molecular Devices instrument into SOFTmax PRO for Lmax Note that only plate data can be imported Selecting Import from the Import Export menu under File causes the Import dialog box to appear Lookin 4 SOFTmax PROS tor Lmax 1 0 iw amp Al etl Gafil esceekdald File name Ce Fesdhp Teirra S Cancel Figure 6 15 Import Dialog Box Choose the file you wish to import and click the button Note that data to be imported must contain the appropriate header information Anew experiment is created in the open SOFTmax PRO for Lmax file for the imported data New Plate sections are created for each data set within the file Plate pre read data can also be imported Note that the time date stamp for a section containing imported data shows imported data rather than a date or time Importing and Exporting Templates Template information saved as an ASCII text file can be exported from Plate sections and can also be imported into such sections A template file consists of seven columns of data that are separated by tabs Each line of the file pro vides information for one well in a plate When template information is exported the information
107. ROS pees TA Gray VAG T ae go oe Purple hoc T gecs Yellow en Pink 515 RTT Blue Clicking anywhere in an inactive section makes it active Selecting an experi ment deactivates any individual section within it that may have been active and vice versa Activating a section also activates the experiment containing that section To activate multiple sections at the same time hold down the Shift key and click in the tool bars of the sections you wish to activate Doing this allows you to move copy duplicate print and delete multiple sections simultaneously 3 8 SOFTmax PRO for Lmax User s Manual Chapter 3 SOFTmax PRO for Lmax Overview The last item farthest right in the SOFTmax PRO for Lmax menu bar changes to reflect the section type that is currently active If a Plate section is active for example the last menu item will be Plate If an experiment is selected this menu will not be shown New Plate Notes or Graph sections can be created whenever you need them and more than one of each type of section can exist in a single SOFTmax PRO for Lmax experiment Group sections are created automatically when you define new groups in the Template Editor or may be created with the Duplicate command A section can be deleted if it is no longer needed The Delete command in the Edit menu changes depending upon which section is active For example if the section titled Plate 1 is acti
108. SOFTmax PRO for Lmax User s Manual Version 1 0L Software for Macintosh and Windows Operating Systems _ z Molecular Molecular Devices Corporation Devices Sunnyvale California 94089 Part 0012 0074 Rev A Molecular Devices Molecular Devices Corporation SOFTmax PRO for Lmax User s Manual Copyright Copyright 2000 Molecular Devices Corporation All rights reserved No part of this publication may be duced transmitted transcribed stored in a retrieval system or translated into any language or computer language in any form or by any means electronic mechanical magnetic optical chemical manual or otherwise without the prior written permission of Molecular Devices Corporation Trademarks SOFTmax is a registered trademark and Lmax and MAXline are trademarks of Molecular Devices Corporation All other trademarks or registered trademarks are the property of their respective owners Lmax is one of Molecular Devices MAXline Microplate Readers Disclaimer Molecular Devices Corporation reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Molecular Devices Corporation assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information
109. Type from the Graph menu or by click ing the Graph button Jin the section tool bar Graph Options Graph i Height E e WB Scatter Sample Text Ob C2 Cluster bar Sample Text Sel hil C Stack bar Legend m Connect points 5 le Text a be Plot symbols Plot O Flat z As Std Dey Standards Yo Yales Standards ecEFFors fo Error Y Errors Ma Error Figure 5 39 Graph Options Dialog Box The default options are shown in Figure 5 39 but you can alter these as desired you may change the font and type size used for the title legend and or axes of the graph and you can choose the color and type of symbol used on a scatter graph to show the plot The graphs shown in preceding Fig ures reflect the default graph options Connecting the points of a scatter graph can be useful in determining the closeness of the fit Figure 5 40 below shows a scatter graph with points connected compared to a linear fit NOH The Connect Points option connects the points in order of appear ance by sample name not the appearance of points from left to right on the graph 5 44 SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data Graph Options Button vows C o X and Y Axis Buttons E concentration y mE Boe B CQ Pb Croupi concentration ve Heans alus Os Figure 5 40 Scatter Graph with Points Connected Linear Fit Applied Other options in this dialog box allow you to pl
110. a Chapter 3 SOFTmax PRO for Lmax Overview Using Help Allows you to choose from an indexed list of topics or to search for the type of help you re looking for About SOFTmax PRO for Lmax Brings up the About screen splash screen which gives the serial number ROM version and the instrument type connected to the computer as well as the software version for SOFT max PRO for Lmax Help Menu Macintosh About Help Brings up a dialog box explaining how to use the Apple Guide which gives detailed information about the help function on the Mac intosh About Balloon Help Show Balloons SOFT max PRO Help Formula Help Show Hide Balloons Depending upon whether or not you have balloon help turned on or off this menu presents the choice to reverse the setting SOFTmax PRO Help Opens the help section devoted to SOFTmax PRO for Lmax Items are presented in alphabetical order Formula Help Opens the formula help section which provides information about how to use formulas including formula examples Interprocess Messaging i 1 This feature is available on the Windows Operating System only soa Many operations found in SOFTmax PRO for Lmax can be controlled by other Windows applications by means of an ASCII string based messaging system Commands are available for opening and saving files controlling instruments selecting sections exporting data from Plate and Group sections and importing T
111. a hierarchy as follows the template is composed of samples and groups A set of one or more replicate wells makes up a sam ple A set of related samples forms a group For example you may have a group named Standards that consists of seven samples named STD01 STD02 STD07 Group names may be up to 32 characters long while sample names may be 300 characters long Samples may be assigned one at a time using the Assign button or several at the same time using the Series button If you create a group in the Template Editor but do not assign any wells to the group a section for that group will still be created automatically when you close the template You can remove the group both its section in the window and its group name in the template by making that Group section active and choosing Delete Groupname where Groupname is the name assigned to that group in the template from the Edit menu Using the Macintosh you can also delete the selected Group by pressing 3 within Windows you can press CTRL Note that deleting the wells assigned to a group from the tem plate does not delete the corresponding Group name from the drop down list The only way to remove a group name from this list in the Template Editor is to delete the corresponding Group section from the experiment Selecting Wells in the Template Editor In the Template Editor for a Plate section you can select one or more wells by clicking or dragging on the grid Mul
112. acintosh to the printer or modem port and the 25 pin connector to the Lmax Connecting to the modem port of the Macintosh is preferred allowing the printer port to remain available If you do connect the Lmax to the Macintosh printer port first inactivate AppleTalk using the Chooser desk accessory in the Apple amp menu NOJ If you are running other applications that use AppleTalk it may be necessary to quit them before making AppleTalk inactive Figure 2 1 8 Pin DIN to DB 25 Macintosh Straight through Serial Cable USB Connection Although the Lmax requires a serial connection through the use of a USB to serial adapter you can connect a serial cable between the Macintosh and the Lmax instrument Molecular Devices has tested and can recommend the Key span USB Twin Serial Adapter not the PDA adapter Connect the USB con nector from the adapter to an open USB connection and choose the printer port from the Preferences in SOFTmax PRO for Lmax the Keyspan software only supports direct access to the emulated printer port and may cause the Mac to freeze if the modem port is chosen Connect the serial cable to port 1 of the adapter not port 2 and plug the other end of the serial cable into the recep tacle on the back of the Lmax instrument SOFTmax PRO for Lmax User s Manual 2 3 Chapter 2 Installation ae ws Compatible Computer IN Windo it The type of cable required to connect your computer and the Lmax will depen
113. after data is collected In addition it is recommended that you define a template the reduction param eters and display parameters prior to reading the microplate as these param eters determine how data is displayed and analyzed but these last parameters may be set up and or modified after data collection A typical process for preparing collecting and analyzing data is as follows Start SOFTmax PRO for Lmax Create Plate section s as needed Define instrument settings use the button in the Plate section tool bar or the command in the Control menu Define the template reduction parameters and display parameters Prepare the microplate to be read and place it in the Lmax Initiate the reading Save the data file Modify the template reduction parameters or display parameters as you wish them to be or if you didn t set them up in Step 4 do so now Save the data file Analyze the data using the Group section tables and Graph sections Save the data file when you are finished This chapter discusses how to define instrument settings create a template and initiate a reading Data analysis is covered in Chapter 5 Displaying and Analyzing Data SOFTmax PRO for Lmax User s Manual 4 3 Chapter 4 Reading Microplates BO Instrument Settings You must specify the instrument settings prior to actually reading a micro plate These settings include e The read mode endpoint dual read long kinetics or fast
114. al 5 17 Chapter 5 Displaying and Analyzing Data BO 5 18 Endpoint Dual Read All of the read modes offer one or more predefined choices for reduction The predefined reductions are described below under the appropriate read mode heading In addition with any read mode you can create a custom reduction formula to better suit your needs Since all custom reduction formulas are defined the same way regardless of read mode they are described together after the individual read mode sections The default reduction for endpoint readings is MValue You can also choose Custom in order to create a different reduction formula The default reduction for dual readings is MValue Other reductions avail able from the pop up menu are PValue MValue PValue PValue MValue IMValue PValue and PValue MValue You can also choose Custom in order to create a different reduction formula Long and Fast Kinetics The default reduction for kinetic readings is MValue If you do not wish to use the default reduction you can choose Custom and create a different reduction formula Kinetic Limits The Limits settings for the display of kinetic data are MaxRLU MinRLU Lag Time and End Time The display of RLU values is shown relative to the first point mea sured for each well Negative kinetic values decrease with time and limits should be set accordingly below 0 to view negative kinetic data Limits define the data that will be vie
115. alon BOX cacidauntiak a daria dra qn Pari ira RH UR RH RAD USER UR 6 18 Import Dao DOXew eus ismadte um pua Ta Sea eta d mea y co TEE PETER REFERS 6 20 Error Message Generated When Pasting Mismatched Data 6 23 Det Password Dialog BOX uo o ua uicem gm ptm iem RR d ACRI CRUR E E UR S 6 26 Password Required Dialog Box 0 0 ec eens 6 27 Tutorial File Blank with Notes 1 and Plate 1 Sections Open 7 5 Instrument Settings Dialog Box Integration and Pre read Section Open 7 6 Instrument Settings Dialog Box Injection and Delay Section Open 7 7 Instrument Settings Dialog Box Injection and Delay Section Revised 7 8 Instrument Settings Dialog Box Injection Wells Section Open with injector Enc Pled C s 7 9 Instrument Settings Dialog Box Injection Wells Selected 7 10 Template with Three Columns of Wells Selected and New Group Demis e i e e ce deo ded ew repeoenupereneedeeageenuneauseweaeneeeceaeanes 7 11 Instrument Settings Dialog DOX ameti m rim UR RR RR RI UR 7 1 Template with Standards Assigned l l eese 7 12 Template with Group Associated Blank Included with Standards 7 12 Detles Wid OP DOLES ee eo des S awe ri xs Td tSUETUTPE RATER E NEUEER REA EUER 7 13 Template with CTRL Shift Keys Depressed 0 0 0 eee eee eee eee 7 13 First Unknowns Added to the Template selle ee eee eee
116. ame groups may be used in different templates After groups are defined in the template and you return to the Plate section the data display will show the group areas in different colors Button Equivalent Template in the Plate section tool bar Reduction Opens the Reduction dialog box Options available in the Re duction dialog box depend on the type of reading performed end point dual read or kinetic The Reduction dialog box is used for initial reduction of the data collected from the microplate reader The luminescence information collected is reduced to a single number per Export Template Import Template Section Mame New Window 3 28 SOFTmax PRO for Lmax User s Manual Chapter 3 SOFTmax PRO for Lmax Overview well More information regarding reduction options can be found in Chapter 5 Displaying and Analyzing Data Button Equivalent in the Plate section tool bar Display Opens the Display dialog box Allows you to decide how the infor mation in the Plate section will be displayed More information re garding display options can be found in Chapter 5 Displaying and Analyzing Data Button Equivalent B Display in the Plate section tool bar Graph The Well Graph commandcan be activated both during and after data acquisition with a kinetic reading You can get a close up view of the plot shown in one or more wells by first selecting the well s and then choosing the
117. ample TEM crot__I genes m Concentration n1 4 5 Cancel Figure 5 23 Standards and Unknowns Groups Created 2 These Group sections are created using the Basic column format which by default generates columns for values concentration and mean values The example below shows the Group section for GA native v ff cacnativey Le Be GA nathwe uM Al poer er D Me T coer rer rer G z 33515 33515 Ei Di 4 426ls dabl LOOD ELLE s 100 GO GOs HI e 48M 40594 tooo Figure 5 24 GA native Group Section 3 Anew Graph section is created by selecting New Graph from the Experiment menu This causes the Plots dialog box to open automatically Clicking OK opens the Graph Options dialog box which allows you to choose the graph type modify the fonts used for the graph and so on Choices should be made as follows GA native for the group to be plotted the concentration of GA native for the X axis and values of GA native for the Y axis No error bars are added SOFTmax PRO for Lmax User s Manual 5 31 Chapter 5 Displaying and Analyzing Data 5 32 4 5 wy Plott x Hame Flot 1 Source iment 1 een 7 pa iem Size necat 5 CHOMOB native Error CHOMOG amp h Color G5A native PY UAXES eee m a iA native Style Error Mo Error bad Circle w Cancel Figure 5 25 Graph Options Dialog Box A second plot is added to this graph
118. and printer can be connected to an A B switch box one 8 pin DIN input port with two 8 pin DIN output ports available from MacWarehouse 1 800 255 6227 part number BND0199 Din 8 A B switch box This switch box can then be attached to the single serial port of the com puter The A B switch of the switch box is used to physically switch the con nection between the instrument and the printer Because of the limitations of the single serial port Macintosh the Autoprint feature of SOFTmax PRO for Lmax cannot be used unless you use the ethernet port of the computer for printing To print from SOFTmax PRO for Lmax for Macintosh using the single serial port Macintosh connect to an AppleTalk compatible printer such as the Hewlett Packard DeskWriter C or 550C using an AppleTalk cable connection and the AppleTalk printer driver Serial printers such as the Apple StyleWriter which use an Apple 8 pin DIN to 8 pin DIN peripheral cable and a serial printer driver cannot be used with SOFTmax PRO for Lmax for Macintosh on a Macintosh with a sin gle serial port Configuration for Reading The computer SOFTmax PRO for Lmax for Macintosh and switch box must be configured as follows in order to establish communication with the Lmax AppleTalk Inactive in the Chooser dialog serial port set to modem in the SOFTmax PRO for Lmax Preferences dialog and the switch box set to com municate with the Lmax to access the Chooser or Preferences dialogs refer to T
119. aph in the Opens the Export Graph Graph menu As dialog box 3 18 SOFTmax PRO for Lmax User s Manual 8S Chapter 3 SOFTmax PRO for Lmax Overview Instrument Settings You must specify the type of reading to be performed prior to actually read ing a microplate Instrument settings allow you to choose a read mode endpoint dual read long kinetics or fast kinetics timing for a kinetic run and so on Complete information regarding instrument settings can be found in Chapter 4 Reading Microplates Menus and Commands The menu bar for SOFTmax PRO for Lmax is located at the top of the window just beneath the title bar File Edit View Experiment Control Assays Graph Help File Edt View Expenment Control Assays Plate Window Help Figure 3 13 Menu Bar Macintosh menu top Windows menu bottom You may access SOFTmax PRO tools and features in several ways menus tool bars dialog boxes and keystrokes Figure 3 14 shows all of the menu items that are available in SOFTmax PRO for Lmax When menu items are unavailable they are dimmed shown as gray rather than black available choices depend on the action you are performing with the software An ellipsis three dots following a menu item indicates that choosing this item will bring up a dialog box Menu items followed by a trian gle are hierarchical menus choosing such an item causes a submenu to appear to the right of the original item Certain menu items
120. apter presents a general description of the features and use of SOFT max PRO for Lmax It describes the parts of the SOFTmax PRO for Lmax window including experiments and sections e the menus and tool bars found within the SOFTmax PRO for Lmax pro gram the basic steps required to perform a reading complete information regard ing reading using SOFTmax PRO for Lmax can be found in Chapter 4 Reading Microplates and e the basic types of data analysis detailed information regarding data analy sis can be found in Chapter 5 Displaying and Analyzing Data SOFTmax PRO for Lmax employs standard Macintosh and Windows conven tions for using menus dialog boxes windows and the mouse This manual assumes that you are familiar with the basic operation of your computer While some general instructions are given here and many of the terms used in SOFTmax PRO for Lmax are defined in Chapter 1 Glossary of Terms if you need additional assistance regarding the basic use of your computer please review the documentation accompanying it The SOFTmax PRO for Lmax Window When you start SOFTmax PRO for Lmax a title screen will appear showing the information entered during registration along with the SOFTmax PRO for Lmax version the instrument type if connected the ROM version installed in the instrument if available and copyright information Version 1 0L Written by Duane Boman Duff Caldewewy Tae Kang and Tom Smith
121. are Dilution Factor Specifies the amount of dilution for a given sample This factor can be multi plied against interpolated values to determine concentration of undiluted sample Dimmed Unavailable or disabled A dimmed button or command is displayed in light gray instead of black and it cannot be chosen SOFTmax PRO for Lmax User s Manual 1 13 Chapter 1 Welcome to SOFTmax PRO for Lmax BQ Double Click To rapidly press and release the mouse button twice while the pointer is on an object Double clicking carries out an action such as opening an icon Drag To move an object on the screen by clicking it holding down the mouse but ton and then moving the mouse Also to select an item in a list by dragging through the list or to select multiple items by dragging across them Dual Read An endpoint reading this mode is designed to perform two separate integra tions one after each of two possible injections in any or all wells of a 96 well microplate no injection is possible in a 384 well plate Duplicate A command available in the Edit menu that allows you to create identical cop ies except data of all sections and experiments The name of the duplicated item will be the same as the previously active item with the word Copy appended Endpoint A single integration of samples in 96 or 384 well plates with two injections possible if desired in any or all wells of a 96 well plate no injection possible in 3
122. are used in this manual Format Meaning Menu Item Menu titles and menu items Text Entry Key Name Entries that you type from the keyboard or keys that you press on the keyboard Information shown in this font should be entered exactly as it appears Text Entry Placeholders for information you must provide for Example if you are asked to type filename you should type the actual name for the file instead of the word shown in this other font Button Buttons shown on the screen Note that sometimes an actual representation of the button may be shown E The accompanying text applies only to SOFTmax PRO for Lmax for Macintosh 71 The accompanying text applies only to SOFTmax PRO for Lmax for H Windows SOFTmax PRO for Lmax User s Manual 1 5 Chapter 1 Welcome to SOFTmax PRO for Lmax BO Keyboard Formats Key designations combinations and sequences appear in the following formats Format Meaning 3 Key Selections on the Macintosh can be made using the mouse but often shortcuts are available using the keyboard Shortcuts com monly use the command 3 key in conjunction with another key and are shown with the command key symbol 3 followed by the accompanying key To use a shortcut you must press and hold the command key while pressing the second key H Keyl Key2 A plus sign between key names means to press and hold down the first key while you press the second key within Windows For example press A
123. are using certain group names and associated sample descriptions may have been created You can use these predefined groups and or create new groups to suit your needs When creating new groups you may enter any name you wish Once entered this name will be available for use in that experiment thereafter New groups can be created independently from selecting or assigning any wells in the Tem plate Editor Each well designated as being part of a group has associated with it a group name a sample name or replicate ID a sample descriptor optional and a default column format group type such as Standards for example When a group is applied to selected wells the group name is displayed in bold type over the wells and the wells bear the same color Clear Edit Sample Series Assign Drop Down List Button Button Button Button Sampe or v Concentration a unital my Expenment 1 Piate 1 Edit AS e ole a a A Frint Figure 4 11 Template Editor Dialog Box SOFTmax PRO for Lmax User s Manual 4 13 Chapter 4 Reading Microplates BO Groups are always visible both in the Template and in the data display of the Plate section Even though they are grouped single wells are still selectable in either the Template Editor or the data display Samples and groups may exist across multiple Plate sections as well as within a single Plate section Each template is built upon
124. ata v Plated1 Copy 2 Ymax units per eec Plate 1Copy Fast Kinetic Integrate 1 sec 20 P Injection Ott M Injection Ott Lag Time 0 00 End Time 0 20 RLU Min 0 RLU Max 50 max Pte 20 20 Wavelength Combination Malye Plate Last Read 10 03 AM 5 21 00 0 1 0 2 3 4 5 s 0 B 0 foe 0 B 0 g zz Figure 5 5 Fast Kinetics Data Reduced Displayed as Ranged Gray Scale The Gray Scale option presents the raw data in eight shades of gray changing from light for values less than or equal to the low limit to dark for values greater than or equal to the high limit Figure 5 6 shows reduced endpoint data displayed as Gray Scale v C3 rer i Plate 1 1 2 3 4 5 B T 5 3 10 11 12 A e IS T E ENW EW Eoo cE C C O MEME TE TTE TT I Id I Peet om a lh ME o0 See _ LITT Wavelength Combination iw alue s 14 BN 25 Jz 38 E 6 5 H 78 H P 52 H 92 Figure 5 6 Endpoint Data Reduced Displayed as Gray Scale SOFTmax PRO for Lmax User s Manual 5 11 Chapter 5 Displaying and Analyzing Data BO 5 12 Well Graphs of Kinetic Wells You can enlarge the display of the wells shown with either long or fast kinetics mode to get a close up view of the data This is done by either double clicking the particular well in the data display or by selecting the well and then clicking the Well Graph button in t
125. ata collected prior to the set Lag Time The value for Lag Time can be changed after the plate is read The default is 0 End Time This setting specifies the time at which you wish to stop showing data in the display Any values occurring after this limit will not be reported in the dis play The default setting is the total assay time The value for end time may not be greater than the total assay time Absolute Values This setting causes the plots shown in the data display to be drawn at their absolute RLU as opposed to the default with the box unchecked which off sets the first point to the 0 0 coordinate Enabling Absolute Values may cause some data points that do not fall within the default limits for the plots to dis appear To see more data yet still display absolute values increase the limits for MinRLU and or MaxRLU found in the Reduction dialog box Figure 5 12 ws Reduction x a Wavelength Combination Slo RN ea noo NIGER EHS Anno poo In SOI EAN dng ASAE Doe Ee Door Ro Sale mood IsSoE ISSR ae rInCHe nISE IE SoenIr Hoo nie noon goer noon ninoernooernoocinsosansanensonrmccinsnearoneneonanrecrias i RLU Limits bo Iv Absolute Values S600 Figure 5 12 Limits in Reduction Dialog Box If you increase the MaxRLU setting to be at least as large as the largest lumi nescence value expected most or all of the data plots should appear in the data display Kinetics Reduction The default kinetics reduc
126. ata files of PC origin as SOFTmax PRO for Lmax files you need to enter the asso ciated three letter extensions that correspond to these files in the File Exchange Add Mapping dialog box The table below lists the type of file and its corresponding extension that should be entered in the dialog box Table 6 1 PC File Name Extensions and Macintosh Document Types for Use with File Exchange Tvoeof File 3 Letter Macintosh TE Extension Document Type SOFTmax PRO for Lmax LPR sPFd Protocol File SOFTmax PRO for Lmax LDA SPFd Data File For example if you will be working with SOFTmax PRO for Lmax protocol files created on the PC enter LPR in the box under Extension Then click the application icon currently shown as File Type in the cen ter of the box under Application Program From the menu choices navigate to the folder where the SOFTmax PRO for Lmax application resides High lighting this application will enable the Macintosh to start SOFTmax PRO for Lmax and read the corresponding protocol file whenever you double click a PC file bearing the LPR extension Also select the appropriate Macintosh document type shown in the table above Clicking OK causes this dialog box to close and updates the earlier dialog box to show the extension and associated program you just entered Repeat steps 3 through 5 for other PC file name extensions with which you will be working SOFTmax PRO for Lmax User s
127. ate section in which the data has been collected Display and reduc tion parameters can be set up either prior to collecting data or after data has been collected The display and reduction parameters may be modified at any time Data analysis and the display of analyzed data is also performed in the Group sections created when wells are assigned to groups in the Template Editor and the Graph section s Notes sections may be used in conjunction with any other section to provide text commentary and or a summation of data through the use of summaries Notes sections are used most commonly to provide an introduction to or sum mary of results for a data file All data files have a minimum of one Plate section containing data Multiple Plate sections may be present Most files will have a template and Group Graph and Notes sections However depending upon the assay and how you wish to report the data only some of these sections may need to be present in a file Some simple assays allow you to calculate concentration in the Plate section In such an example Group and Graph sections would be necessary only if you wished to display the data graphically Standards and unknowns are defined in a template Unknowns are then inter polated from a standard curve The following diagram shows the relationship between the different sections in SOFTmax PRO for Lmax Standard Group Table lt 4 Reduced Number from Plate Reported Here
128. ate sections all or selected and or Group sections all or selected After choosing a name for the exported data file click OK to save the exported data SOFTmax PRO for Lmax User s Manual 6 17 Chapter 6 File Management and Printing Expat Data bo s Save ri E SDF T rax FRU fer Less 1 0 ia wl cl E3 F9 rid Fie pare alris giir kia Ed ERA oe T eit Fies 7 4 Ceod T Fise Sector A Geop Sachont e aM ou Selected C betected Figure 6 14 Export Dialog Box Exported tab delimited ASCII data files have a minimum of five lines and in most cases will be considerably longer The first line of an exported ASCII data file includes one field indicating the number of data sets in the file This single field includes the text BLOCKS followed by a number that equals the number of data sets The second line of a file for a Plate section includes 19 fields that provide all of the information needed to determine the type of test that was run to measure the first data set The table following describes each of these fields 6 18 SOFTmax PRO for Lmax User s Manual 8S Chapter 6 File Management and Printing Table 6 2 ASCII Data File Fields and Descriptors Section name Defined in Section dialog Export version 1 2 if there are headers Export format PlateFormat or TimeFormat set in Preferences Read mode Endpoint dual read long kinetics or fast kinetics
129. ates can be copied and pasted to other regions of templates within the same or to a different experiment All template settings are pasted into the new template except the group name the new group is given the name Group X where X is the next greater number than that used already for group names To copy a region of a template highlight the region and then select Copy from the Edit menu or press C Macintosh or CTRL C Windows Only the high lighted wells will be copied To paste the region into another area of a template highlight the region and select Paste from the Edit menu or press 3 V Macintosh or CTRL V Win dows Only the highlighted wells will have template information pasted into them Data is pasted using row priority all wells in a row of highlighted wells have information pasted into them before proceeding to the next row from left to right If fewer wells are highlighted for pasting than were copied the data SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microplates will be pasted until all paste highlighted wells are filled If more wells are highlighted for pasting than were highlighted for copying the extra wells are left empty NOF If you try to paste template information between Plate sections hav ing different plate format settings an error will result Plates must be of the same type to copy and paste template information Reading a Microplate When you read a microplate using a
130. ation of the current ver sion This can cause SOFTmax PRO for Lmax to default to using the old directory for assay files rather than the directory associated with the most current program version Follow the instructions given above to choose the correct folder for the default assays folder SOFTmax PRO for Lmax User s Manual 2 15 Chapter 2 Installation 2 16 SOFTmax PRO for Lmax User s Manual Chapter 3 SOFTmax PRO for Lmax Overview iaia abote UO 6 a 2505 t ENEE E E AA pA IM A tae Cae Saree MC ALIE 3 3 The SOFTmax PRO for Lmax Window eese 3 3 Window 2 Se tenses sso tase Ave ae ne stesso a Aa eve dow tan END UE eee ae 3 5 SLL 108 Fel Sea a Sea Se ST eS ean aE 3 6 Experiments and DECHONS 475 505 stay ye tod anand SAU APA anne res 3 7 Per Te Biba oio pU Anton te toe tbe am Ae UPS 3 7 DOCLHOPIS 3 599 PAD tare ane toe ne aoe ke dow Baw daw nw arene dom tong wes 3 8 Notes SECU ON ao PADS nary ae oe ane ee eg aoe ge 3 12 Plate SECTIONS vs o doo DS aos Ae oe OO oq esed 3 13 SOUP SECON SAPE IUD ae eee ee b t ttu 3 15 Graph SeChOn 4 224245 9s n i PEPLUPPPUPHPUPP AMA Ep 3 17 Dnstrupmic nb Se CES S Los dpo APPORT atone tt ta tort 3 19 Menus and Commands eee 3 19 IDIGTISEOCOSS Messac IBID sob dori dote E A ORA PRECES PEE 3 33 Chapter 3 SOFTmax PRO for Lmax Overview 3 2 SOFTmax PRO for Lmax User s Manual 2 Chapter 3 SOFTmax PRO for Lmax Overview Introduction This ch
131. ature set point After a reading however the temperature set point range and aver age temperature are recorded in the saved file Cover status This icon displays the status of the cover over the microplate tray of the Lmax closed or open You must close the lid prior to reading a micro plate SOFTmax PRO for Lmax User s Manual Chapter 3 SOFTmax PRO for Lmax Overview 96 or 384 well plate setting display This icon shows whether or not a micro plate is present in the reading chamber and also if the Lmax is currently set to read 96 well or 384 well plates when no plate is present an X appears over the icon as shown The Lmax requires that two changes be made when switching from reading one plate size to the other you must physically recon figure the position of the Lmax read head and you must change the software setting for the plate size in the SOFTmax PRO for Lmax Control menu Select ing the 384 well plate size in the Instrument Settings without configuring that setting in the Control menu will produce an error Experiments and Sections Experiments Each SOFTmax PRO for Lmax file contains at least one experiment and can contain more than one Within an experiment are one or more sections dis cussed below Experiments define a naming scope for the sections within them and provide you with quick access to different types of information without opening or referring to multiple files An experiment can be se
132. ax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data e Interleaved In this display the wells are shown in a format that skips every other well as follows all odd columns and rows begin in the upper left corner of the plate display and are followed by all even columns and rows So well A1 is still in the upper left corner but it is followed horizon tally by well A3 A5 A23 A2 A4 etc and vertically by well C1 E1 O1 B1 D1 etc This display is most useful when the 384 well plate is composed of 4 daughter plates of 96 wells each gt O ruit ke a E M D I j F a a a a a a Endpoint Fiscion Cnt Last Read resale mec Minecti n OH bk Daji Vier h Cimon Elus Reduced Number The reduced number is based on the settings defined in the Reduction dialog box It is a combination of plate blank subtraction and or endpoint reduction or kinetic reduction The information acquired from the microplate reader is reduced to a single number or value The reduced number will be reported in the Group section when a template has been defined To view by reduced number alone select the Number option the first choice to the right of the default just after the dividing line to see a reduced num ber as well as another type of display click the box next to the option that says With reduced number in the lower left of the dialog box to enable it Chang i
133. ay settings for the current plate nor mally Raw Data Example NameCurSection Plate O02345 A 4 SOFTmax PRO for Lmax User s Manual 8S Appendix A SOFTmax PRO for Lmax Remote Command Language New Have SOFTmax PRO for Lmax send a New command This creates a new doc ument and reads in the values contained in the Default Protocol lpr proto col file OpenAssay XXX Have SOFTmax PRO for Lmax open a protocol file The file should be in SOFTmax PRO for Lmax s Assays folder which is set using the Assays menu Protocols can also be stored in subdirectories of the Assays folder Notes e Only protocol files can be opened e SOFTmax PRO for Lmax will append lpr to any filename passed in e If the file is not found SOFTmax PRO for Lmax does nothing Examples OpenAssay myFile OpenAssay mySubDirectory myFile Read Select a plate section and send SOFTmax PRO for Lmax a read command Notes e If more than one plate section exists in the current document the first selected plate will be read If no plate is selected the first plate after the cur rently selected section will be read If nothing is selected the first plate in the document will be read typical case e If the plate already has data the data will be overwritten without confirma tion e If Autosave is turned on the document will be saved normally after the read is complete e Pre reads of plates should not be performed Ret
134. be calculated and displayed according to user defined formula settings Reduction Limits Sets limits on data to be included in data reduction for kinetic readings SOFTmax PRO for Lmax User s Manual 1 19 Chapter 1 Welcome to SOFTmax PRO for Lmax BO 1 20 Hu 5 gf 448 199 P 19 9 P 19 9 9 119 9 9 toad fag Zu LT T TIT oad Restore Box Windows The small box containing both an up and a down arrow at the right of the title bar The Restore box appears only after you have enlarged a window to its maximum size Mouse users can click the Restore box to return the window to its previous size Keyboard users can use the Restore command on the Con trol menu Rotated Display Scroll A display option for 384 well plates that shows the plate rotated 90 degrees To move up down right or left through text or graphics in order to see parts of the file or list that cannot fit on the screen A scroll bar appears at the bot tom and or right edge of a window whose contents are not entirely visible Each scroll bar contains a scroll box and two scroll arrows A scroll box is a small box that shows the position of information currently in the window or list box relative to the contents of the entire window A scroll arrow is an arrow on either end of a scroll bar that you can use to scroll through the con tents of the window or list box Semi Log Curve Fit The semi log function fits the best straight line to a set of
135. can also be chosen by pressing keys on the keyboard H Within Windows menu items contain an underlined letter in their names Pressing the ALT key will activate the menu bar pressing a letter key that cor responds to the underlined letter of a menu name will then activate that menu Subsequent menu items can be activated chosen by simply pressing the underlined letter in their names you do not need to press ALT again For example to open a file from within SOFTmax PRO for Lmax using the key board shortcut you would press ALT F O which will first activate the menu bar then select the File menu then choose Open from the File menu T iq When available a shortcut key combination will be shown to the right of the command in the menu On the Macintosh begin by pressing and holding down the command 3 key followed by the key shown in the shortcut Win dows users should press and hold the control CTRL key followed by the key shown in the shortcut Wau wee EEE SEEERED BEBEBEEE BBBEEEE 4soad Some menus are divided into groups of items that perform related functions Dividers are shows as thin horizontal lines between menu items File Items in this menu relate to entire SOFTmax PRO for Lmax files Com mands to open print apply password protection and save files are located here SOFTmax PRO for Lmax User s Manual 3 19 Chapter 3 SOFTmax PRO for Lmax Overview BO 3 20 Edit This menu contains featur
136. ce sepes vete EEE SEP EE 7 3 Hard and Soft Parameters 7 3 Description of the Assay Example 7 4 Define the Protocol 0 02 eee e ee eese 7 4 Start SOFTmax PRO for Lmax and Open the Tutorial Plees Gute cs EPUPERMU PIDE EVE 7 5 Define the Instrument Settings 7 6 Define the Template 0 0 00 eee eee 7 11 Set the Display Parameters lusus 7 17 Save the Protocol 2 cece e eee eens 7 18 Read the Plate e654 Se44 yes ERES Ove ee OPERE 7 19 Data Analysis Group Sections 7 21 Data Analysis Standard Curve 7 29 PANEER PO ace Ginn cio Sue EE ED ODE Oe 7 31 Chapter 7 Tutorial 752 SOFTmax PRO User s Manual 2 Chapter 7 Tutorial Introduction The hands on written tutorial included in this chapter is designed to take you through the basic features of the SOFTmax PRO for Lmax program while creating and running a simulated quantitative endpoint assay We suggest that you read and follow the steps in this hands on tutorial before using SOFTmax PRO for Lmax for the first time Going through this tutorial will help you learn to use the basic features quickly To learn more about SOFT max PRO for Lmax you will want to read through Chapter 3 SOFTmax PRO for Lmax Overview Chapter 4 Reading Microplates and the intro duction to the Formula Reference Guide NO
137. cific types of assays for example These files can be named in any way you choose Folder name s after dividing line Choosing any of these folders will open a submenu showing protocol files residing in this subfolder Select one of the protocol files to open it File name s after dividing line The files that appear here are protocol files that reside within the folder determined using the Set Folder com mand You can quickly open and use one of these protocol files by se lecting it from the Assays menu Plate Menu Plate This menu item changes to show Plate Notes Graph or Group depending E nee on the type of section that is currently active If no section is active this menu E item does not appear ERE Template Opens the Template Editor Eyes The Template Editor allows you to describe the samples that are in the T wells thus providing the link between the raw data and the analysis groups You can create new groups in the template or assign wells to Copy Template already established groups Paste Template If your experiment contains multiple Plate sections the template for each Plate section must be defined using the Template Editor in that section You can create anew template or modify an existing one using the Template Editor Each Plate section requires that a template be created in order to identify groups within that plate However two or more Plate sec tions may use the same template or the s
138. cintosh computer requirements 2 5 Windows compatible computer 2 6 Standard curve 5 43 7 29 Standards minimum number 5 43 Stationery file 7 18 Status bar 3 4 3 6 4 29 4 30 Summary 5 27 5 28 adding editing 5 22 5 27 aligning 5 28 formula 5 27 line up with column 5 28 moving 5 28 plot 5 29 Suspend Recalculation command 3 25 5 22 SOFTmax PRO for Lmax User s Manual T Temperature display 3 6 3 7 Template 3 8 4 14 clear 1 12 6 23 command 3 28 copy paste 4 27 6 21 eroups 4 13 4 14 hierarchy 4 14 import export 6 20 samples 4 14 selecting wells 4 14 series 4 21 fill direction 4 23 Template Editor 3 28 4 13 4 14 7 11 Group area 4 15 tool bar 4 15 Text format 6 13 Text Style command 3 25 6 13 Threshold display 5 9 5 10 Time at Half Maximum 1 21 5 20 Time at Maximum 1 21 5 20 Time at Minimum 1 21 5 20 Time to Vmax 4 10 4 11 5 20 Title Bar 1 21 Title screen 2 9 3 3 Tool bar 3 9 Troubleshooting Technical Services 1 10 U Undo command 1 21 3 24 Uninstall 2 8 V Vertical Display 1 21 View menu 3 26 Vmax kinetic reduction 5 20 Vmax Points 5 12 5 20 W Well graph 5 12 5 15 printing 5 15 6 11 Wells graphing 5 12 masking 5 15 selecting in template 4 14 Window active 1 11 3 22 3 30 Page Setup command 3 5 scroll bar 3 9 size 3 5 3 10 sizing 5 15 Windows compatible computer requirements 2 6 1 5 X X axis command 3 31 5 46 Y Y axis command 3 31 5 46
139. ck the Minimize button to reduce a window to its icon Keyboard users can use the Minimize command on the Control menu A minimized application continues running and you can select the icon to make it the active application M Modem Port Macintosh A serial communication port on the back panel of the Macintosh marked with the icon of a telephone handset Normal Display The default display for 384 well plates SOFTmax PRO for Lmax User s Manual 1 17 Chapter 1 Welcome to SOFTmax PRO for Lmax BO 3 NVRAM Refers to the Non Volatile Random Access Memory of the instrument Infor mation stored in the NVRAM is not lost when power to the instrument is turned oft Onset RLU The change in signal required to compute the onset time for kinetic read ings Onset Time The time it takes for a given increase in signal called the onset RLU to occur Open Close Triangle A small triangle located on the left hand side of section and experiment tool bars Used to open close sections and experiments within a SOFTmax PRO for Lmax file Option Button Radio Button A small round button that appears in a dialog box or window Within a group of related option buttons you can select only one Parallel Port Windows A connection on a computer usually LPT1 to plug in the cable for a parallel printer Windows supports parallel ports LPT1 through LPT3 Paste A function that copies the contents of the clipboard an
140. ction is commonly used to suppress outliers Masked wells are shown with diagonal lines across them and the word Masked is shown over each masked well MaxRLU The limit for the maximum value you wish to use for displaying and analyz ing kinetic data The default is 20 000 RLU Any values that are above this limit will not be shown and will be excluded from data reduction H Maximize Button Windows The small box containing an up arrow at the right of the title bar Mouse users can click the Maximize button to enlarge a window to its maximum size Key board users can use the Maximize command on the Control menu Menu A list of items most of which are commands Menu item names appear in the menu bar at the top of the window To choose an item in a menu place the pointer on the menu heading and drag to the selection you desire then release the mouse button Menu Bar A list of items most of which are commands Menu item names appear in the menu bar the horizontal bar that appears below the title bar at the top of the window MinRLU The limit for the minimum value you wish to use for display and analysis of kinetic data Any values that are under this limit will not be shown and will be excluded from data reduction The default is 0 To display negative Kinet ics the value should be set below 0 zero H Minimize Button Windows The small box containing a down arrow at the right of the title bar Mouse users can cli
141. ctions changing the order in which sections will print and add ing text describing each experiment in Notes sections You can also create summaries consisting of reduction formulas and associated text in Notes and Group sections The size and content of Group sections can also be changed Including or Excluding Sections When sections are created they default to being included in the printed report You can exclude sections from printing by clicking the print setting button on the tool bar of that section it is a toggle so it will change from enabled E to disabled m or vice versa Changing the print setting option for the experiment will change the state of all sections within that experiment Changing the print setting option for one or more sections within an experiment will cause the print setting button for the experiment to change to reflect an undecided status with regard to the sections within it amp Toggling the experiment s print setting button will override any changes made to individual sections and set all of them to printing When Print is chosen from the File menu selecting Print All will override the print settings made to sections or experiments Customizing the Report In addition to choosing which sections will print in the report by toggling their print status you can choose to print all items in a file or to print only the report from the dialog box that appears when you choose Print from the
142. custom groups created groups created by a user Figure 4 13 The Group Drop Down List Depending on the default template used when opening SOFTmax PRO for Lmax version 1 0 initially no groups may appear above the gray line As you create new groups they will be posi tioned above the line e Blank Creates a plate blank in the Plate section Note the name blank is reserved for use by the SOFTmax PRO for Lmax program and should not be used when creating custom groups e New Creates a new group The Group Settings dialog box will appear see Figure 4 14 showing default settings This dialog box allows you to define the name for a group of related samples what type of descriptor will be associated with these samples and the initial column format for the Group section Whenever a new group is created whether or not wells are selected in the template a Group section table will also be created To delete a group you must delete the Group section Simply clearing a group from the Template Editor will remove the assignment of wells to that group name but the name will still show in the group pull down menu and the Group sec tion will still exist Group Settings Groups L1 Sample Deser iptar Concentration Units units rl gt Cx Figure 4 14 Group Settings Dialog Box Using Default Settings 4 16 SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microplates Name
143. d command displays a dialog box asking you to select which Plate section to read The default Plate section se lected by pressing the Return key is chosen on these criteria e The selected Plate section if one is selected e The next unread Plate section e The first plate if all have been read Once a reading is in progress menu items are no longer available until the reading is finished Button Equivalent in the Status bar Incubator Allows you to turn the incubator on or off When on you can en ter a setting for regulation of the temperature in the microplate cham ber Button Equivalent Ir in the Status bar Wash Injectors Performs a manual wash of either the P or M injector or both for a specified number of injections default is 30 Choosing this op tion causes a dialog box to appear which contains a note that reminds you to change the reagent bottles if necessary Choose the settings you desire and click the Wash button to start the wash process Prime Injectors A fter changing reagents you should prime the lines to re move any air that may be present and to ensure that the lines are filled with the new reagent Choosing this menu option opens a dialog box that allows you to prime the P or M injector line or both You can choose the number of injections to perform to prime the line the de fault is 7 The dialog box reminds you to change reagent bottles if necessary prior to starting the priming p
144. d from one part of a template to another within the same or to different templates and from one experiment to another Plate Section Data and Templates Copying and Pasting Data can be copied from one Plate section to another within SOFTmax PRO within SOFTmax PRO for Lmax Make the source section active by clicking on it and then choose for Lmax Copy 38C CTRL C from the Edit menu Create a new section if necessary and make the destination section active Choose Paste V CTRL V from the Edit menu to paste the data into this section SOFTmax PRO for Lmax User s Manual 6 21 Chapter 6 File Management and Printing BO Copying from Other Programs 6 22 When copying Plate sections all well data is copied It is not possi ble to copy and paste the data from only certain wells Even if you select only one well prior to choosing the Copy 3 C CTRL C com mand all plate data will be copied and pasting will replace all data in the destination plate If the Instrument Settings of the target section do not match the settings from the section being copied the Instrument Settings for the target section will be updated appropriately to match Templates can also be copied from one Plate section to another within SOFT max PRO for Lmax However the effects of this action depend on whether you are pasting the template to another section in the same experiment from which it was copied or to a section in a different experiment Pasti
145. d inserts this copied information at the current selection or cursor position The paste function is enabled by pressing 36V on the Macintosh and CTRL V in Windows Path Windows Specifies the location of a file within the directory tree For example to specify the path of a file named ASSAY BAT located in the SOFTmaxPRO directory on drive C you would type c eoftmaxproNaseay bat Photomultiplier Tube PMT A vacuum tube that detects light especially from dim sources through the use of photoemission and successive instances of secondary emission to produce enough electrons to generate a useful current Plate Section The Plate section is used to display data to specify how microplates will be read and to define how the data received from the instrument should be reduced The data display in this section is a matrix that corresponds to the well format of the microplate in use Plate Type A choice in the Instrument Settings dialog box to distinguish between 96 and 384 well microplates The specification of the plate type depends on the well format and manufacturer of the microplate SOFTmax PRO for Lmax User s Manual Chapter 1 Welcome to SOFTmax PRO for Lmax Point Using the Mouse You point to an object on the screen by moving the mouse so that the pointer is positioned on that object When the pointer is an arrow the tip of the arrow must be on the object Point to Point This curve fitting option fits a linear equat
146. d reduced displays this button must be set to either Scale to Limits or Scale to Data for both raw and reduced views of the Well Graph Graph Options button Opens the Well Graph Options dialog box which allows you enable or dis able connected points or plotted symbols the graph Graph Options xf connect Points wf Plat Symbols C Figure 5 7 Graph Options Dialog X Axis Settings button Y Axis Settings button Opens the Well Graph Axis Settings dialog box for the particular axis allowing you to set Auto Range parameters choose the minimum and max imum values for the selected axis cause the axis to be auto ranged add or remove gridlines and add or remove axis tick marks When a Well Graph is opened the default settings are to show the reduction limits for that axis with Auto Ranging off and gridlines and tick marks selected Only the Well Graph being viewed is affected by changes in these dialog boxes Done Closes the Well Graph dialog box SOFTmax PRO for Lmax User s Manual 5 13 Chapter 5 Displaying and Analyzing Data 5 14 mi hee SL ee ee ee Hoe rx ap Ap 3 i H m E zn IDE i E L Baa ie j i m D T B A i d a y a D 3 4 t 5 a 1 10 11 1z 1 12 LS 1E 18 15 a E 4 s e J nane Figure 5 8 Well Graph for a Single Fast Kinetics Microplate Well Raw Data Multiple wells from the same Plate section can be graphed together in one
147. d upon the type of serial port s available on your computer The end of the cable that will attach to the Lmax must be a 25 pin connector The cable itself should be a straight through serial null modem cable The connector for your computer may be a 9 pin or 25 pin connector or you can use an adapter that converts from 9 pins to 25 pins see Figure 2 2 9 pin to 25 pin pi 9 pin connector 25 pin connector p adapter Figure 2 2 25 Pin DIN to 25 Pin or 9 Pin Serial Cable 2 4 SOFTmax PRO for Lmax User s Manual 8S Chapter 2 Installation Computer System Requirements The following computer hardware and operating system software specifica tions are recommended to ensure proper operation of SOFTmax PRO for Lmax The minimum configuration required is also given Macintosh Computer Recommended System Configuration e Apple Power Macintosh G3 e Operating software System 9 0 e Sony 17 inch Multisync Color Monitor e 64 MB RAM 6 GB hard disk drive e CD ROM drive e Extended keyboard e Keyspan USB Twin Serial Adapter e Epson 7401 color printer Minimum System Configuration e Apple Macintosh PowerPC e Operating software System 7 5 5 e Apple 14 inch color monitor e 24 MB RAM 270 MB hard disk drive e 1 44 MB HD high density 3 5 inch floppy disk drive e Access to a CD ROM drive e Available serial communications port 1 Macintosh hardware tested for the SOFTmax PRO for Lmax release iMac with OS
148. difficult and or time consuming Wiese Creating new folders or subdirectories must be done outside of SOFTmax PRO for Lmax using the Finder for Macintosh or the File Manager or Windows Explorer within Windows for example Creating New Files Selecting New 3N CTRL N from the File menu causes a new untitled file to open creating a new window on the screen The initial contents of this win dow are based on the default protocol file chosen in the Preferences dialog box NOI You can create and or open more than one SOFTmax PRO for Lmax file at a time Subsequently opened files will appear in separate win dows in a cascading diagonal order SOFTmax PRO for Lmax User s Manual Chapter 6 File Management and Printing New files can be saved as protocol files at any time either before or after data is acquired To create a new protocol file enter all the instrument settings for the reading and then choose Save or Save as from the File menu The dialog box that appears presents the option of saving a file as either a protocol or a data file the default On the Macintosh click the option button next to Pro tocol and enter a name for the file in the text box type over Untitled In Windows choose the appropriate format from the list in the box under the heading Save File as Type Also remember to choose the folder or subdi rectory in which to save this file When you have entered all the information click or O
149. duet Ree ios a baa rb pos du ds 8 1 Introduction 0 cee eee eee eee eee eee e eee eeeeee 8 3 Wessace Boxes and Aleris Ls aee anpim te ari po aren m OR LAC UREA CURRUS RA 8 3 Oneor More Points Lost VPBSSHBO s oucsucaueon uir quin p o ctor p qw A n 8 4 LHOPMSSSQsP POX OSs ces quee QUSE ERR RUEECA ATA SFO ERN ORARE THAURAT DACIA RII 8 4 Communication Problems 0 00 eee RR 8 5 Macintosh Cable Connections 0 0 00 cece cee e 8 8 PC Cable Connections 0 0 0 0 e eee I 8 9 Display FOD i P 8 10 Pantao PODION xai ward npe not qoa 30 008 879 E S 8 10 General Problems lseeeeeeeeeeee RR RR RR RR has 8 11 Appendix A SOFTmax PRO for Lmax Remote Command Language 8 1 Introduction 0 0 cee eee hee A 3 SOFTmax PRO for Lmax Responses 1 0 0 0 cee ees A 3 SOFTmax PRO for Lmax Commands 0 0 0 00000 eee eee A 4 Visual Basic Excel Macro Example 0 e eee eee eens A 7 MFC C Interface to SOFTmax PRO for Lmax Remote Commands that Return Values eee A 8 SOFTmax PRO for Lmax User s Manual vii viii SOFTmax PRO for Lmax User s Manual 2 Figures Figure 1 1 Figure 1 2 Figure 1 3 Figure 1 4 Figure 1 5 Figure 1 6 Figure 1 7 Figure 2 1 Figure 2 2 Figure 2 3 Figure 2 4 Figure 2 5 Figure 2 6 Figure 2 7 Figure 2 8 Figure 2 9 Figure 3 1 Figure 3 2 Figure 3 3 Figure 3 4 Figure 3 5 Figure 3 6 Figure 3 7 Figure 3 8 Figure
150. e appropriate icon in the Instrument Set tings dialog box Endpoint A single integration of samples in 96 or 384 well plates with two injections possible if desired in any or all wells of a 96 well plate no injection possible in 384 well plates The minimum integration time is 0 1 second A pre read of the microplate may be made before injection of buffer or reagent and or read ing of the samples The signal is integrated for each well over a user selectable reading time maximum 5000 seconds You can program a delay after each injection if desired up to 1000 seconds with a minimum of 1 6 seconds for the P injection and you can read all or only some of the wells in the plate Values are reported as relative luminescence units RLU the default reduced value is MValue Read Mode Buttons Figure 4 5 Instrument Settings for an Endpoint Reading Dual Read Also an endpoint reading this mode is designed to perform two separate integrations one after each of two possible injections in any or all wells of a 96 well microplate no injection possible in a 384 well plate Integration times for both readings must be the same minimum integration time is 0 1 second maximum is 1000 seconds You may program a delay after each injection of up to 1000 seconds a minimum of 1 6 seconds for the P injector and you can read all or only some of the wells in the plate Pre reading of the microplate is not possible with this mode Values are
151. e click the name of the Group section Standards to the right of the Group section icon New Window Opens the active section in a separate window Mouse Equivalent double click the icon in the Graph section H Window Menu Tile If you have several windows open choosing the Tile command will re size and arrange all open windows so that they are all visible at the same time generally side by side Cascade lIf you have several windows open choosing the Cascade command will resize and layer the open windows so that each title bar will be visible Arrange Icons If you have several files open but reduced to icons choosing the Arrange Icons command will move all icons to the bottom of the SOFTmax PRO for Lmax window and put them in order Beneath the line will appear the names of all open files within SOFTmax PRO for Lmax The active file will have a checkmark next to it Choosing one of the file names shown here will cause that file to come to the forefront of the screen and will restore the file to its previous size if it had been minimized H Help Menu Windows Contents Displays the contents of the Help screens allowing you to choose from the listed topics Clicking a topic opens the help screen associat ed with it Index Shows the alphabetical listing of all items that can be viewed within the Help function Clicking a topic opens the help screen associated with it SOFTmax PRO for Lmax User s Manual
152. e color as that group ZH In order to save changes you have made to the template in the Win Ht dows version of the Template Editor be sure to click the button to close the window rather than clicking the Control Menu box Click ing the Control Menu box located in the upper left of the window performs the same function as the Cancel button the window will close but any changes you have made to the template will not be saved Copying and Pasting Templates Copying Complete Template Information Two template actions Copy and Paste can only be activated from the Plate menu when a Plate section is active rather than from the Template Editor If you have created a template in a Plate section and wish to use it in another Plate section you may copy that template and paste it into the new section The results of copying and pasting a template depend upon whether the action is performed within the same experiment or between experiment sec tions Copying and Pasting within the Same Experiment Section To copy a template make the section containing that template active and choose Copy Template from the Plate menu the menu name changes to reflect the type of active section Then activate the destination Plate section and choose Paste Template from the Plate menu SOFTmax PRO for Lmax User s Manual 4 27 Chapter 4 Reading Microplates 4 28 Plate Template Reduction Display Graph Mask
153. e content of Group sections changes depending on the wells that are assigned to these groups in the Tem plate Editor Before a plate is read these sections show no data for the wells After a reading Group sections contain information about the values for the wells contained within the groups SOFTmax PRO User s Manual 7 21 Chapter 7 Tutorial BO Scroll down until the Standards Group is visible It should look like this H Standards E Ey fi Standards pq group blank 1 210 stl 500 000 H1 6 300 r000 0 100 14 H2 TOO H3 7 100 ote ew O00 r1 3 300 6 000 0 100 Li G 6 000 G 6 10 s s n al stid 62 500 3 900 4 000 0 100 2S 4 00 410 sth 31 250 a d 3 000 0 100 33 3 00 3 10 15 625 1 90 ALT 0 100 50 AE 2 10 7 DU 0 90 10 0 100 Figure 7 26 The Group Section for Standards l I malest standard value 1 000 Largest standard value 7 000 The columns in this group are set up as follows from left to right e Sample the seven sample names assigned for the Standards group Concentration the concentrations assigned to each sample in the template e Wells The wells that are assigned for each sample in triplicate in the template e Values raw RLU values obtained for each sample e MeanValue the mean of data from the Value column for each sample e StdDev standard deviation for each sample value e CV coefficient variation percent for each sample value In the following part of t
154. e experi ments in a file simultaneously use the Expand and Minimize commands in the View menu Selecting Minimize 3 M for Macintosh CTRL M for Windows will close all sections leaving experiments open to show only section title bars Selecting Expand E CTRL E will open all sections whose If you close or minimize an experiment the tool bars in that experi ment will be hidden see Figure 3 7 If all sections are presently expanded the Expand command is dimmed unavailable 3 10 SOFTmax PRO for Lmax User s Manual Chapter 3 SOFTmax PRO for Lmax Overview m 5UFTmax PRO for Lmax Ele Edit View Expenment Control Assays Plate Window Help ae ve EG Untitled ka m Experiment zi b Bj netesitt 24 b E Plated lt M Setup ES Template E Reduction fy Display 7 b EJ Plate Eg Display 7 Figure 3 7 Result of Minimizing an Experiment Tool Bars Hidden You may also open a section in a separate window Double clicking the sec tion icon in the tool bar or selecting New Window from the active section menu either Plate Graph Notes or Group causes the currently active sec tion to open as a separate window a copy of the tool bar still remains in the original experiment but the section is closed clicking the closed section places the new window behind the main window the original section remains closed Clicking on the triangle in the section tool bar closes the new window and causes the section in the exper
155. e settings for the active group currently visible in the group drop down list If you wish to edit a group that is not currently active first choose the group from the drop down list and then click the Edi t button The fig ure below shows the Group Settings dialog box for the active group called Patient 1 broup Settings Name Patient 1 Name of Group Sample Descriptor PD Sample Descriptor Dilution Factor Units mg ml Wr 1 Units for Sample Descriptor P Column Format Column Format Original Unknowns W Setting Choices are Calumn Format i v Basic Standards Unknowns Unknowns Dilution I Figure 4 15 Group Settings Dialog Box for the Group Named Patient 1 The name sample descriptors and column format can be changed for the group that you are editing When you edit a group you will notice that the information shown for the Column Format in the Group Settings dialog box changes to show the name of the original group used to define the group columns see Figure 4 16 Group Settings Name Standards P Sample Descriptor 2 units mar rw Column Format Original Standards Y When you are editing Group Settings rather than creating a new group the word original precedes the Column Format Figure 4 16 Groups Settings Dialog Box Showing Original Column Format SOFTmax PRO for Lmax User s Manual 4 19 Chapter 4 Reading Microplates BO
156. ect the Hardware 0 cee eee eee eee ee ees 2 3 Computer System Requirements 0 0 0 0 eee 2 5 Macintosh Computer 0 0 0 c eee cece eee eee 2 5 Windows Compatible Computer 2 6 Install SOFTmax PRO for Lmax 0 0 0c eee eee 2 7 What Is Installed 2 0 e eee eee eee eee 2 8 Enter Registration Information 0 0 0 0 0 0 ees 2 8 Communicating with the Lmax 0 00 e ee eee ee 2 10 SOL DIGITIS Ceca ie topo Oe Ohne oe bibe brio bud bo Seen 2 11 De alr on alas Gane ae bem bor bud beo ee 2 11 LXDOLIUEOU Nal sete pe be oe Canes ae bote boe Set 2 12 PATO 30 LEE 2 12 VV ONG0 0 da Generate ea a ea tare eee ara a kere eae ee ea 2 15 Bn gVSROIdSI RE petu Cte tn tutu Gey Gute Gute outu ewes Gus 2 15 Chapter 2 Installation 2 2 SOFTmax PRO for Lmax User s Manual 8S Chapter 2 Installation Connect the Hardware A KANNO Turn OFF the power to both the Lmax and the computer before connecting or disconnecting any components or cables Not doing so could potentially result in damage to components in either or both machines m Macintosh NOF If your Macintosh has a single serial port refer to Chapter 8 Trou bleshooting for information regarding connection to the Lmax Serial Port Connection Use an 8 pin DIN to DB 25 Macintosh modem cable to connect the Macintosh to the Lmax Connect the 8 pin round DIN connector to the M
157. ed name BL appears below the gray line the sample name BL is the name reserved by SOFTmax PRO for Lmax for group associated blanks The item above the gray line is the sample ready to be or currently being assigned After being assigned it will be shown below BL below the gray line alm EB Concentration lo mg Figure 4 18 The Sample Drop Down List When this list first appears the only selec tion shown below the dividing line is BL the active sample name that is ready to be assigned appears above the line When samples are assigned to a group the default name is the first 3 letters of the group name followed by a number For a group called Standard for example the default sample names would be Sta01 Sta02 Sta03 and so on SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microplates Sample names can be 300 characters long however only the first five or six characters will be displayed in the template and in the Plate section You can type a sample name directly into the box next to the arrow for the drop down list by highlighting and typing over the existing sample name If one or more wells were highlighted typing a differ ent name will replace the sample name shown in the template with the name you have typed and if the name is new will add this name to those that can be chosen in the drop down list When wells have been selected in the template and sample names have been defined t
158. ed to cre ate the group you are editing Four selections are offered Basic Standard Unknowns and Unknowns Dilution Different types of columns will be created for a new group or changed in an existing group depending on which of these column format settings you select Table 4 2 shows the default columns created with each selection type NOF If you are creating a new group for unknowns that have a dilution factor use the Unknowns dilution column format as a base rather than the Unknowns group included in the default protocol SOFTmax PRO for Lmax User s Manual 4 17 Chapter 4 Reading Microplates Table 4 2 Columns Created and Their Formulas Name Values Values R Outside Standard R Outside Standard Range Range Formula WellValues WellValues If Values gt MinStd If Values gt MinStd Standards and Standards and Values lt Values Max MaxStd Standards Std Standards R un ug Formula InterpX STD Standard dardCurve Values Curve Values Formula Average Result Formula Stdev Result Formula Cv Result Cv Result Dilution Name Formula Factor Name A dj Result lt Sk Formula MeanResult Factor IConcentration Factor and SampleDescriptor return identical information 4 18 SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microplates The Button When you click the Edi t button the Group Settings dialog box will appear reflecting th
159. eed to know this information if you call for technical support Other items in the Apple menu depend on the settings of your Macintosh New 38N CTRL N Opens a new file window allowing you to begin a new or different data file Information from each SOFTmax PRO for Lmax window which can consist of one or more experiments with their sub sections is saved to a separate file Open 380 CTRL 0 Opens an existing SOFTmax PRO for Lmax file or an older SOFTmax file When you choose Open from the File menu a di alog box appears To open a file from the list of available files double click the filename to be opened or click once on the filename and then click the Open button Close dW CTRL W Closes the front most active window If you have only one file open this file will be closed If you have changed the file in any way since it was opened a dialog box will open asking if you want to save the file before closing This menu item is dimmed if no win dow is open Save 8 5 CTRL S Saves the active window to a file The active file name is shown at the top of the status window protocol files are always shown as Untitled To save the file in a different folder subdirecto ry or under a different name use Save As If the file has not yet been saved and given a name is Untitled the Save As dialog box will appear and you will be asked to name the file This menu item is dimmed if the file has alread
160. ell plates This setting is useful when looking at large numbers if you have a larger 17 inch or greater monitor Linear Curve Fit The linear function fits the best straight line to the data The equation for this fit has the form of y A Bx where A is the y intercept of the line and B is the slope A linear fit should be used whenever the values appear to lie on or scattered around a straight line More information regarding curve fits can be found in Chapter 5 Displaying and Analyzing Data Log Logit Curve Fit The Log Logit is also called a two parameter curve fit The equation used to generate this curve fit is y A D 1 X C D More information regarding curve fits can be found in Chapter 5 Displaying and Analyzing Data Long Kinetics During long kinetics readings data is collected over time with multiple read ings made at regular intervals The values calculated based on raw kinetic data are Vmax Vmax per Sec Time to Vmax and Onset Time 1 16 SOFTmax PRO for Lmax User s Manual Chapter 1 Welcome to SOFTmax PRO for Lmax Luminescence The emission of light by processes that derive energy from essentially non thermal changes the motion of subatomic particles or the excitation of an atomic system by radiation Mask Lets you hide the data in selected wells of the Plate display so that the data stored there will not be used for calculations and will not be reported The Mask fun
161. emplates The commands for interprocess messaging are described in Appendix A SOFTmax PRO for Lmax Remote Command Language along with examples for programming these com mands SOFTmax PRO for Lmax User s Manual 3 33 Chapter 3 SOFTmax PRO for Lmax Overview 3 34 SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microplates OO GIU HOTI oS Os ocean t ee Sa HUP DEEP IN QUUD PES 4 3 Instrurnenb Setups ose te e Sig BR odor door qe Erde dore Eoo dos ie 4 4 Instrument Settings Options 00 0085 4 9 Neil ate BOO 99st te St Sete he o he Cedo Soe Ee Eoo 4 13 Selecting Wells in the Template Editor 4 14 The Template Editor Tool Bar sese 4 15 BOM sese nS EE ots DEDE EPI Dee boe 4 24 Copying and Pasting Templates 4 27 Reading a Microplate e pe ve bor be Eo Seo ude nce ee Gute o 4 29 Chapter 4 Reading Microplates 4 2 SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microplates Introduction 1 2 3 4 5 6 7 8 9 10 11 Reading a microplate using SOFTmax PRO for Lmax is a three part process e Prepare the software to collect data e Prepare the microplate s to be read and e Read the microplate s Before reading a microplate you must create the Plate section s and define the instrument settings for the reading Instrument Settings must be defined prior to collecting data and may NOT be changed
162. eplicate Fill from Left two replicates Fill from Right four replicates Fill from Top three replicates Figure 4 23 Fill Options and Replicate Settings SOFTmax PRO for Lmax User s Manual 4 23 Chapter 4 Reading Microplates a 4 24 7 Click to save the series and close the dialog box While still in the Tem Blanking plate Editor you can view the value assigned to any well by clicking that well The value will be displayed at the top of the Template Editor Holding down the Ctrl Shift keys both Mac and PC will display the values for all wells Sometimes you may wish the names of a series of samples to incre ment but may not have a concentration or dilution factor that you wish to enter or you want the concentration or dilution factor to remain constant This can be done using the Series button setting the starting value to 1 or another constant value and either multiply ing by 1 or dividing by 1 In this way the names will increment but the value of concentration or dilution will not change If you don t have a concentration or dilution factor simply deselect the sample descriptor checkbox if it is selected If sample names are set to increment automatically using the Series function be aware that SOFTmax PRO for Lmax will automatically truncate the sample name to three or four characters including the incrementing number if the sample name starts with letters If the sample name is entirely numer
163. er bar Sample Text _ Connect points sample Text Set axes De Plot symbols Dla E Plot 5 Concentration Standards Y MeanaluemStandards Errors Wo Error Y Errors Mo Error ces CC Figure 7 38 Graph Options Dialog Box This dialog box allows you to rename the graph choose the type of graph that will be shown and to format the text used in the graph You can add or remove plots click the New or Delete buttons and you can also edit existing plots See Chapter 5 Graph Section for more information regarding the graphing options that are available SOFTmax PRO User s Manual 8S Chapter 7 Tutorial Step 9 Print a Report SOFTmax PRO for Lmax allows you to print several types of information In addition to printing a copy of the template you can print the active section the sections that have been designated to be included in the report or all sec tions The default setting in SOFTmax PRO for Lmax is for all sections to be included in the report You can exclude sections from printing as described in Chapter 6 File Management and Printing Otherwise choosing Print from the File menu and choosing Print report will print all of the sections that are designated to be included in the report regardless of whether or not they are open A standard Print dialog box will appear the appearance of this box depends upon the type of computer and printer you are using Se
164. eral users You may also choose the file type for the Autosaved file SOFTmax PRO for Lmax file or ASCII text file choose to append the date to the autosaved file name and if you select the Text file option you can then choose either the Create a new file or Append to file option When Autosave is enabled in SOFTmax PRO for Lmax file mode within files containing multiple experiment sections each experiment section will be autosaved to a separate file 6 10 SOFTmax PRO for Lmax User s Manual 2 Chapter 6 File Management and Printing Printing eee eee BEBEEEE BEEEBEEBE n BELEEEE unuunu HJ Five printing options are available for an experiment 1 You can print a report at any time by choosing Print 3 P CTRL P from the File menu You can determine which sections or whether all sections will be printed in this report see The Printed Report below 2 Acopy of the template can be printed while you are in the Template Ed itor by clicking the Print button in the bottom left corner 3 Areport can be printed automatically at the end of a reading To enable this function choose Preferences from the Edit menu and select Au toprint 4 You can print the active section or selection This option is available within the File menu If only one section is selected the name shown in the menu will reflect the name of the active section For example if the active section is named
165. ere This menu item is Test Style Ctrl T dimmed if no selection has been made or if the selection cannot be Preferences copied Suspend Recalculation Parts of an experiment can be copied within the same experiment or Recalculate Nowy Ctrl to another application using the Copy and Paste commands You may also use this command to copy information from SOFTmax PRO for Lmax into other programs Paste V CTRL V Pastes information from the clipboard just to the right of the insertion point in the document Can also be used to import tab delimited data into SOFTmax PRO for Lmax This menu item is dimmed if nothing has been copied to the clipboard Clear Clears or erases highlighted text or data without storing it on the clip board This menu item is dimmed if nothing has been selected You can remove one or more columns from a Group table or text or formu las from a Notes section by highlighting the column or text formula and then choosing Clear from the Edit menu Columns and text or for mulas can also be removed by highlighting them and pressing the De lete key Select All 6A CTRL A The Select All command can be used in Notes Plate and Group sections when a section is active it is not available in Graph sections In a Notes section if no summaries are present it will select all text If summaries are present at the top or bottom of the Notes section this command will select all text but not the summaries
166. eriment the group Unknown will be created but all information regarding the group Standards in the destination experiment will remain un changed If you copy and paste the template with the same experi ment no new groups are created Group names can be edited in the pasted template these changes will be reflected in the original tem plate also Export Template Opens the Export Template to dialog allowing you to ex port a template to an ASCII text file Import Template Opens the Import Template to dialog allowing you to import a template from an ASCII text file Section Name Allows you to change the name of the Plate section Mouse Equivalent double click the name of the Plate Plate 1 Jin the Plate section tool bar to the right of the Plate section icon SOFTmax PRO for Lmax User s Manual 3 29 Chapter 3 SOFTmax PRO for Lmax Overview BO New Window Opens the active section in a separate window Mouse Equivalent double click the icon in the Plate section tool bar Notes Menu me This menu item changes to show Plate Notes Graph or Group depending on the type of section that is currently active If no section is active this menu item does not appear Create Summary Edit Summary Create Summary When the Notes section is active choosing Create Sum mary opens a dialog box allowing you to give a title to a new summa ry and then enter a formula to be used for the summary
167. eriod 6 6 SOFTmax PRO for Lmax User s Manual Chapter 6 File Management and Printing Table 6 1 found later in this chapter lists the three letter extensions that are associated with data and protocol files from SOFTmax and SOFTmax PRO 3 Insert a PC formatted disk into the Macintosh floppy drive It will appear as an icon on the Macintosh desktop 4 Drag the files renamed with 3 letter file extensions onto the IBM disk icon to copy the files to the disk Drag the disk to the trash to eject it Certain special characters will not translate accurately across plat forms Characters that are created on the Macintosh using the option key such as C TV or e bullet character for example will be shown on the PC as other letters or characters Also if you align items using spaces these may or may not align when viewed on a different platform since fonts on the different platforms have different charac ter widths Transferring PC Files to a Macintosh Third party software is available for the Windows compatible PC that allows the PC to read Macintosh 3 1 2 inch disks However if you need to transfer files between Macintosh and PC platforms and do not have such software for the Windows compatible PC you can use PC disks for both platforms since the Macintosh will be able to read them Macintosh computers are equipped with software that allows you to read and transfer files from PC disks If you are using System
168. es along with the formula for the fit at the bottom of each Graph section for each different fit All plots on a graph must have the same type of fit When a curve fit is chosen the values for the coefficients of the equation A B C etc will be shown as will the square of the correlation coefficient R 2 The correlation coefficient describes how well a change in x values correlates with a change in the y values Please see Judging a Good Curve Fit on page 5 43 for more information The R 2 value should only be used for curve fits having linear axes Linear The linear function fits the best straight line to the data The equation for this fit has the form of Y A B X SOFTmax PRO for Lmax User s Manual 5 35 Chapter 5 Displaying and Analyzing Data BO where A is the y intercept of the line and B is the slope A linear fit should be used whenever the values appear to lie on or scattered around a straight line This equation is shown on the screen as y A Bx A linear fit should be used whenever the standard values appear to lie on or are scattered around a straight line Figure Figure 5 30 shows an example of a linear fit xz 7 Standard Curve Fit Standard Curve T n c ba T zx 30 Concentration J Bx A o Std Standards Concentration vs Mean value 0 058 Figure 5 30 Graph with Linear Fit simulated data Semi Log The semi log function fits the best straight line to
169. es Show All Show Formulas Show Formulas Command Group Settings New Window Figure 5 17 The Show Formulas Command in the Group Menu Choosing this option causes an additional row to appear beneath the head ing for each column which shows the formula used to calculate the data in each column To see formulas briefly hold down the shift and control Ctrl keys CHOMO6 native uM Formulas HoampleN Well Index WellVal Averagel Yahi Factor 1 443 1 443 0 000 Figure 5 18 Formulas Shown in Group Columns Modifying Column Formulas To modify the formula for an existing column double click the column or click the column once and then click the formula button in the section tool bar The dialog box associated with the column formula will open allowing you to edit the formula and the name associated with the column SOFTmax PRO for Lmax User s Manual 5 25 Chapter 5 Displaying and Analyzing Data BO 5 26 Create Column Button Create Summary Button Formula Button Meanvalue Average ales Pel Eq fef CHOMO6 natre uM DEEDEE Concentration anpleN Well Index Wien Valen EUM Factor NEP 1 1 0000 33 566 114 175 0300 CHOS D 4 171485 171 485 203 082 CHOS JEF 5250423 IEEE e40 792 CHO JH 8 Edo 53 542 100 000 HT cono ive Figure 5 19 Formula Button Activated and Column Formulas Shown For a complete discussion of custom formu
170. es Technical Service 8 3 3 N New command 3 22 6 4 New Window command 3 30 3 31 3 32 Non Volatile RAM NVRAM 1 17 1 18 Normal Display 1 17 Notes menu 3 30 Notes section 3 8 3 12 5 28 text format 6 13 Notes Settings command 3 30 NVRAM 1 18 O Onset Time 4 10 4 11 5 20 Open command 3 22 6 6 Option button radio button 1 18 P Page Setup command 3 5 3 23 5 26 Parallel Port Windows 1 18 Partial plate read 4 12 Password protection 3 23 6 25 changing 6 26 setting 6 26 Paste 1 12 6 21 command 3 24 6 21 Group columns 6 23 Plate blanks 1 11 4 25 Plate menu 3 28 Plate section 3 8 3 13 5 4 data display 4 30 5 4 7 19 instrument settings summary 4 4 menu commands buttons 3 14 reduction options 5 4 Plate Settings command 3 29 Plots 5 28 5 35 command 5 28 Point to Point curve fit 1 19 5 41 Port 1 19 Preferences 2 11 command 3 25 6 9 Pre read plate blanks 1 11 4 24 4 25 Print command 3 23 6 14 Print Section command 3 23 Printing 6 11 Autoprint 6 11 options 6 11 problems 8 10 report 6 11 6 12 7 31 including excluding sections 6 12 section 6 11 order 6 12 setup 6 11 template 6 11 well graph 5 15 6 11 Protocol file 6 4 6 5 Default 1 13 6 3 locating 6 4 saving 7 18 Q Quadratic curve fit 1 19 5 38 Quit command 3 23 R Radio button option button 1 18 RAM Random Access Memory 1 19 Ranged display 5 9 5 10 Read 4 29 7 19 button 3 6 command
171. es for changing items within SOFTmax PRO for Lmax Commands to select copy paste delete and duplicate items are located here This menu also contains the Preferences command which allows you to set options regarding printing communication with the Lmax and saving files View Choices regarding what is shown and how it is shown are available here The last section in this menu provides a navigational tool allowing you to choose the particular section you would like to view a section chosen in this manner will open if it was closed and will be brought into view within the window Experiment Choices in this menu allow you to create new sections within the current experiment as well as to create a completely new experiment Control Items in this menu allow you to set up and activate Lmax functions Assays This menu allows you to choose the folder containing the protocols you have saved and shows those protocols below the divider making it easy to open one or more of them A second level of folders can be stored within the Assays folder and will appear on the Assays menu allowing you to sepa rate protocols further as desired Notes Plate Group Graph The name of this menu item changes to reflect the section type that is currently active If a Plate section is active for example the last menu item will be Plate An active Graph section causes this menu to show Graph If an experiment is selected this menu will not be shown Windo
172. escribes how rapidly the curve makes its transition from the asymptotes in the center of the curve A large value of B describes a sharper transition Typically B has a magnitude of about 1 Both the log logit and 4 parameter equations are shown on the screen as y A D 1 k C B D SOFTmax PRO for Lmax User s Manual 5 39 c ny S Q 69 N gt xq rS gt 8 69 e SS a C LS Y banay a S O v L4 Graph x aux op B3 1 E Tana 34TA 249 B Q 6a amp 0 o v C A DW e CERT e Ex Poli 13 integealion palm s Meanvalue Parameter Fit simulated data I i i i i i 4 i i i i i i i i L i i EM e ana uei Figure 5 34 Graph with 4 Concentration s D T1252 0308 1 247 i ye DORT eC PEO Pio Ground concentration ws Means siue Logit Fit simulated data Graph with Log Figure 5 35 Some publications show the log logit equation in another form blog c m TET bx log where log X A different algebraic expression of these is D A D 1 x C SOFTmax PRO for Lmax User s Manual 5 40 Chapter 5 Displaying and Analyzing Data The difference between the log logit fit and the 4 parameter fit are in the way the coefficients A and D are calculated For the log logit method the standard values for the lowest and highe
173. ettings and asks for plot information Group Not available Section Password Protection Replicates text and summaries from source Notes section Replicates Instrument Setup Reduction and Display parameters from last Plate section cre ated Replicates all Graph settings including any plots that were defined in the source Graph section Creates a replicate of the source Group section table The new group will be in the template Group drop down menu but no wells will be assigned to this group Individual protocol and or data files can be protected by adding a password to them When a protocol file is password protected you may read data into the file but you may not change the file in any other way When a data file is password protected you may view the file but are unable to change it The correct password must be entered to make changes to the files The table below shows the various actions that are protected when a pass word is assigned to a SOFTmax PRO for Lmax file SOFTmax PRO for Lmax User s Manual 6 25 Chapter 6 File Management and Printing Table 6 5 Actions Protected by Password Buttons File gt Page Setup Setup Read Summary Moving a Moving a formula in a Notes section Filelmpot Instrument Instrument Setup Resizing columns in a Resizingcolumnsina Group um RM AS Editor Moving sections ELA experi ments Edit gt Undo Typing in sections Notes and Plate
174. f the ANSI character set and most other 8 bit character sets The ASCII character set is the most universal character coding set Autosave Enabling the Autosave feature in the Preferences dialog box will save a data file automatically each time a plate is read You can Autosave to one of two different file formats SOFTmax PRO for Lmax used to save your data auto matically in SOFTmax PRO for Lmax format or Text File used when you want to have the data available for use with a spreadsheet or other program for data analysis external to SOFTmax PRO for Lmax Blanking Pre Read Blanking subtracts background signal caused by the microplate itself as well as by solvents reagents or samples This feature is available only for endpoint mode The individual blank reading from each well can be subtracted from the final reading from the same individual wells choose Pre read plate from within the Instrument Settings dialog box Plate Blanks are created by entering a Blank group in the template After a reading the average of the blank values obtained from the Blank group is subtracted from all wells in the plate Plate blanks can be used with all read modes Group Associated Blanks are created by placing blank wells within a group in the template The values of group blanks are averaged and subtracted from the values of the wells within that group only SOFTmax PRO for Lmax User s Manual 1 11 Chapter 1 Welcome to SOFTmax PRO for L
175. f the window beyond the last section or the right edge beyond the edge of the sections the intervening space will be gray You can also reduce the window to an icon by clicking the Minimize button in the upper right corner of the window When the window is shown as an icon it is still functional double clicking the icon will restore the window to its pre vious size Other options are available for resizing windows trefer to the doc umentation that came with your Windows software for more information iY H The width of the experiment and sections shown in the window is 5 constrained by the width set for the printed page size The dimen sions and orientation of the printed page and consequently the dis play size may be changed by selecting either Page Setup Macintosh or Print Setup Windows from the File menu H BELELER A BEEEERE BEBEEEE SOFTmax PRO for Lmax User s Manual 3 5 Chapter 3 SOFTmax PRO for Lmax Overview BO Status Bar 3 6 21 2 E A Near the top of the SOFTmax PRO for Lmax window is a status bar that starts with an icon representing the Lmax instrument see Figure 3 3 Current Chamber Click Instrument Temperature to start Incubator Cover Plate Icon Display a reading Button Status Setting Figure 3 3 Status Bar NOF The status bar can be hidden from view by selecting Hide Status from the View menu Selecting Show Status from the view menu will restore it to view
176. fault is enabled Graphs may be dis played as bar graphs or scatter plots The Graph section is divided into three areas e The tool bar The body of the Graph section e Plots information below the graph You may create more than one Graph section within an experiment Plots in the Graph section must be defined from groups within the same experiment section as the graph with one exception if you duplicate an experiment that contains data in the Plate section and has a plot defined in a Graph section that plot will remain in the duplicated Graph section and new plots may be added SOFTmax PRO for Lmax User s Manual 3 17 Chapter 3 SOFTmax PRO for Lmax Overview BO The Graph menu contains the following items shown with their tool bar or mouse equivalents These menu items and buttons are active only when a Graph section is active Table 3 4 Graph Section Menu Commands and Tool Bar Buttons Menu Command Ee New Window in the Opens the Graph section in doed Graph menu a new window Name of Graph sec Opens the Section dialog Graph tion box double click Graph Settings in the Opens the Graph Settings Graph menu dialog box Drop down menu allows you to choose a curve fit X Axis in the Graph Opens the X Axis Settings menu dialog box Y Axis in the Graph Opens the Y Axis Settings E menu dialog box is Includes excludes section Graph Options in the Opens the Graph Options Graph menu dialog box Export Gr
177. fy or change a formula SOFTmax PRO User s Manual 7 23 Chapter 7 Tutorial 24 B Resize and then Autosize a Column Position the pointer on the line dividing the columns named Concentration and Wells The pointer will change to a double headed arrow Click and drag the line dividing these columns to the right Figure 7 29 shows these actions and the resulting wider Concentration column or f Sanas HH Ea zn enne ce I siers H EA ere enitn a Double Staredai dtp tll Headed Samia ds pga group blank 121 oun SE Arrow E LETITE t Pointer Dividing Line Dragging Right gt IT Saras n 24 Aa merito Acme nue Sramdardes pali group blank 1210 Gimpera Jelly Vae TN WII 7 eat Wr e Tn Resulting BE Column is Wider To shrink this column to the minimum size needed to display the complete title click once in the Concentration column to highlight it Then choose Autosize from the Group menu The column will be reduced in width as shown in Figure 7 30 Several columns can be Autosized at one time by highlighting dragging across multiple columns in the Group and then choosing Autosize SOFTmax PRO User s Manual Chapter 7 Tutorial FTT Standards Hs 24 Mean value Average valles Standards pg ul group blank 1 210 mm sio EI 7 6 400 DI o 14 E Th H3 T10D G2 6 000 C3 E10 Before TTE ESO Fl 430 s00D a 20 5 D
178. g Box 0 cece cece eens 8 11 SOFTmax PRO for Lmax Info Dialog Box lees 8 12 Notes Section Menu Commands and Tool Bar Buttons 3 12 Plate Section Menu Commands and Tool Bar Buttons 3 14 Group Section Menu Commands and Tool Bar Buttons 3 15 Graph Section Menu Commands and Tool Bar Buttons 5 3 18 Features Available in the Instrument Settings Dialog Box 4 4 Columns Created and Their Formulas eeeeeeeeeeeeeeee 4 18 Minimum Number of Standards Required for Curve Fits 5 43 PC File Name Extensions and Macintosh Document Types for Use with File Exchange slseeeeeeeeeeeeeeeeeeeeeee 6 8 ASCII Data File Fields and Descriptors 0 0 0 0 cece cece eee 6 19 ASCII Template File Columns and Descriptions 0 0 00004 6 21 New vs Duplicate within an Experiment 0 0 0c cee eee eee eee 6 25 Actions Protected by Password 0 0 cece ccc eee ees 6 26 SOFTmax PRO for Lmax User s Manual Chapter 1 Welcome to SOFTmax PRO for Lmax WTOC HODIE erir Bodo ps 6a divs ave Aon ae Ea Ann og tr d 1 3 About the Documentation lesse sees 1 4 Conventions Used in This Manual 1 5 GO LB eels tie aia baronies 1 7 Macintosh On Line Help 1 7 Windows On Line Help Lsuuue 1 8 Troubleshooting and Technical Support
179. he Plate section you de fine a template to be used for analysis determine the instrument settings choose how the data will be displayed and define the type of reduction if any that will be performed on the raw data received from the instrument Nu Introduction Group sections are created automatically when groups are defined in the Template Editor The example shown at left is for a group named Standard Protocol files generally contain default groups you can create others as need ed Each Group section contains default columns of data which can also be changed if desired Within a Group section you can resize columns modify existing columns to use different analysis formulas add columns containing new formulas and or add one or more summaries at the bottom of the sec tion Graph sections are used to plot information from groups defined in the Tem bd plate Editor Values associated with the X or Y axes depend on the type of group selected for graphing More about Graph sections can be found in Chapter 5 Displaying and Analyzing Data General Information about Sections Sections are either active or inactive Multiple sections can be active at the same time although it is typical to have only one active section The tool bar of an active section is dark gray Inactive sections are color coded to make it eas ier to discern a section s type Inactive Experiment or Section Color EXPONEN E cioe isis arte PS P
180. he Plate section tool bar Well Graphs plot the data for the individual wells in a microplate and show the well ID and reduction information including the goodness of fit R 2 value if appropriate The default display for the X axis shows time RLU is displayed on the Y axis Positioning the cursor within a Well Graph shows X and Y values for the cur sor location allowing you to determine more precisely the location of data points Well Graph Options Along the bottom of each Well Graph dialog box are eight buttons that per form the following functions e Print Opens the Print dialog box e Reduction Opens the Reduction dialog box By default the ranges on the enlarged plot are set to the limits defined in the Reduction dialog box and the information included near the bottom of the window is based on the reduction settings currently in use You may change these settings type of reduction used minimum and maximum RLU limits lag time and end time and number of Vmax Points More information about the reduction options can be found later in this chapter Changes to the reduction settings made within well graphs will be applied to all wells in the Plate section When viewing Raw data you can scale the graph to see data outside the reduction limits line s are included on the graph indicating the limits and the area outside of them will be shaded slightly This view should assist you in deter mining the best limits for
181. he options by clicking on OK SOFTmax PRO for Lmax User s Manual 6 13 Chapter 6 File Management and Printing BO 6 14 The Print Dialog Box Choosing any of the Print commands brings up a dialog box The contents of this box will vary depending upon the type of printer you have selected or the particular printing option you have chosen but the basic elements are the same The dialog boxes shown below represent those displayed when you choose the Print command from the Macintosh and from Windows 85 Printer Printer Destination Pages amp All 2 From To Paper Source amp All pages from Auto Select 3 First page from Cassette 250 sheets Remaining from Cassette 250 sheets rm Figure 6 10 Print Dialog Box for the Macintosh You can choose to print all of the sections regardless of whether they are included in the report by clicking the option button next to Print all at the bottom left of the Print dialog box SOFTmax PRO for Lmax User s Manual Chapter 6 File Management and Printing Figure 6 11 Print Dialog Box for Windows Epson Stylus 800 Shown If more than one printer is available to your computer make sure you select the desired printer prior to selecting one of the Print options in the File menu If you start to print and discover the wrong printer is selected choose to exit from the Print dialog box Select the correct printer using the Ch
182. he sample menu will look like the one below The checkmark indi cates the sample that is currently being named alm 2B Concentration lo mg Figure 4 19 Sample Drop Down List Sample Names Defined and Wells Selected Sample Descriptor Field The field associated with the sample descriptor only appears in the Template Editor tool bar if the group column type that has been selected requires this type of information Generally the information entered in this field is a dilu tion or concentration Position the cursor in this field and enter a number The label and units assigned to the sample descriptor in the Group Settings dialog box are shown on either side of the field Assign Area The Assign area contains the Series and Assign buttons ES TI Concentration hoo mg m Figure 4 20 Assign Area of the Template Editor Tool Bar Assign Button and Function Click this button to apply a sample name to one or more wells that have been selected in the template You may also perform this operation by using the Enter key on your keyboard Series Button and Function This button is used to define several samples as a series allowing you to easily enter incremental sample descriptors for example dilutions or concentra tions and sample names to the template as long as the increment can be expressed as a mathematical operation Replicate Samples and Samples in a Series You can assign the same group or sample definition
183. he top of the switch box indicating the identity of each output port To Switch from Reading to Printing Configure the computer SOFTmax PRO for Lmax and the switch box as described above for reading a microplate Read the microplate Set the switch box to communicate with the printer Quit SOFTmax PRO for Lmax In the Finder select Chooser from the Apple menu to bring up the Chooser dialog Set AppleTalk to active In the box on the left side of the Chooser dialog choose the AppleTalk driver for the printer The printer name should appear in the box on the right side of Chooser dialog Close the Chooser dialog Start SOFTmax PRO for Lmax Print the desired sections SOFTmax PRO for Lmax User s Manual 8 7 Chapter 8 Troubleshooting a To Switch from Printing to Reading 1 Configure the computer SOFTmax PRO for Lmax and the switch box as described above for printing 2 Print the desired sections 3 Select Chooser from the Apple menu to bring up the Chooser dialog 4 Set AppleTalk to Inactive 5 Set the switch box to communicate with the instrument 6 Select Preferences from the Edit menu 7 Without changing any settings click OK 8 Read the microplate Macintosh Cable Connections For an Lmax connected to a Macintosh one end of the cable should end with an 8 pin DIN connector This connector is small and round and should be con nected to either the modem or printer port of the Macintosh
184. hichever occurs first The User may terminate the License by returning to MDC all Software documentation the original Software all backup and archival copies of the Software and all demonstration copies of the Software The License agreement will terminate automatically without no tice from MDC if the User fails to comply with any provision of this License Agreement It is incumbent upon the User to take all reasonable measures to effect termination only in accordance with this License Agreement SOFTmax PRO for Lmax User s Manual 77 General 1 This License Agreement shall be governed by the laws of the State of California 2 The provisions of this License Agreement are severable and if any one or more of such provisions are judicially determined to be illegal or otherwise unenforceable whether in whole or in part those remaining provisions or portions of the License Agreement unaffected by such determination shall be binding on and enforceable by and between the User and MDC 3 In the event it becomes necessary to retain the services of an attorney to enforce any provisions of this License Agreement the non prevailing party to such legal action an or litigation agrees to pay the prevail ing party s costs including reasonable attorney s fees and court costs if any 4 The User acknowledges that by opening the media package the User has read this License Agreement understands it and agrees to be bound by its terms and cond
185. his tutorial you will view formulas associated with columns and summaries resize individual columns autosize all the columns add a column and hide replicates 522 SOFTmax PRO User s Manual Chapter 7 Tutorial A Viewing Formulas To view the formulas associated with all columns in the Standards Group section hold down the CTRL Shift keys while the Standards group is active Another row will appear beneath the column titles showing the formula for each column Depending on the size of the column all or only part of each formula may be shown Tl Standards 244 24 Standards pq group blank 1 210 gan eomeenteaion_ Wels ahes d Meanitahe Stiler Eve a ai O00 a 6 100 E wr 0 wt He DOLI ER 7 0l 5 4900 C nnn Figure 7 27 A Portion of the ou Group Section with Formulas Shown by Pressing the CTRL Shift Keys If column formulas are long as in this tutorial example they may be seen in their entirety in the default column size To see the complete formula for a par ticular column click once on a column to highlight it The formula for that col umn will be shown in the tool bar T 2 standards uu 24 hMean alue Average values Standards pqiul group blank 1 210 Mean Value 501 SODODU H1 6 900 NU 1 14 FRUI 6 000 0 100 7 100 Figure 7 28 Standards Group T with Formula Shown for Highlighted stil 250 000 Bi d 900 Column MeanValue Click the ta EM button to modi
186. hown automatically unless you choose a different dis play option Reduction options allow you to display the raw data received from the Lmax in a reduced analyzed form Which reduction options are available depend on the read mode custom settings using formulas you create are available for all read modes and read types Reduction settings do not affect the raw data but simply cause it to be dis played differently allowing you to change reduction settings without affect ing the raw data from the plate reading in any way Custom reduction formulas require the use of accessors and operators that are understood by SOFTmax PRO for Lmax Information regarding custom formulas can be found in the Formula Reference Guide The Data Display 5 4 After a plate has been read the information sent from the Lmax is shown in the data display in the active Plate section The default display for data from endpoint readings for the Lmax is raw RLU numeric values Data from kinet ics readings is displayed in plots format as raw RLU values with respect to time While a kinetic reading is in progress the display updates dynamically to show the data collected so far All choices except those defined in the Instrument Settings affect only the manner in which the data is displayed the raw data is not changed permit ting you to optimize the display after data is collected Data reduction changes the way in which data is displayed and also affects
187. ic it will not be truncated If sample names are very long and they must be kept long for reference you will have to set up your replicates manually and will not be able to use the series function Blanking Pre Read Plate The blanking selection found in the Instrument Settings dialog box for enpoint mode only is known as pre read plate blanking but pre reading the plate is only one of five types of blanking available in SOFTmax PRO Under standing the differences between the types of blanking functions will enable you to choose the type of blanking that is appropriate for your assay Note that all types of blanks can be used in combination and have cumulative effects Pre read plate blanking for endpoint mode is discussed here for a description of the other types of blanking that are available and how these blanks work in combination see Blanking on page 4 24 Integration and Pre read Integration Time Sec pw Pre Fead plate PO rae re error creer eter Normal Pre read same az Measurement Special Integration Time sec LRRRERRRERERRERREHRRRERRHRERRERERERRRERERRREEHRRHREREHRRHREERHRERREHRRERRRHREEREHRRERERRRRERRRERERREREEHREEREHRREEREERREREREREERREREEREEREREEEE Figure 4 24 Pre Read Plate Blanking Section of the Instrument Settings Dialog Box Endpoint Mode Only SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microplates The purpose of pre read plate blanking is to correct for well
188. ick in a new window Introducti S TEE E Opens the Section dia niroduction log box double click New Window in the Opens the section in a Notes menu new window Section Name in the Opens the Section Name Notes menu dialog box Create Summary in the Opens the Calculation Notes menu dialog box allowing you to create a summary Edit Summary in the Notes menu or double Opens the Calculation click the summary dialog box allowing you pointer changes to dou to edit the summary ble headed arrow Shows hides the calcu lations used to derive summaries Includes excludes sec Printer icon tion from printed report SOFTmax PRO for Lmax User s Manual Show Hide Formulas in the Notes menu 8S Chapter 3 SOFTmax PRO for Lmax Overview Plate Section Tool Bar jew E3 Piste SB Setup E Template Eisa Z n PRE Endpoint Integrate 1 sec Data Display P Injection Off M Injection Off Instrument Settings Reduction Settings Wavelength Combination Ihyalue 3 1 Figure 3 10 Plate Section The Plate section is used to collect data from the instrument define how the data is displayed and what the initial reduction of the data will be In addi tion the Template Editor in the Plate section is used to create a map of the contents of the microplate Choosing a particular function or command in the Plate section can be done in several ways You can use the mouse
189. icroplates can be compared within the same experiment SOFTmax PRO for Lmax can collect data from one or more microplates and store it in a single data file using the same or different instrument settings for different microplates Four read modes are possible using SOFTmax PRO for Lmax e Endpoint Allows a single integration of samples in 96 or 384 well plates with two injections possible if desired in any or all wells of a 96 well plate no injection possible in 384 well plates A pre read of the microplate may be made before injection of buffer or reagent and or reading of the samples The signal is integrated for each well over a user selectable reading time maximum 5000 seconds You can program a delay after each injection if desired no minimum delay for the M injector the P injector has a mini mum delay of 1 6 seconds both have a maximum setting of 1000 seconds and you can read all or only some of the wells in the plate Default values reported are relative luminescence units RLU Dual Read Also an endpoint reading this mode is designed to perform two separate integrations one after each of two possible injections in any or all wells of a 96 well microplate no injection possible in a 384 well plate Inte gration times for both readings must be the same minimum integration time is 0 1 second maximum is 1000 seconds You may program a delay after each injection if desired no minimum delay for the M injector the P
190. iment to open and closing the separate window opens the section within the experiment again All of the tool bar options applicable to the section are available in the separate window except the triangle indicator IB Experiment x C3 Pieter m Plate 1 A i 2 3 4 5 B 7 8 8 10 n 12 esol L 7 Se oe 2 ical Wavelength Combination Mv alue Plate Blank Used Lind 1 6 Ei Hz Figure 3 8 Separate Section Window SOFTmax PRO for Lmax User s Manual 3 11 Chapter 3 SOFTmax PRO for Lmax Overview BO Notes Section n Tool Bar A Results 24 f This nates section can be used to display summaries that indicate the results of the reading Body Figure 3 9 Example Notes Section Named Results The Notes section is used to record information that pertains to the experi ment You can enter text of any kind and add summaries containing reduction formulas The Notes section is divided into two areas e The tool bar e The body You may create multiple Notes sections within the same experiment The tool bar contains the following items shown with their tool bar or mouse equivalents These menu items and buttons are active only when a Notes sec tion is active Table 3 1 Notes Section Menu Commands and Tool Bar Buttons Mena Command Description Tool Bar Button Mouse BE adu CR Notes section icon Opens Opens the Notes section NO UGNGECHOM double cl
191. injector has a minimum delay of 1 6 seconds both have a maximum setting of 1000 seconds You may read only some or all of the wells in the plate pre reading the microplate is not possible with this mode Default values reported are relative luminescence units RLU SOFTmax PRO for Lmax User s Manual 1 3 Chapter 1 Welcome to SOFTmax PRO for Lmax a e Long Kinetics Allows repeated measurements on one or more wells of a 96 or 384 well microplate Pre reading the microplate is not possible with this mode One or two injections can be made in any or all wells of a 96 well plate at the beginning of the reading no injection possible in 384 well plates An important application for this mode is cellular luminescence see page B 5 Default data reductions for kinetic data are Vmax per Sec Vmax Time to Vmax or Onset Time FastKinetics Allows repeated measurements of one or more wells of a 96 or 384 well microplate up to a 100 point maximum integration Pre reading the microplate is not possible with this mode One or two injections can be made in each well of a 96 well plate at the start of the reading no injection possible in 384 well plates Integration time can be set from 1 to 100 sec onds Default data reductions for kinetic data are Vmax per Sec Vmax Time to Vmax or Onset Time About the Documentation The SOFTmax PRO for Lmax documentation set consists of this manual an on line help system and a formula reference ma
192. ion estimates the area under the curve as defined by the data plots in wells within the reduction limits The data plots are treated as a series of trapezoids with vertices at successive data points and at the X axis coordi nates of the data points The areas defined by each of the trapezoids are then computed and summed Custom Reduction Formulas If the predefined reduction formulas do not meet your needs you can create one or more different reduction formulas for any or all read modes endpoint dual read or kinetic Choosing Custom from any of the menus or dialog boxes causes a Formula button to appear an example of this button in an endpoint Reduction dialog box is shown in Figure 5 13 m Reduction X aaa Wavelength Comnbinatiorr OC BGOoOOGebasecoanceaosceonaaeaooncancoaseosceeoonooaada neonsaosceenaenasencon ia asenoscee enoadcaneaesacescnecoonacanconcansacencieosensoaaoascaeoccecoeoacnaenen i MERRRRRRRRRRRERRRRRRRRRERRRRRERERRRTERERRRRERERRRERRRRRERRERENSRTERRRERRRSERSSERSRRRRSRSSERSRRSRSSRSRRRRXS RSS HR P na uuu uu ro EIREREXRRRXRRRHRRRERRRRRERRRRRRRRRRERRRRRTRERERERRERERERERERRERERERERRRRRRRRERERRRHSRSRRRRNRERESRERRE Formula Button Figure 5 13 Custom Selected Showing Formula Button Endpoint Clicking the Formula button opens the Calculation dialog box in which you can name and define a new reduction formula W Calculation Formula dalne Accept Cancel Figure 5 14 Calculation Dialog Box
193. ion to each pair of data points Port A connection or socket on the computer Ports are common used for connect ing devices such as printers or monitors to your computer and for sending information to and or from your computer to such devices Protocol File Contains the template s instrument settings and reduction parameters Pro tocol files are useful if you repeat a particular type of assay frequently Quadratic Curve Fit A mathematical description of a parabola y A Bx Cx RAM Random Access Memory The memory that can be used by applications to perform necessary tasks while the computer is on When you turn the computer off all information in RAM is lost Ranged Data Display Raw or reduced data is assigned proportionally to integer values from 0 through 9 based on user defined limits Values above the high limit are dis played as plus and values below the low limit are displayed as minus Raw Data Signal reported as RLU from the Lmax with no alteration Read Interval The interval of time between the start of one reading and the start of the next reading during a kinetic run Read Button or Command Starts the reading process and shows the endpoint dual read or kinetic data in the data display of the Plate section Read Mode The method used to read the microplate endpoint dual read or kinetic Reduced Data Data reduction causes the raw signal values reported by the instrument to
194. iplicate The easiest way to start setting this up is to create a series by first highlighting the remaining Standards wells and then clicking the Series button in the upper right of the Template Editor In the dialog box that appears leave the default values for the First Sample name Std01 and Sam ple Descriptor Concentration in pg uL Choose to have this series start from the bottom and enter 500 for the starting value choose the function and enter 2 for the increment of the series These choices will cause the series of wells you have selected to be assigned concentrations of 500 250 125 62 5 31 25 15 625 and 7 813 pg uL respectively in triplicate from the bottom up The choices you have made should look like this Start From First Sample Sto C Top lv v Sample Descriptor 1t i Botte Eoncenrsien Unts c I ECC Left Starting value cost Right Step by Replicates Cancel Figure 7 11 Series Dialog Box Click the button to close the dialog box and return to the Template Edi tor The wells you selected should now be assigned the values entered for the series You can check the concentrations assigned to these wells by pressing and holding the CTRL Shift keys m Expenment 1 Platel Ex meer Inl ses Group Ses n Clear Concentration T013 paul ps Aer ram S00 sal Cancel Figure 7 12 Template with CTRL Shift Keys Depressed SOFTmax PRO User s Manual 7 13 Cha
195. is active the Edit menu will show Delete Experiment 1 Using the Macintosh you can also delete the selected experiment by pressing within Windows you can press CTRL Take care when deleting experiments that they do not contain infor mation you wish to save You may also choose to delete multiple experiments simultaneously by hold ing down the Shift key and activating the tool bars of the experiments you wish to delete Having done this the Edit menu will now show Delete Selec tion instead of Delete Experiment SOFTmax PRO for Lmax User s Manual 3 7 Chapter 3 SOFTmax PRO for Lmax Overview BO You can add new sections to the active experiment using the New Section command under the Edit menu Sections SOFTmax PRO for Lmax contains four types of sections Notes Plate Group and Graph A brief synopsis of these sections is given below Notes sections are used to record information pertaining to the experiment You can enter text in this section as you desire in addition you can create summaries containing formulas for displaying reduced data Text in Notes sections can be formatted font size and style any way you choose Each Plate section shows a grid of cells which corresponds to the wells of the microplate To the right of or underneath this grid is a box showing the current instrument settings except incubator settings which are always shown at the bottom of the Plate section after data is acquired In t
196. it showing that it is ready to move left or right see Figure 5 15 x Standardz Double headed Sample Concentra Aes Meanvalue Std Dev CY arrow cursor Smallest standard value Largest standard value Figure 5 15 Cursor Positioned on a Line between Columns Reducing Column Size and Hiding Columns You can reduce a column to a size smaller than the width of its title in which case the title will not be completely visible You can also hide a column by reducing its size completely drag one edge of the column on top of the other or by selecting that column and choosing Hide from the Group menu If you hide a column in this way and then wish to view it again use the Show All command in the Group menu All columns that were hidden will again be visible There is no way to show only one hidden column SOFTmax PRO for Lmax User s Manual 5 23 Chapter 5 Displaying and Analyzing Data BO Divider being dragged to the left Enlarging Column Size You can enlarge the width of columns to any size Increasing the size of col umns may make the width of the Group section increase beyond the page width boundary If this occurs the columns will disappear off the edge of the screen but are really still there To bring these columns back into view reduce the width of the columns that are still visible until the others reappear Standards al e tan ee ee ee Std De Figure 5 16 Dragging a cM Divider Line
197. itions The User further agrees that this License Agreement constitutes the full and complete agreement between the user and MDC with respect to the Soft ware and License which agreement supersedes any proposal or prior agreement whether oral or written and any other communication between the User and MDC relating to this License Agreement Trademark Information SOFTmax is a registered trademark of MDC and MAXline is a trademark of MDC these are not to be used in any type of promotion or advertising without written permission from MDC iv SOFTmax PRO for Lmax User s Manual e Contents Suo FTT MM V labio NN Mr ix j e T ix Chapter 1 Welcome to SOFTmax PRO for Lmax eeeeeeeee ee ee 1 1 IBtEOOUP HOSE aed ode teca vtedtetebrE eb need etudes etude deni 1 3 About the Doc mentaHoN e eoe oe cedetetetecetedeseteneteeedesetesetaseee 1 4 Conventions Used in This Manual 0 0 cee e eee eee 1 5 CCG s e aicedesgeuaaeteesyeeagagstaeenatanaeaaagenaeaueuaegaennayaen 1 7 Macintosh On Line Help 0 0 eee cet eens 1 7 Windows On Line Help esee 1 8 Troubleshooting and Technical Support 00 cess eee eee 1 10 Clessaty Ol ICUS tccctaecuncenecesauonaeeeaeataa eau eaaeseaeseaereaucs 1 11 Chapter 2 Installation 562299999 PFERDE PRESE SUR D RESO KR PES 2 1
198. kinetics e Timing for kinetic runs e Plate type number of wells e Blanking pre read plate e Wells to be read e Integration time Settings for the reading are made in the Instrument Settings dialog box which can be accessed three ways 1 Choose Instrument Setup from the Control menu 2 Click the Setup button in the tool bar of the active expanded Plate section Or 3 Double click anywhere in the settings box located to the right of the well grid in the active expanded Plate section You may create more than one Plate section in a single experiment and the instrument settings associated with each Plate section can be different Instrument Setup can only be chosen while a Plate section is active and the settings you choose will affect only that section A new Plate section that is created in an experiment that contains an active Plate section will contain the same instrument settings found in the previously active section The settings for the new section can be modified as desired Table 4 1 lists all possible instrument settings choices based on instrument type When you have defined the instrument settings click the OK button to close the Instrument Settings dialog box After closing a summary of the instrument settings will appear in the box to the right of the data display in the Plate sec tion The Lmax will use these settings to read the plate Table 4 1 Features Available in the Instrument Settings
199. l for appli cations including coagulation chemistry where the changing concentration of the reagents does not change Vmax but rather will change the time at which the reaction reaches the maximum rate Onset Time This is another method for analyzing non linear kinetic reactions Onset Time reports the time required for a kinetic reaction to change by a specified amount of luminescence onset RLU above the initial RLU reading for the well Time at Minimum This setting reports the time at the minimum RLU that falls within the reduc tion limits Time at Maximum This setting reports the time at the maximum RLU that falls within the reduc tion limits Time at 1 2 Maximum This setting reports the time at the half of the maximum RLU that falls within the reduction limits To calculate this reduction SOFTmax PRO for Lmax first determines the kinetic point within the reduction limits that has the maxi mum signal level It then scans the kinetic plot from left to right until it finds two points having signals that bracket half of that value A linear interpola tion between these two points is used to estimate the Time at 1 2 Maximum SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data Slope This setting determines the slope of the combined plot i e the slope of the line using linear regression This setting uses all visible timepoints in the reduction window Area Under Curve This reduct
200. lace Figure 4 29 Alert Regarding Replacing Data for Plate Section After beginning a reading the status bar will show the actions that are occur ring in the Lmax along with a box showing the elapsed time for each action SOFTmax PRO for Lmax User s Manual 4 29 Chapter 4 Reading Microplates BO 4 30 in minutes and seconds The Read button will change to Stop allowing you to terminate a reading if necessary An example of the appearance of the status bar at the start of a kinetic reading is shown below 4341212 Fe dms GS raten Wel C7 Readingi lof 13 Clo o0 09 Figure 4 30 Status Bar during a Long Kinetics Reading The values read by the Lmax will appear in the data display of the Plate sec tion as they are received in real time Depending upon the read mode end point dual read or kinetic the plate type and the display options selected the data display will show one or more numbers or plots You can enlarge the display of one or more wells while a kinetic reading is in progress Double click a single well to enlarge its display to a Well Graph or hold the Shift key while selecting more than one well and then choose Graph from the Plate menu or click the graph button on the tool bar to dis play the data from multiple wells in one enlarged Well Graph The data in the enlarged Well Graph will update to show new data points as they are received from the Lmax Other choices and settings within SOFTmax PR
201. las see the accompanying Formula Reference Guide Adding Columns You can add a new column to a Group section by clicking the Create Column button in the Group tool bar or by choosing Create Column from the Group menu which will open the Column Formula dialog box Calculation Namej terf Decimal Places 3 Figure 5 20 Column Formula Dialog Box In the Column Formula dialog box you can change the name of the new col umn from the default which simply lists the column by number to reflect the data that it will contain If no existing column is selected when you click the Create Column button the new column will be created to the right of all existing ones If you select highlight a column and then add a new column it will be positioned to the right of the one you selected If the table is currently as wide as the sheet of paper specified in Page Setup when a new column is added the columns will disappear off the right side of the window but are still there To bring these columns back into view resize visible columns smaller New columns can contain references to other columns both in the current section or in a different one within the same experiment For example if you wanted to subtract the mean values in one group from those in another you SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data could create a new column in either group to do this If the two groups were
202. lates and then click the up or down arrow until the desired value is reached Alter natively you can type over an existing value using the keyboard After choosing a total run time and an interval between readings the total number of readings that will result is Shown below the run time If you set the interval time to be shorter than the instrument s minimum read interval the program will alert you with a warning message Fast Kinetics Repeated integrations are made for a specified number of data points up to a 100 point maximum integration in a single well of a 96 or 384 well micro plate This process is then repeated for each of the rest of the selected wells One or two injections can be made in each well of a 96 well plate at the start of the integration no injection possible in 384 well plates Integration time can be set from 1 to 100 seconds Data is collected over time with readings taken at regular intervals The default reduced values calculated for kinetic data are Vmax per Sec RLU Sec Time to Vmax seconds Onset Time seconds Time at Minimum Time at Maximum Time at 1 2 Maximum Slope and Area Under Curve irge criaa lec a P amp 1 5n Eon BS E mari BE AS E33 4 5 V Schedin Pinjact 25 uL Daley 1 5 zac M inici 10D uL niaga rec wri mcew Xl beer baira rampit tha red wall 5 Fell nrc ci Tha obi bree fo reed the eleched wollr iz apgecornateby 21 21 Lema e Figure
203. lay parameters Save the protocol Read the plate start the simulator and then open the Tutorial file Analyze data Standard Curve Analyze data Standard section Print the report ro ue xo ergo oe qs9 mi SOFTmax PRO User s Manual 7 3 Chapter 7 Tutorial a Description of the Assay Example Following are the details about the quantitative assay settings we will use to create a protocol file using SOFTmax PRO for Lmax Type of Assay Endpoint quantitative assay with Standards and Unknowns with and without dilution factor Standards in pg uL 500 0 250 0 125 0 62 5 31 3 15 6 and 7 8 each standard run in triplicate Blanks Group associated blanks with Standards in triplicate Integration 1 second Injection P injector volume 100 uL delay 3 seconds Standard Curve Fit Linear Reduction Default IMValue Define the Protocol The following steps will guide you through the process of creating a protocol file for a quantitative assay Reading an actual plate will not be required data will be acquired using a simulator program that is part of SOFTmax PRO for Lmax 7 4 SOFTmax PRO User s Manual 2 Chapter 7 Tutorial Step 1 Start SOFTmax PRO for Lmax and Open the Tutorial File SOFTmax PRO User s Manual Double click the SOFTmax PRO for Lmax icon to start the program Choose Tutorial Ipr from the Assays menu You should see a new file labeled Unti tled which should resemble the o
204. lect the appropriate settings for your printer and then click Print or OK 5 6 Printer Printer Destination copies 1 Collated Pages amp All 2 From To Paper Source amp All pages from Auto Select T D First page from Cassette 250 sheets Remaining from Cassette 250 sheets Save Settings Figure 7 39 Print Dialog Box for Macintosh SOFTmax PRO User s Manual 7 31 Chapter 7 Tutorial Figure 7 40 Print Dialog Box for Windows Epson Stylus 800 Shown You have completed this tutorial but SOFTmax PRO for Lmax has many other features that you will find useful The next chapter describes in detail items only briefly discussed up to this point and will provide information regarding customizing and using the rest of the features of SOFTmax PRO for Lmax 7 32 SOFTmax PRO User s Manual Chapter 8 Troubleshooting HOO GLO CHO in On aa Oe Ge ne ee Se eo De Ee orbes 8 3 Message Boxes and Alerts cese pee Co Erw Gedy Se REN EP int 8 3 One or More Points Lost Message 8 4 Error Messa e DOXES svi ex stereo Os ney Doe Se e ope ped 8 4 Communication Problems 0 0 0 0c eee ee eee eee eee 8 5 Macintosh Cable Connections 200 8 8 PC Cable ConnechOns zs sobre Er Do EN doe Suu PPAR 8 9 Display FO Deis se sene Sie Goa aie is be ine Soe is E PEN 8 10 Prine PODES ese Guy bad oie Ge OPES HIP Ss a ie Se Se 8 10 General Problems s
205. lected by clicking its tool bar the area that shows the experiment name with the mouse Selecting an experiment causes its tool bar to change from light gray to darker gray and the experiment title to be shown in boldface type Selecting is different from activating an experiment Selecting an experiment allows you to perform actions on the entire experiment such as deleting or duplicating it Multiple experiments can be selected by holding down the Shift key while clicking the individual tool bars of the experiments A single click anywhere within an inactive experiment activates it Activating an experiment allows you to add new sections to it or to perform other opera tions within that experiment With sections however selecting and activating are identical functions Clicking anywhere within an inactive section activates and selects it Creating a new experiment by choosing either New or Duplicate from the Edit menu creates an identical copy of the experiment excluding data The new experiment retains the same name as the original with the word Copy appended when you choose to Duplicate e g the duplication of Expt1 pro duces Exptl Copy and an increment of number when you choose New e g Exptl becomes Expt2 An experiment can be deleted if it is no longer needed The Delete command in the Edit menu changes depending upon which experiment is active For example if the experiment titled Experiment 1
206. ll A1 in the upper left corner Columns 1 through 24 are along the top and rows A through P are shown from top to bottom The wells are smaller and data is shown with fewer digits less precision 5 6 SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data Plates e Vertical the plate is shown in the same orientation as Normal except that columns 1 through 12 all rows are shown above columns 13 through 24 all rows SOFTmax PRO for Lmax User s Manual 5 7 Chapter 5 Displaying and Analyzing Data BO e Rotated Same as the Normal view except that it is rotated 90 degrees clock wise well A1 is in the upper right corner v ime E ee sehen tue is 8 Za Ug Le n BD Large Same as the Normal view except larger This display can be seen in its entirety only if you have the page size set for legal paper 8 5 x 14 inches in landscape orientation set this in Page Setup or Preferences under the File menu In the example below the display is shown as it appears if you do not change the page size from letter size to legal or 3 Pistes Lg Seta Ex Terehe E Fearin rises i ux X r E C T amp cmo m OC CO d od ih Ernipiint P iector Offi Lai Resi iieyaa j zer Mineo Of Ho Costes SOFTm
207. m If you need additional assistance or if you encounter a problem with SOFT max PRO for Lmax that is not listed here in the U S call Molecular Devices Technical Services group at 1 800 635 5577 elsewhere call your local repre sentative For problems with an instrument that do not relate to the use of SOFTmax PRO for Lmax please see the instrument manual for more informa tion Message Boxes and Alerts Message boxes typically appear to inform you that the action you are attempting needs adjustment in some way Message boxes contain only an button that you can click to remove the message after you have read the information Alerts inform you that the action you are about to take will have conse quences that may not be reversible or may make a significant change within the program Alerts are indicated by an alert symbol N typically alerts do not prohibit an action but warn you about a condition that may cause a prob lem One common example of an alert occurs when you are defining instrument settings for a kinetic assay and the read interval is too short The message might look like this Read interval is too short Minimum 9 seconds Figure 8 1 Example of an Alert Message SOFTmax PRO for Lmax User s Manual 6 3 Chapter 8 Troubleshooting One or More Points Lost Message m SOFT mar Pro for Linax One ar more points may have been lost during this read Please check your data A problem may e
208. mary can be posi tioned in a different place in the Group section if desired To delete a summary highlight it and press the Delete key or choose Cut from the Edit menu A summary can also be copied or cut and then pasted in a different location You can also change the format of the text in Group section columns and or summaries By selecting the column or summary and then choosing Edit and then Text Style Ctrl T you can change the font size and or style of the text Notes Sections The Notes section allows you to enter text describing the experiment and one or more summary lines that contain formulas To create a summary click the Create Summary button in the tool bar or choose Create Summary from the Notes menu The Calculation dialog box that appears see Figure 5 21 allows you to enter a description of the summary accept the default title for the summary change it or hide it and to enter a formula including the number of decimal places the reduction should show if appropriate Detailed information regarding operators and accessors can be found at the end of this chapter If you wish to see the formulas associated with the summaries without calling up the Calculation dialog box for each one you can choose Show Formulas from the Notes menu To see these formulas only briefly hold down the shift and control Ctrl keys while that Notes section is active The summaries will expand to show the complete formula alo
209. max BO Checkbox A small square box that appears in a dialog box and that can be selected or cleared When the checkbox is selected an X appears in the box A checkbox represents an option that you can turn on or off Clear Within the Template Editor Clear removes information that was designated previously for selected wells By highlighting wells and then choosing Clear any information regarding those wells will be erased the wells will return to their default status of empty Note that if you clear the entire template the groups themselves will not be deleted and are still selectable Keeping the groups allows you to reassign wells without having to recreate groups Click You click an object on the screen by pressing and quickly releasing the mouse button while the pointer is on that object When you click once on an object such as an icon or file name it becomes highlighted which means it is selected Whatever action you choose next will affect a selected object Clipboard A temporary storage location within the operating system used to transfer data within and between files and applications Concentration The amount of a substance present per unit volume Used as the default sam ple descriptor for a series of samples Control A sample that is run as an internal check of the assay The control has a pre dictable or known assay result against which other results can be compared H Control Menu Box Windows te The icon
210. mmary Edit Calumn Edit summar Autasize Hide Ctrl H Hide Replicates Show All Show Formulas Group Settings New Window This menu item changes to show Plate Notes Graph or Group depending on the type of section that is currently active If no section is active this menu item does not appear Create Column lIf a Group section is currently active you can add a column to the display by choosing this command If no columns are highlight ed the column will be added at the end of the group If you select a column first the new column will be added to the right of the high lighted column Button Equivalent pm in the Group section Create Summary Allows you to create a summary and enter a formula for this summary for the Group Summaries appear at the bottom of the group table Button Equivalent 24 in the Group section Edit Column Selecting a column makes this command active Choosing Edit Column allows you to change the formula associated with the column and to change the column name Mouse Equivalent Double clicking the column performs the same function Button Equivalent in the Group section Edit Summary Allows you to edit the formula associated with the summary as well as to change the summary name This command is dimmed if asummary has not yet been created First highlight the summary and then select this command Mouse Equivalent Double clicking the summary performs the same function
211. n 5 10D FF Standards 24 Concentration Concentration Standards paul group blank 1 210 ample Concentration OT H1 6 100 r000 0 100 14 He F000 H3 7 100 250 000 51 5 3f 6 000 0 100 Me G3 6 100 125 000 7 4500 5 000 0 100 F000 F100 be AQ 5 3 500 4000 0 100 en Ee 4000 Figure 7 30 Columns Before and After Autosize C Add a Column The columns that are created by default with various Group sections may not always meet all the needs of a particular experiment You can add a column to any Group section and define the purpose of this column in your data analysis By default new columns are added to the right of any selected highlighted column or if no columns are selected after the last col umn in the Group To add a new column to the right side of the Standards Group make sure this section is active Then deselect any highlighted columns by clicking outside the column area Click the Create Column button or choose Create Column from the Group menu A dialog box will open asking you to enter information for the new column W Calculation x hlarmm Colt Formula Decimal Places Accepi Cancel Figure 7 31 Calculation Dialog Box SOFTmax PRO User s Manual 7 25 Chapter 7 Tutorial e 26 D In this tutorial example the new column will show the maximum value obtained for each sample Type over the default column name entering MaxValue in the box next to Name Now e
212. n Lmax the information sent by the instrument to SOFTmax PRO for Lmax is luminescence data which is the measurement of light given off by a chemical or biochemical reaction You can start a reading at any time after defining instrument settings It is not necessary to define groups and assign wells within the Template Editor first The values received from the Lmax are raw luminescence readings and are not affected by settings in the Template Editor Only instrument settings must be defined prior to reading a microplate To start a reading insert the prepared microplate in the drawer and then click the Read button in the status bar or choose Read from the Command menu If you have created more than one Plate section and no Plate section is active choosing the Read command will cause a dialog box to appear asking you to choose which section to read see Figure 4 28 Select the correct section and then click to close the dialog box If a Plate section is active choosing the Read command will read that plate Select Section Sections Plate 1 v Cxj Figure 4 28 Select Section Dialog Box If you select a Plate section that already contains data and choose to Read again an alert appears asking you to confirm replacing data in the destina tion plate see Figure 4 29 If you select Replace the reading will proceed and the previous data will be replaced with new data f Replace data in Plate i Cancel Rep
213. n for the file use the pull down menu at the top of the dialog box to select a different folder To change the name overtype the existing file name Click when you have entered all the necessary information and you are ready to save the file SOFTmax PRO for Lmax can be configured to save a copy of your data file automatically Autosave after a reading This function is enabled in the Pref erences dialog box choose Preferences from the Edit menu SOFTmax PRO for Lmax User s Manual 6 9 Chapter 6 File Management and Printing wm Preferences Serial Port COM Ww Export Form at oo z Iv Autazave File Panu cava eteee vem ren e Y REST EE SNR CENT ER NER NR EN ED Y TEDIN NT EE VETTER YT EIER i IFE Time ale Plate a SOF Tmax PROS for Lmax 1 0 t 2 M Include Labels E P File Pref Data Append Date f SOFT max for Lmas file C Text File Reader Fes Autoprint Cancel Figure 6 7 Preferences Dialog Box When you enable the Autosave function by clicking its checkbox in the Pref erences dialog box the textbox below it becomes active allowing you to accept or change the default location for saving data files The default location is the folder containing the SOFTmax PRO for Lmax program You may accept the default file prefix or change it to another prefix of your choice For example you may wish to use a prefix that is specific for a certain group of experiments or one of sev
214. n in the Group menu or dou ble click the column Show Hide Formulas in the Group menu Autosize in the Group menu Hide Show All in the Group menu Hide Replicates in the Group menu Printer icon Opens the Calculation dia log box allowing you to edit the summary Opens the Calculation dia log box allowing you to edit the column Shows hides the formulas used in columns and sum maries Enlarges or reduces col umn size to fit the largest piece of text Hides one or more selected columns or shows all col umns that were hidden Hides data from all repli cates Includes excludes section from printed report Table 3 3 Group Section Menu Commands and Tool Bar Buttons SOFTmax PRO for Lmax User s Manual 8S Chapter 3 SOFTmax PRO for Lmax Overview Graph Section Tool Bar v axis Body 0 0 1 0 2 0 3 0 4 0 5 0 6 o f 0 6 0 9 1 x axis Plot Information Use c Piot 1 Groups Result vs values i Figure 3 12 Graph Section example shown before data collection To create a graph section choose New Graph from the Experiment menu The Plots dialog box opens automatically and a default plot called Plot 1 is selected The plot will default to the first group available in the experiment Once a graph has been created new plots can be added and deleted the axes can be customized and the size displayed area of the graph can be changed Grid lines can be enabled or disabled de
215. named Group 1 and Group 2 for example and both groups contained a column entitled Mean you could create a column within Group 1 that would subtract the mean values in Group 2 from those in Group 1 The column formula needed to do this would be Mean Mean Group2 Numerous possibilities exist for creating and editing column formulas to dis play the data you desire For a complete discussion of formulas and opera tors see the accompanying Formula Reference Guide NOF To delete a column highlight it and press the Delete key or choose Cut from the Edit menu Adding or Editing a Summary in a Group Section To add a summary at the bottom of a Group section click the Create Sum mary button in the tool bar or choose Create Summary from the Group menu The Calculation dialog box will appear allowing you to change the name of the summary or hide the name if desired as well as to enter a description of the summary and to create a formula for it including the number of decimal places for the result if appropriate Double clicking an existing summary causes the same dialog box to appear allowing you to modify summary infor mation Calculation Name Summary 1 C Hide Name Decimal Places 3 ene Figure 5 21 Calculation Dialog Box Information entered in this dialog box will be placed at the bottom of the Group section as follows Name Description Formula Result You can click the checkbox next to Hide Name
216. ne shown below m SUFTmax PRO for Lmax nix Ele Edit View Experiment Control Assays Window Help oe alc Be IS Untitled Bel E3 v L5 Experiment A m Natesi 24 v E Pate em Setup E Template E Reduction Egbispteu ke ZZ E Plate 1 1 2 3 4 5 5 T 5 J 10 11 e j jl S S S ne tT tT tT tT Ll LII g 1 1L E E E E LL p Pea LL OO Wavelength Combination value Figure 7 1 Tutorial File Blank with Notes 1 and Plate 1 Sections Open 7 5 Chapter 7 Tutorial Step 2 Define the Instrument Settings Click on the Plate 1 section to make it active and then click the but ton to open the Instrument Settings dialog box The option button next to Endpoint at the top is automatically selected no change is required On the left side of this dialog box are main section headings followed by descrip tions of the current settings for those headings The right side of the dialog box shows the options available for the selected heading on the left The first section Integration and Pre read is selected automatically showing that the Integration Time is set to the default of 1 second and Pre Read plate is dis abled These settings are fine for this assay 9o 4 Dual Read Fast Kinetic Integration and Pre re Figure 7 2 Instrument Settings Dialog Box Integration and Pre read Section Open 7 6 SOFTmax PRO User s Manual Chapter 7 Tutorial Click the next heading on
217. ned using SOFTmax PRO for Lmax so limits for the display of information in graphs must be entered manually Cross Platform Files If you have SOFTmax or SOFTmax PRO for Lmax files that were created on a platform other than the one you are currently using you can transfer these files to your computer and use them provided you have the required soft ware and or hardware to do so Transferring Macintosh Files to the PC Third party software is available for the Windows compatible PC that allows the PC to read Macintosh formatteed 3 1 2 inch disks However if you need to transfer files between Macintosh and PC platforms and do not have such software you can use PC disks with both platforms since the Macintosh will be able to read them Macintosh computers are equipped with software that allows you to read and transfer files from PC disks If you are using System 7 5 or later you will need to make sure that File Exchange is available in the Control Panels The process of using File Exchange is described below 1 If you have not yet enabled File Exchange on your Macintosh select Control Panels from the Apple menu From within the Control Panels choose the Extensions Manager and turn File Exchange on by placing a checkmark next to it Restart your computer File Exchange will now be enabled 2 Rename the Macintosh files you wish to transfer to PC disks so that their names have appended the proper 3 letter extension preceded by a p
218. ng a tem plate to a section in the same experiment from which it was copied is useful for cross plate analyses since corresponding wells in each plate will be assigned to the same group and sample Pasting a template to a section in a different experiment will create new groups in that experiment if those groups have not been created previously in that experiment If you are pasting templates between experiments and groups from the source template do not exist in the destination experiment pasting a template will create them Existing groups of the same name will not be changed by the pasting process For example if the template being copied contains the groups Standard and Unknown and the group Standard already exists in the destination experiment the group Unknown will be created but all information regarding the group Stan dard in the destination experiment will remain unchanged Experiments define a naming scope for the sections within them which is especially important for Group sections it allows you to have identical groups name and column format within two or more different experiments in the same file Having multiple experiments in the same file also provides you with quick access to different types of information without having to open or refer to multiple files Plate section data and Notes section text can be copied from programs other than SOFTmax PRO for Lmax and pasted into a Plate section or Notes section For
219. ng the reduction parameters after choosing a reduced data display mode will cause the display to update accordingly More information about reduction formulas and how to customize them follows later in this chapter Threshold Ranged and Gray Scale To display Threshold Ranged or Gray Scale data high and low values must be entered When you choose one of these display options SOFTmax PRO for Lmax provides default values for the high and low limits by means of an autoranging function encompassing all the data from lowest to highest value You can change the high and low limits by highlighting the existing value and entering a new value An example of the Display dialog box for a kinetic reading with the Threshold display option chosen is shown in Figure 5 3 SOFTmax PRO for Lmax User s Manual 5 9 Chapter 5 Displaying and Analyzing Data 5 10 ws Display Ej o Faw Faduced Ymax units per sec Malte Example Options eee eee eee eee eee Low High Display Threshold wr Rowe Limit Boxes Tesauauuuusuususuesuuusuuuseusuususussuuuusess Show With reduced number Figure 5 3 Display Dialog Box for Long Kinetics Threshold Display Threshold The Threshold display shows the raw data as a plus for values above the high limit an asterisk for values within the limits and a minus for val ues below the low limit Figure 5 4 shows an endpoint reading displayed using the Threshold option alo
220. ng with the other summary infor mation They will resume their previous appearance when you release the option key Graph Section The Plot X Dialog Box Whenever you create a new Graph section or add a plot to an existing graph the Plot X dialog box opens automatically X starts with 1 and incre ments each time a new plot is added to a Graph section In this dialog box you can rename the plot assign specific information to be plotted on the X and Y axes and choose which icon and color will be used If you rename the plot the title of the dialog box will change to show the new plot name All groups that have been created or assigned within the file are listed on the left side of the Source field in the Plot X dialog box The first group in the file is automatically selected Other groups can be selected by highlighting them When a group is selected the pull down menus in the X axis and Y axis SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data subfields change to show the group columns that are available for graphing Group columns can be assigned for both the values to be plotted and any error bars to be included for both axes Summaries and user defined formulas can also be graphed Pushing the button for either the X axis or Y axis opens a dialog box which allows you to enter formulas To plot a summary enter the summary name in this dialog box Formulas can be plotted for comparis
221. ng with the reduced number v C3 Peter El Plate 1 1 2 3 4 5 B 7 8 8 40 441 42 A 44001200 1 300 1400 1 500 1 600 1 700 1 800 1 300 2 000 2 100 2 200 estate 1 sec B 2100 2200 2 300 2 400 2 500 25600 2700 2800 2300 3000 3100 3 200 M Iniection Off C 3400 3200 2 200 3 400 3500 3500 3700 3800 3 800 4 000 4 100 4 200 10 01 AM 5 21 00 D 4400 4 200 4 300 4 400 4 500 4 500 4 700 4 800 4 300 5 000 5 100 5 200 E 5400 5 200 5200 5 400 5 500 5600 5 700 5 800 5 300 6 000 6400 6 200 F 6400 6 200 6 200 6 400 6 500 6 600 6700 6 800 6 900 7 000 7400 7 200 T T T T tr t 7100 7 200 7 300 7 400 7 500 7 600 7 700 7 800 7 900 8 000 6 100 6 200 H a100 6 200 8 300 6 400 8 500 6 600 8700 8 800 6 900 9 000 8 100 9 200 Wavelength Combination Malie 08 14 1112032 p 82 Figure 5 4 Endpoint Data Displayed as Threshold with Reduced Number Ranged When the Ranged display is chosen raw data that falls between the high and low limits is assigned proportionally to integer values from 0 through 9 Val ues above the high limit are displayed as a plus and values below the low limit are displayed as a minus Figure 5 5 shows reduced kinetic data dis played in Ranged format SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing D
222. nter Max Values in the box next to Formula and click OK Be sure to enter the formula exactly as shown The dialog box will close and a new column will be shown at the right of the previous columns showing the maximum value obtained for each sample HT Standards 42 24 Max alue Maxi alles Standards pg ul group blank 1 210 6 900 7 000 0 100 14 7 100 r000 T 100 5 900 6 000 0 100 L7 6 100 6 000 6 10 3 000 aie 3 100 1 40 e UON 0 100 2 100 2 2 10 0 900 1 0010 0 100 10 0 1 100 1 00 1 100 malest standard value 1 000 Largest standard vale 7 000 Figure 7 32 New MaxValue Column Appended to Standards Section Create an If Statement in a Column Formula An If statement in a for mula defines a condition that if found to be true causes a certain result and if not true causes a different result The basic structure of an If statement is An Tf statement in a formula defines a condition that if found to be true causes a certain result and if not true causes a different result The basic structure of an If statement is If condition result1 result2 We are going to create an If statement formula within the Unknowns section that will compare data from the Values column in that section against calcula tions found in the Standards section Here s the formula we will create If Values gt MinStd Standards and Values lt MaxStd Standards Outlier This statement says that
223. nual Additional documents on disk may also be provided to cover the latest information regarding the software An outline of the contents of this manual follows along with intro ductory information that should help you get started using SOFTmax PRO for Lmax Screens and parts of screens represented in this manual may depict either the Macintosh or the Windows version of the software The two interfaces are quite similar but bear subtle differences such as where the Help menu is located etc Depending upon which com puter platform you are using which operating system you are using and the type of screen that is depicted your screen may look different from the one shown Note that Macintosh screens are depicted using OS versions 7 through 9 The rest of this chapter Welcome to SOFTmax PRO for Lmax provides introductory information regarding the program and its features A glossary of terms is included at the end of this chapter e Chapter 2 Installation contains basic information about installing the program on your computer and making sure the correct connections are made between the computer and the instrument Chapter 3 SOFTmax PRO for Lmax Overview explains how SOFTmax PRO for Lmax is organized and how it is used with the Lmax e Chapter 4 Reading Microplates contains instructions regarding how to prepare for and conduct a reading using SOFTmax PRO for Lmax Chapter 5 Displaying and Analyzing
224. o Switch from Printing to Reading below Configuration for Printing The computer SOFTmax PRO for Lmax for Macintosh and switch box must be configured as follows in order to print from SOFTmax PRO for Lmax AppleTalk active in the Chooser dialog serial port set to modem in the SOFT max PRO for Lmax Preferences dialog and the switch box set to communi cate with the printer to access the Chooser or Preferences dialogs refer to To Switch from Reading to Printing below SOFTmax PRO for Lmax User s Manual Chapter 8 Troubleshooting 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 Connecting to the Switch Box These instructions are for connecting to the A B switch box available from MacWarehouse 800 255 6227 part number BND0199 DIN 8 A B switch box Attach one end of the 8 pin DIN to 8 pin DIN serial cable provided with the switch box to the single 8 pin DIN serial port of the computer Attach the other end of the cable to the input port I O of the A B switch box Attach one end of the 8 pin DIN to 8 pin DIN serial cable provided in the SOFTmax PRO for Lmax software package to one of the two output ports A or B of the A B switch box Attach the other end of the cable to the Lmax Attach one end of the 8 pin DIN to 8 pin DIN AppleTalk cable to the remain ing output port of the switch box Attach the other end of the cable to the AppleTalk printer Place a note on t
225. ocess of adding reagent and or buffer to a microplate well Lmax has two injectors that may be used wiht the 96 well plate format injection not available with 384 well plates Instrument Icon Located in the status bar the instrument icon shows the status of the connec tion between the computer and the instrument If the icon has an X through SOFTmax PRO for Lmax User s Manual 1 15 Chapter 1 Welcome to SOFTmax PRO for Lmax BO it the connection is not functioning or no instrument is connected if the instrument icon appears without an X the connection is working properly Instrument Setup Defines the parameters read mode run time read interval etc used to inte grate data from the microplate Interleaved Display A display choice available with 384 well plates is useful when four 96 well daughter plates are combined into one 384 well plate Interprocess Messaging Messages that can be sent to and received from other applications This fea ture is very useful for interfacing Lmax with robotic systems and for sending and receiving data from LIMS systems Lag Time Lag time is the period of time in a kinetic assay of very slow growth microor ganisms or rate of reaction that may precede the rapid or linear phase of reaction When a lag time is specified in SOFTmax PRO for Lmax data col lected prior to the lag time is not included in data reduction Large Display A display choice available with 384 w
226. on with other data For example if the data is assumed to follow a specific model the model formula can be entered as a plot and then the data can be chosen for another plot within the same graph You can also use this feature to plot kinetic data in Graph sec tions For example to plot kinetic data from well A1 in a Graph section you can enter the formula A1Lm1 for the Y Axis and the TimeRun accessor for the X axis The entered formulas are also shown in the Source field of this dialog box The size color and style of the icon used for a plot can be selected on the right side of this dialog box Four sizes nine colors and nine styles are available from drop down menus The Graph Options Dialog Box The Graph Options dialog box allows you to change the name of the Graph section change the height of a graph select the type of graph to be displayed change the fonts used on the title legend and axes access the Plot X dialog box for editing plots entering new plots and or to delete plots This dialog box is opened after pressing in the Plot X dialog box when a new Graph section is being created This dialog can also be accessed by pressing the Graph Options button fi on the Graph section toolbar or by choosing Graph Options from the Graph menu Scatter plots cluster bar charts and stack bar charts can be created Error bars can be used with all graph types You can also choose whether to connect the points and or plot the s
227. ong and fast kinetics Other choices available in this dialog box include dis playing the data as a single reduced value number in threshold ranged or gray scale formats or any combination of display choices with the reduced number the reduced number display choice cannot be used with the reduced number checkbox SOFTmax PRO for Lmax User s Manual 5 5 Chapter 5 Displaying and Analyzing Data Endpoint By hapla Dual Read By apley pa z Rowe FLU thea h reduces panier Long and Fast Kinetics my heals F z Fini Erlari Ree FAU Shaw Raw RLU defau It ma Raw Data Plot RLU VS Time default wa CO Raw Data Plot RLU VS Time default _twed x mm Hum l Reduced engai Display Grgtcade Choices ETT L FRUSTUESS EI afz Reduction AD aie uimpe Catia EMM eum 1 Reduced mei Display sexa Choices my apley Ea ajjz eb bilari E um m mo Reduced Display Choices Figure 5 2 Endpoint Dual Read and Long and Fast Kinetics Display Dialog Boxes Data in the Plate section is displayed in a grid array microplate format With all plates other than 384 well plates the display is always shown as a stan dard microplate with well A1 in the top left corner If you are reading 384 well plates additional display options can be chosen to make the display of data clearer e Normal shows the data in standard left to right format with we
228. ooser for the Macintosh or the Print Setup dialog box for Windows and then repeat the print pro cess Additional information regarding the general print functions available in these dialog boxes can be found in the documentation that accompanies your computer and or printer SOFTmax PRO for Lmax User s Manual 6 15 Chapter 6 File Management and Printing BO Exporting Importing Information 6 16 Exporting Data for Later Reimport 1 2 3 4 5 If you are exporting data so that you can manipulate it using another program and then reimport it into SOFTmax PRO for Lmax the data must be displayed as raw data without a reduced number prior to being exported Follow the steps below to ensure that the exported data will be able to be reimported properly into SOFTmax PRO for Lmax Complete instructions regarding how to perform each of these steps can be found in the following section Check that the correct plate format and export options are specified in the Preferences dialog box Make the Plate section from which you wish to export data the active section click somewhere in it or on its toolbar Check that the reduction settings are as you wish them to be by clicking the Reduction button on the Plate section toolbar Click on the Display button and verify that the display settings are set to show raw data without a reduced number Choose Export from the File menu and save the exported data to a file on
229. open allowing you to choose a new location for saved files Select Directory c COMPUTER Figure 2 8 Autosave Location Dialog Box Windows SOFTmax PRO for Lmax User s Manual 2 13 Chapter 2 Installation ae Highlight the folder where you want files to be saved The Select button at the bottom of the dialog box will reflect this choice clicking the Select but ton will set this folder as the default location for saved data files You can change the location at any time by repeating this process To change the file format from SOFTmax PRO for Lmax to ASCII text select the Text File format from the list If the Autosave function is reporting an error or if files are saved in the wrong location it may be that a previous version of SOFTmax PRO was not uninstalled prior to installing the newest version Fol low the instructions given above to choose the correct folder for saved files Create New File Text only After each Plate section is read the data will be saved in a new file This file will contain all the data from the current and previous readings for example if you read a Plate section andthen another Plate section File1 will contain data for Plate1 File2 will contain data for Plate1 and Plate2 This option is available for both file formats SOFTmax PRO for Lmax and ASCII text OXGIONA If you Autosave to ASCII text format you will not have a SOFTmax PRO for Lmax version of the file unless you
230. or leave the default to print all pages Make sure you have selected the correct printer prior to choosing one of the Print options in the File menu If you start to print and dis cover the wrong printer is selected choose Cancel to exit from the Print dialog box select the correct printer using the Chooser or the Print Setup command and then repeat the printing process Quit 3 CTRL Q Closes SOFTmax PRO for Lmax as well as any SOFTmax PRO for Lmax files you may have open If you have open files that have changed and have not yet been saved a dialog box will ask if you want to save them before quitting SOFTmax PRO for Lmax User s Manual 3 23 Chapter 3 SOFTmax PRO for Lmax Overview BO Edit Menu Undo 3Z CTRL Z Restores the last text operation to the condition that exist ed before the editing was done used primarily when entering text in Notes sections This menu item is dimmed if no actions have been Cut Ctrl taken or if undoing the last action is not possible Undo Lez C Ctrl C ind iy Cut 3X CTRL X Removes highlighted information text or data from the Clear document and stores it on the clipboard so it can be pasted elsewhere Select Al Chh This menu item is dimmed if no selection has been made or if the se lection cannot be cut Delete Notes 1 Ctrl Copy 6C CTRL C Copies selected highlighted text or data and stores it on iL jus ee the clipboard so it can be pasted elsewh
231. ot the data as a bar graph with data shown in clusters or stacked Figure 5 41 shows both of these bar graph options E 1 Pell Urea pne Seep ep binnen m Poll Orca Seep n ier nde lll Poe reer Seep or inni Cluster Bar Graph Stack Bar Graph 7 14 amp EU PEUT pia own Emp sd eost Pin Deep Tamil e ey B MEL EST DI TI Figure 5 41 Bar Graphs Cluster and Stack NOH Bar graph options for multiple plot graphs are only available when each plot contains the same number of data points and has matching X axis coordinates NOH When choosing the Connect Points option the order in which points are connected is sample name dependent SOFTmax PRO for Lmax User s Manual 5 45 Chapter 5 Displaying and Analyzing Data BO Axes Options Double clicking either graph axis clicking the X axis or Y axis button in the Graph section toolbar or selecting either X axis or Y axis from the Graph menu opens a dialog box concerning the configuration of that axis Figure 5 42 shows the dialog box for the X axis X AXIS Settings amp Linear Q Log Figure 5 42 X Axis Dialog Box The default settings are shown in the Figure above You can change these as desired for either or both axes Axis Settings Enabling the Auto range option causes the graph to be drawn such that all data points are shown by automatically adjusting the minimum and maxi mum range settings to encompa
232. ould have the name Data 01 31 2000 1 Checking this box will enable automatic printing of a report after each plate is read This report will contain information from each section that is designated to be included in the report the default is for all sections to be included For more information on reports and including or excluding sections see Chapter 3 SOFTmax PRO for Lmax Overview The Assays menu is used to choose the folder that will contain protocol files you wish to access most often After installation of SOFTmax PRO for Lmax the protocol files shown under the Assays menu will be the basic protocols shipped with the software Other folders may be installed with SOFTmax PRO for Lmax that contain different protocol files for other applications you may select one of these folders instead if desired or create a new one of your own To change the default folder choose Assays and then choose Set Folder A dialog box will appear asking you to choose the folder or directory con taining the protocol files you wish to be shown in the Assays menu Select one of the other folders within the SOFTmax PRO for Lmax directory folder or if you have other protocol files in a different location choose that location and then click Select If you do not see any files in the Assays menu or if the files shown are not correct it may be that another or previous version of SOFT max PRO was not uninstalled prior to install
233. ph sections that are duplicated include all settings for plots fit etc Notes sections are duplicated exactly including any summaries and all text New sections and new experiments can be created by selecting New from the Section or Experiment menu If an existing section or experiment is active when this command is chosen all of the information in the section or experi ment except data is copied to the newly created section or experiment The name of the new section or experiment is the same as that of the original with the addition of an incrementing number e g OldName 2 A new section is created immediately below the active section A new experiment is created immediately below the active experiment New Group section tables can also be created but are devoid of any data New Graph sections include all settings for plots fit etc but contain no data New Notes sections are exact copies of the active Notes section including any summaries and all text same as duplicated Notes sections except for the name SOFTmax PRO for Lmax User s Manual 2 Chapter 6 File Management and Printing Table 6 4 New vs Duplicate within an Experiment Nw e Notes Section Plate Replicates Instrument Setup Reduction Section and Display parameters from last Plate section created if no Plate section exists default values are used Graph Section Empty Notes section Creates a new Graph section with default s
234. ppropriate for your assay Judging a Good Curve Fit The correlation coefficient is also known as the goodness of fit value because it shows how good a fit exists between the computer curve and the standard data points It measures the degree to which the points fall on the computer curve and may give an indication as to what residuals are to be expected The correlation coefficient is popularly known as R and is between 1 and 1 inclu sive When the data points lie on a perfectly straight line with negative slope then R 1 If the correlation coefficient is not suitably close to 1 or 1 a per SOFTmax PRO for Lmax User s Manual 5 43 Chapter 5 Displaying and Analyzing Data BO fect fit you can simply apply one of the other types of curves without having to reread the plate For nonlinear curve fits SOFTmax PRO for Lmax performs transformations that cause the curve to become more linear The correlation coefficients are derived from that transformed data Because of this you should be cautious about using correlation coefficients as an indicator of goodness of fit Do not let this single value for goodness of fit sway your intuition about which fit is best Some curve fits may seemingly give a good fit e g fit values close to 1 but when inspected by eye show a poor fit along a major portion of the plot Graph Options Graphs can be customized further through the Graph Options dialog box which is accessed by choosing Graph
235. pter 7 Tutorial e 7 14 E F This tutorial assay uses two groups of unknown samples one with and one without dilution Each unknown is run in triplicate Start by highlighting wells A4 through C5 Select New from the Group menu and enter Unknowns for the name Unknowns for the Column For mat and then click OK The selected wells will now show the default first sample name for the Unknowns group Un01 The template should look like the one below m Expernment 1 Plateiti Xe Edit unt j i Group Unknowns ww sample Line v Series Assign ear B Unknowns Undt Ongi Ungt Und Ongi UnOd Ln T Ln T Und Ongi UnOl Ln T Ln T Uno Figure 7 13 First Unknowns Added to the Template These unknowns need to be identified with different sample names The shortcut to doing this is to make them a series but change only the name To do this click the Series button and in the dialog that results leave all set tings as they are but enable the Sample Descriptor section by clicking in the checkbox Leave the information in this section as it is starting with 1 and dividing by 1 This will not change the group in any way except to increment the name of each sample from left to right When finished click to close the dialog box The template should now look like this SOFTmax PRO User s Manual G H Chapter 7 Tutorial W Expenment 1 Plate 1 Group Smeh I
236. r Lmax file C Text File Reader IN Autoprint Figure 2 6 Preferences Dialog Box Windows The instrument icon should now appear normally without an X through it If the port setting is correct and the Lmax is on yet communication is still not functioning properly quit SOFTmax PRO for Lmax turn off the power to both the computer and the Lmax and check that the cable connections between the instrument and computer are secure Turn both machines on again and restart SOFTmax PRO for Lmax If an X still appears over the icon refer to Chapter 8 Troubleshooting for more information Set Preferences It is a good idea to set the SOFTmax PRO for Lmax preferences before pro ceeding to use the program The preferences include certain configuration set tings such as which port is used for the connection to the instrument as well as settings for automatic printing and saving of files and the format for exported data To make these selections choose Preferences from the Edit menu to bring up the Preferences dialog box see Figure 2 6 Serial Port Check that the setting for the serial port matches the physical port used to connect the computer and the instrument A selection of Neither can be made for the serial port which can be selected if communication with an instrument is not required or if no instrument is connected to the computer When this option is selected the Status Bar in SOFTmax PRO for Lmax will reflect it by
237. racter Set 1 11 AppleTalk 2 3 8 6 8 7 8 8 Area Under Curve 1 11 4 10 4 11 5 19 5 21 ASCII Character Set 1 11 file 1 14 1 15 2 14 3 14 3 22 3 29 string based messaging 3 33 Assays folder 2 15 3 20 6 3 6 4 A 5 menu 2 15 3 20 3 28 6 3 6 4 A 5 Autoprint 2 15 3 25 6 11 8 6 Preferences setting 2 11 Autosave 1 11 2 12 2 14 3 25 6 9 6 10 A 5 Append to File 2 14 Create New File 2 14 directory A 6 file prefix 2 14 Preferences setting 2 11 Autosize command 3 16 3 32 5 24 7 22 7 24 B Blanking 1 11 1 21 4 24 4 25 blanks in combination 4 26 group associated blanks 1 11 4 26 plate blank 1 11 4 25 8 10 pre read plate blank 1 11 3 27 4 24 C Cable connections 2 11 8 5 Macintosh 2 3 8 6 8 8 PC 1 18 1 20 2 4 8 9 Calculation dialog box 3 12 3 15 5 21 5 27 5 28 hierarchical 5 16 Checkbox 1 12 Chooser 2 3 3 23 6 11 6 15 8 6 8 7 8 8 Clear button 4 13 4 15 4 20 Clear command 1 12 3 24 Close command 3 22 A 6 SOFTmax PRO for Lmax User s Manual Column adding 5 22 5 26 adding deleting 3 24 5 26 Autosize 3 16 3 32 5 24 7 24 copy paste 6 23 Create command 3 31 creating new 3 15 deleting 5 22 5 27 editing 3 16 3 31 5 22 5 24 5 25 enlarge 5 24 format 4 17 formulas 5 27 copy paste 6 23 editing 5 24 modifying 5 25 hiding 3 32 5 22 5 23 reference to other 5 26 resizing 5 22 5 23 showing 3 32 showing formulas 5 22 showing hiding formulas 3 16 text fo
238. raphs but these graphs can be exported as either PICT or EMF files see Exporting Graphs on page 6 21 for more information Group Columns You can copy a Group column and paste it within another Group in the same experiment or into a Group of another experiment The data included in the pasted columns will be appropriate for the target group Highlight the column select Copy 3 C CTRL C from the Edit menu then acti vate the section into which you want to paste the column and select Paste 3V CTRL V from the Edit menu If you highlight a column in the destination section the column you are pasting will appear to the right of the highlighted column Otherwise it will be appended to the right of all existing columns Columns and summaries in Group sections can also be copied and pasted to other programs Data from other programs cannot be pasted into Group sec tions within SOFTmax PRO for Lmax however Copying Formulas and Summaries Formulas are used in Group columns custom reductions and summaries Sometimes it is useful to copy an existing formula column or summary from one area of the program to another or from one experiment to another rather than taking time to recreate it Summaries can also be copied and pasted within SOFTmax PRO for Lmax After pasting both formulas and summaries can be edited SOFTmax PRO for Lmax User s Manual 6 23 Chapter 6 File Management and Printing BO Duplicate New 6 24
239. reported as relative luminescence units RLU the default reduced value is MValue SOFTmax PRO for Lmax User s Manual 4 9 Chapter 4 Reading Microplates 4 10 Number of Readings Calculated from the Run Time and Interval pi rectum 5 eara p Foy D a Read Mode Dean Fie lara Falt iE Buttons Figure 4 6 Instrument Settings for a Dual Reading Long Kinetics Reads all of the selected wells of a 96 or 384 well microplate and then repeats this process for as many readings as are specified in the Instrument Settings One or two injections can be made in any or all wells of a 96 well plate at the beginning of the reading no injection possible in 384 well plates The default reduced values calculated for long kinetics data are Vmax per Sec RLU Sec Vmax Time to Vmax seconds Onset Time seconds Time at Minimum Time at Maximum Time at 1 2 Maximum Slope and Area Under Curve An important application for this mode is cellular luminescence ET brochure 5 eblrey rl amp T Figure 4 7 Instrument Settings for a Long Kinetics Reading Timing Enter the total kinetic run time and the time interval between read ings A default value will be shown for the run time to change this value click in the box Up and down arrows will appear enabling you to increase or decrease the value shown Highlight the hours minutes or seconds shown SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microp
240. riptor val ues of zero 0 on a log axis Curve fits affected by this are the semi log log log log logit and 4 parameter curve fits Minimum Number of Standards The number of standards used in a standard curve depends on the particular curve fit selected and on the judgment of the operator The table below gives the minimum number of standards required mathematically by each of the curve fitting algorithms Table 5 1 Minimum Number of Standards Required for Curve Fits ter Qmm 3 mam 3 07 dem 0 3007 AININI NQJ nn deu 0 0 4 hme 000004 0 mem 003 0 Eee 03 e oO N gt A TN For optimal results you should always exceed the minimum number of stan dards required for any given fit There are other requirements on the standards for the log logit and 4 parame ter logistic curves The fits for the 4 parameter logistic curve are based on the assumption that the curve has a high and a low asymptote and a certain steepness for the linear portion around the inflection point between the asymptotes If a sufficient number of points do not define the inflection point and the asymptotes you will receive a no fit error message or an inappro priate fit For best results make sure that the 4 parameter fit is appropriate for your assay and that the entire range of the assay is represented in your stan dards If your standards do not clearly define both a high and low asymptote the spline fit may be more a
241. rmat 5 28 Communication instrument icon 2 10 ports Preferences setting 2 11 problems 8 5 serial port 2 11 Connect Points 5 44 Control menu box Windows 1 12 Copy 1 12 6 21 command 3 24 6 21 formulas 6 23 Group columns 6 23 Plate data 6 21 template 6 21 Create Column command 3 31 5 26 Create Summary command 3 30 3 31 5 27 5 28 Cross platform files 6 6 Cubic Spline curve fit 1 12 5 42 Curve fit 1 13 5 34 5 35 4 Parameter 5 39 Cubic Spline 1 12 5 42 Exponential 1 14 5 42 judging a good fit 5 43 Linear 1 16 5 35 Log Log 5 37 Log Logit 1 16 5 39 minimum number of standards 5 43 Point to Point 1 19 5 41 Quadratic 1 19 5 38 Semi Log 1 20 5 35 5 36 Custom formulas 5 46 reduction 5 21 Cut 1 13 Cut command 3 24 D Data exporting 6 16 6 20 importing 6 20 Data display 1 13 4 30 5 3 5 4 5 22 7 19 Gray Scale 5 9 5 11 Group section 5 22 limits 5 9 Ranged 5 9 5 10 reduced number 5 9 Threshold 5 9 5 10 well graph 4 30 5 12 Data file 1 13 5 3 6 5 locating 6 4 Data points 5 19 5 43 5 46 8 10 one or more points lost 8 4 Data reduction 5 16 calculation hierarchical 5 16 Default protocol 1 13 6 3 Delete command 3 7 3 9 4 14 Delete Section command 3 24 Desktop 1 13 Dialog box 1 13 Dilution factor 1 13 Dimmed 1 13 Display command 3 29 default settings 5 5 dialog box 5 5 options 6 17 parameters 5 3 problems 8 10 reduced number 5 9 Dual Read 1 3 5 18 display defaults 5 5 D
242. rmation such as Unknowns column type Edit Sample Etat hd Group Standards Y Concentration ogra A group is selected for which a concentration has been defined such as Standards column type Group Sample uo Le Dilution Factor o A group is selected for which a dilution factor is defined such as UnknownsDilution column type Figure 4 12 The Appearance of the Template Editor Tool Bar under Different Circumstances The tool bar is divided into three areas the Group area the Sample area and the Assign area The Group Area The Group area contains the Group drop down list which allows you to assign wells to existing groups create new groups or modify using the button existing groups In addition it contains the Clear button which allows you to clear well assignments in the template Group Drop Down List This drop down list consists of two areas divided by a horizontal gray line The two items below the gray line Blank and New are always available Items above the gray line are custom groups that may be created as you wish The Group drop down list will appear differently depending upon the cus tom groups that have been created by default or by the user The figure below shows two different instances of the appearance of the Group list SOFTmax PRO for Lmax User s Manual 4 15 Chapter 4 Reading Microplates Patients Standards y Unknowns mY Blank Mew No custom Example of
243. rmation for the new group Standards SOFTmax PRO User s Manual 7 11 Chapter 7 Tutorial C e Click to close the dialog box and apply the Standards group to the wells you had selected in the template It should now look like this mo Expenment 1 Plate 1 x EX sameEm vl lean xs GG Standards Ww Edit Series f Assign oup Sania E o Concentration 1 2 3 4 5 Standards 2 cun cm Stm sul st S su su 5 atal S01 S s st 5 Su st 5 St S01 S I nm Z2 A O BE suu st S Print Cancel LK Figure 7 9 Template with Standards Assigned The first three wells in row A are to be designated as a group associated blank within the Standards group To do this first click on the template somewhere other than on the Standards group and then highlight wells A1 through A3 Click the arrow to the right of the Sample descriptor in the upper middle of the Template From the drop down menu choose BL The template should look like this mi Experiment 1 Platett Ed Eat J Same vllmmen ae aoe Est eres reet i mex Imm 2 Standards c uo Cancel LK Figure 7 10 Template with Group Associated Blank Included with Standards D The Standards entered into the template are currently all the same However this assay requires the use of standards with different concentrations run in 7 12 SOFTmax PRO User s Manual Chapter 7 Tutorial tr
244. rocedure Reverse Injectors Choosing this menu option allows you to send fluid from the lines back into the reagent bottle for example to preserve costly reagents You can set this option for as many injections as desired default is 7 through either the P or M injector or both Set Instrument to Read 384 96 wells One of two settings that need to be changed in the SOFTmax PRO for Lmax software in order to switch from 96 to 384 well capability and vice versa the other setting is found in the Instrument Settings parameters Additionally a hard ware reconfiguration must be made to change from one plate size to the other See the Lmax User s Manual for more information SOFTmax PRO for Lmax User s Manual 3 27 Chapter 3 SOFTmax PRO for Lmax Overview BO Move to Service Position Moves the read head to the service position al lowing you to perform service related activities such as changing the injector tips More about such functions is included in the Lmax User s Manual Assays Menu E Set Folder Allows you to select a folder or subdirectory containing com Eune monly used protocol files In addition to any protocol files residing within this folder which will be listed below the dividing line and Leluer may be opened by clicking them an additional level of subfolders if ELE esent any will be shown below a center division Protocol files can be used Default Protocal Socr repeatedly for spe
245. rocess to be accessed read only by a process running in another address space All messages must be sent to the SOFTmax PRO for Lmax main window To get the proper HWND hwnd FindWindow SOFTMaxPROMainWnd SOFTmax PRO for Lmax Finally to send the message SendMessage hwnd WM_SETTEXT smaxMsg LFARAM myCma Notes e The emaxMeg tag MUST be sent as the wParam so that SOFTmax PRO for Lmax knows this is an external command and not a Windows generated message e The tag and hwnd should be checked for non zero values before sending the message Opening and or closing the Lmax chamber cover must be done manually and so cannot be automated by means of interprocess messaging SOFTmax PRO for Lmax Responses Most commands that are sent to SOFTmax PRO for Lmax are just that com mands that tell SOFTmax PRO for Lmax to do something However a few SOFTmax PRO for Lmax commands respond with ASCII results Examples are ReturnStatus and ReturnData Two response modes are available In the first mode SOFTmax PRO for Lmax responds by putting the requested information on the clipboard The data can then be pasted into other applications such as into Excel spreadsheets For many other applications however the clipboard mechanism is not appropri ate for retrieving data from SOFTmax PRO for Lmax Because of this SOFT max PRO for Lmax can also send the response strings back in a separate message using the MFC C WinProc
246. rst sample in the series in the box or accept the de fault provided by SOFTmax PRO for Lmax Subsequent replicates use this name as a base and either increment the number within the name or append a number to it In the example above the sample name provided by SOFTmax PRO for Lmax describes the first sample 01 in a group named Standard here shortened to Sta 4 The information shown in the box labeled Sample Descriptor is derived from the information entered in the Group Settings dialog box If the information for 4 22 SOFTmax PRO for Lmax User s Manual Chapter 4 Reading Microplates sample descriptor or units is not correct you may change it here If you did not already do so specify the starting value for the first sample in the series Values for subsequent samples are calculated from this starting value 5 Choose the operator for the series or from the Step by drop down list Enter the starting value for the series in the Starting value box The incre ment is entered in the box to the right of the Step by box and below the Start ing value box Figure 4 22 shows a starting value of 5 with the operator being division and the increment being 2 Thus the series will start with a value of 5 and divide each subsequent sample by 2 to produce the following series values 5 25 125 etc 6 You must assign a fill direction from the top bottom left or right
247. s LX Serial Port Export Format Ic Time Iw Include Labels ea fopend Date v Silelimarntor Loar ble C Oeste MEME e mas Header silere Auto Cancel Figure 7 22 Simulator Choice in the Preferences Dialog Box When the simulator is active the instrument icon in the upper left corner of the window will be blue instead of orange With the simulator active click the Read button in the status bar at the top of the screen to initiate the simu lated reading Values will appear in the data display in the Plate section as if they were being read by the Lmax When the simulated reading is complete the plate section should look similar to the one below SOFTmax PRO User s Manual 7 19 Chapter 7 Tutorial 7 20 A v Carat i Platez1 5 5 T B 9 10 11 12 D Oo O r volume 100 uL Delay 3 Sec a ae M Injection Off 1 NN LJ X p Wavelength Combination M alue Figure 7 23 Plate Section with Simulated Data The display for each well that is defined by a group boundary shows a raw RLU value displayed in a reduced form as a number The reduction formula is shown at the bottom of the display Wavelength Combination MValue In this part of the tutorial you will display the data in various other reduced formats Click the Display button again to open the Display dialog box Earlier in this tutorial we chose to display the data a
248. s a reduced number The other dis play choices for endpoint data are Threshold Ranged and Grayscale You can experiment more with the various display options if desired at a later point For now choose Grayscale from the drop down menu The dialog box will update to show the low and high limits for grayscale display values which are automatically set to the minimum and maximum data values ww Display Ex ax Raw Faduced Reduction h y alue Example Options High 7 9019 High and Low Be a rows 20088 Limit Settings Show E With reduced number Cancel Figure 7 24 Display Dialog Box Updated with Low and High Limit Settings SOFTmax PRO User s Manual Chapter 7 Tutorial Click OK The dialog box will close and the data display in the Plate section will update to show the data represented by shades of gray as shown in Fig ure 7 25 Plate 1 1 2003 4 5 8 7 8 89 10 n 12 AB didi HL ML o f Endpoint B L il Integrate 1 sec c NENNN 1 vw UE NE ee ERE NT Delay 3 Sec E d h Injection Off j ate Last Read G 7 54 PM 6 26 00 H Wavelength Combination This alie W mE 132 2B fe 3 9 m 52 BE 5 8 B 78 H 78 Figure 7 25 Plate Display with Data Displayed as Gray Scale Step 7 Data Analysis Group Sections Group sections are created automatically by SOFTmax PRO for Lmax when you define groups in the Template Editor Th
249. s dialog box for this menu plate Setup l Opens the Template dialog box allowing you to Template in the Plate menu create or edit the template E l emplate Opens the Reduction dialog box containing Double click in a single well or Enlarges selected well s to a Well Graph display when several wells are selected kinetic mode only choosing Graph again Graph in the Plate menu returns to regular display toggle Opens the Display dialog box containing display Mask in the Plate menu Masks the selected wells sacan Name a eraen Opens the Section dialog box allowing you to rename the section New Window in the Plate menu Opens the section in a new window I Cony Template inthe Platemenu Copies the template allowing it to be pasted to another Plate section Paste Template in the Plate menu Pastes a previously copied template to a new Plate section Export Template in the Plate Opens the Export Template to dialog allowing menu you to export a template to an ASCII text file Import Template in the Plate Opens the Import Template to dialog allowing menu you to import a template from an ASCII text file 3 14 SOFTmax PRO for Lmax User s Manual 8S Chapter 3 SOFTmax PRO for Lmax Overview Group Section Tool Bar Body A5 Figure 3 11 Group Section example shown prior to collecting data Group sections are created automatically when you create or select groups for the experiment in the Template Editor A Group
250. section and print again Changing the Section Order When sections are created either automatically by SOFTmax PRO for Lmax or by you their order may not be optimal for either viewing or printing The order of sections in the window determines how they will print in the report and you may find that changing the order of sections makes it easier to view the data When you create a new section it is positioned directly after the currently active section To change the order of a section first make it active by clicking on it then click in its tool bar and drag it to a new position on top of the sec tion or section tool bar you want it to precede Sections may be open or closed during this procedure Changing the Text Format The text in Notes and Group sections can be formatted to change its appear ance The Format Text dialog box accessible by choosing Text Style 3 7 CTRL T from the Edit menu provides a number of different options see Figure 6 9 Format Text Im Underline ABCDEabcde 3 Outline Apply 1234557890 O shadow Condensed E Extended Figure 6 9 Format Text Dialog Box Macintosh First highlight the text you wish to format Then choose Text Style 3T CTRL T and select the options you wish to apply to the selected text You can click on the Appl y button to see the result of the selections you have made allowing you to select more or different options before finally setting t
251. sed in a Group section table as well as its size and style can be changed by first selecting one or more columns in the Group sec SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data tion table and then choosing the Text command from the Edit menu and mak ing the desired choices from the dialog box that appears Settings from the Plate Section Affect Data Display in Group Sec tion Tables Reduction options in the Plate section allow you to display the raw data received from the Lmax in a reduced analyzed form Which reduction options are available depend on the read mode used and reduction settings chosen Reduced number from the Plate section is displayed in the Values col umn of the Group section tables by default Reduction settings do not affect the raw data but simply cause it to be dis played differently You can change reduction settings without affecting the raw data from the plate reading in any way Custom reduction formulas require the use of accessors and operators that are understood by SOFTmax PRO for Lmax A list of these accessors and operators is given at the end of this chapter Resizing and Hiding Columns The columns in a Group section can be resized in several ways One of the simplest is to drag the line dividing the columns to another position moving it horizontally When you point to a line between columns the cursor changes to a double arrow with a vertical line through
252. sees Bar ri B Unknowns Ungi Und unos Unod unos Print Cancel DK Figure 7 14 Unknowns Changed to a Series The next group to add is the Unknowns with Dilution Highlight wells E5 through G9 Choose New from the Group menu and in the Group Settings dialog box enter the name Unk w Dilution choose the Unknowns dilu tion Column Format and enter pg L for the units The Group Settings dialog box should look like the one below When all settings have been entered click OK to close the dialog box mw Group Settings EJ Mame Unk weiDilution jv Sample Descriptor E units paud v Cancel Figure 7 15 Group Settings Dialog Box for Unknowns with Dilution These Unknowns with Dilution need to have names that reflect distinct sam ples run in triplicate and they also need to all show a dilution factor of 10 Choose the Series button again and in the dialog box that appears enter 10 for the starting dilution Leave the rest of the settings in this dialog box as they are the series will progress from left to right and will divide the start ing concentration by 1 Clicking OK will update the template so that all wells in the Unknowns with Dilution now show a dilution factor of 10 and sample names increment from left to right in replicates of 3 as shown below SOFTmax PRO User s Manual 7 15 Chapter 7 Tutorial 7 16 I my Experiment Platettl Unk wverDilution
253. showing an instrument icon with an X through it along with the words No port selected This selection allows you to use the serial ports for other purposes modem connection network connection or printing for example but does not allow you to communicate with a Lmax SOFTmax PRO for Lmax User s Manual 2 11 Chapter 2 Installation Qe Export Format Autosave 2 12 The preferences settings regarding the Export Format allow you to choose whether to export the data as it appears in plate format or linearly over time The option to include text labels along with numerical data can be activated by clicking Include Labels For more information on the Export function refer to File Creation and Management on page 6 3 Enabling the Autosave feature in the Preferences dialog box will save a data file automatically each time a plate is read Autosave saves a copy of the file that is currently open If the file that is open is a protocol file note that the file will still be Untitled i e not named even after an autosave has occurred This allows you to to autosave to a text file format and then save manually as a SOFTmax PRO for Lmax file You can Autosave to one of two different file formats e SOFTmax PRO for Lmax used to save your data automatically in SOFTmax PRO for Lmax format e Text File used when you want to have the data available for use with a spreadsheet or other program for data analysis ex
254. splay button to raw data plot with reduced number Compare the number shown in the Plate display with the number of Vmax Points listed at the right hand side of the Plate section The numbers should be the same If a time point was not collected in a well the Plate display num ber will be lower Error Message Boxes 8 4 Error boxes contain a stop graphic C and text explaining the error if the software can report it Some errors are not recoverable and require that you perform the action or assay again Others such as an obstruction of the drawer of the microplate reader report the problem and allow you to correct it and then proceed with the assay SOFTmax PRO for Lmax User s Manual Chapter 8 Troubleshooting 5 Pasted Text doesn t match plate settings You have attempted to paste values from an external file containing ASCII tab delimited data into a Plate section but the number of fields of data that you have copied does not match those required by the setup of that Plate sec tion The format of the copied information must match the Instrument Settings of the target Plate section and the settings chosen in the Export Format in the Preferences Check that all the required fields are present and that you have highlighted all of these fields prior to copying the data and try pasting again Communication Problems The instrument icon shown 1 Is the Lmax turned on in the upper left corner of the 2 If
255. ss the data You can disable the Auto range function by clicking the checkbox or by changing either or both values for the Min and or Max settings Formulas in SOFTmax PRO for Lmax 5 46 You can enter custom calculations in several places within SOFTmax PRO for Lmax to provide the information you desire e n Plate sections you can specify formulas to be used for reduction e In the Group table sections you can view and edit the formulas associated with the various columns or create new columns containing new formulas The data shown in columns from one group can relate to or contain infor mation derived from columns in another group You can also create sum maries which consist of formulas and text e Notes sections allow you to enter summaries which consist of formulas and related text Use of formulas is described in detail in a separate manual The SOFTmax PRO for Lmax Formula Guide SOFTmax PRO for Lmax User s Manual Chapter 6 File Management and Printing File Creation and Management ssesleeeeeeeess 6 3 Peral PROLOGO S 5 tonio hs ore Eon op EO UP Er De 6 3 Locating Protocol and Data Files 6 4 Creating New Piles eresi tr Sue epee diy Sie RES SS Sas 6 4 ODSIDUS ol ols gee a en eee Ree ROR RN ERR DEN 6 6 saving Files Manually ses Suds Gy Ge Ge Se Sa 6 9 PUTO SAV Cs 6 ote A a Sig cay tease ee eee 6 9 Gee eas Ne Siar LIP Gane Gane Sone ge gs Net Ee 6 11 The Printed Report
256. st software applications that removes a selection of text or graphics and places it on the clipboard The cut function is enabled by pressing X on the Macintosh and CTRL X in Windows Cut and Paste A method of moving selected text or graphics from one location to a different location in the same file or into another file which could be in an application other than SOFTmax PRO for Lmax Data Display Located in the Plate section the data display is a grid that corresponds to the layout of the wells in the microplate This display can show the results of the reading in various formats for example raw signal data RLU reduced data number or plot Threshold Ranged and Gray Scale Data File Contains the raw data collected from the instrument as well as any associated sections or settings Notes Graphs Groups Instrument Setup Display Options and Data Reductions Default Protocol A protocol file included with the SOFTmax PRO for Lmax program or cre ated by a user The Default Protocol file defines the initial information that will appear when you open SOFTmax PRO for Lmax or when you create a new file from within the program Desktop The background on which windows and icons appear Dialog Box A rectangular window that appears temporarily to request information Some dialog boxes have options you must choose before SOFTmax PRO for Lmax can carry out a command and must be closed before you can continue in the softw
257. st values of x are used The corresponding y values are assigned to A and D respectively Based on these fixed values for A and D the algorithm then computes values for B and C This technique works well if there are standard points along the upper and lower asymptotes If this is not true the log logit fit should be avoided in favor of the 4 parameter logistic In SOFTmax PRO for Lmax 2 x the 4 parameter logistic curve fit uses 2000 iterations If the fit does not converge in this number of iterations the pro gram will report a fit error The curve fitting algorithm for the 4 parameter equation was based on the Levenberg Marquardt Method Discussion of this method can be found in Numerical Recipes in C The Art of Scientific Computing by William H Press Brian P Flannery Saul A Teukolski and William T Vetterling published by Cambridge University Press New York 1988 Point to Point A linear equation is fit to each pair of data points Fit parameters are not given The point to point curve fit is a linear fit composed of pieces that assume a linear relationship between each pair of data points The line seg ment defined by each pair is used to interpolate data between those points Since there are multiple line segments fit parameters are not shown on the graph gt LE crepe Pit Pont To point au a Gi aphi z enu 700 en 0 d q1 3 h E Time usc Blane
258. sts SOFTmax PRO for Lmax will automatically overwrite the file with NO warning e If the subdirectory does not exist the file will not be saved Examples OaveAe myFile SaveAs mySubDirectory myF ile SelectAll This is equivalent to pressing CTRL A or choosing Select All from the Edit menu This can be used to select data in group tables for copying not needed for copying plate data SelectSect Select a section by name This can be useful for multi plate protocols or for selecting Group tables for copying Examples SelectSect Plate OO232499 SelectSect Group l SelectSectNum Select a section by its order in the document This can be useful for multi plate protocols or for selecting Group tables for copying Example SelectSectNum 3 Setlemp XX X Send a command to set the current instrument incubator temperature Stop Send SOFTmax PRO for Lmax a command to stop reading This is equivalent to pressing the Stop button on the SOFTmax PRO for Lmax tool bar This should be sent before any Close or Quit command if there is a possibility that the instrument is still reading SOFTmax PRO for Lmax User s Manual 8S Appendix A SOFTmax PRO for Lmax Remote Command Language Quit Tell SOFTmax PRO for Lmax to close all documents and Quit NISNUNGNB If the data has not been saved the application quits anyway with no user warning Visual Basic Excel Macro Example First some
259. t Read Box Mo Deis Plate Vertical Section Scroll Tice ce i i e i Bar Data eength Combination Iv ale Display al ne Figure 3 2 SOFTmax PRO for Lmax Screen and Window for the PC Windows 3 4 SOFTmax PRO for Lmax User s Manual 8S Chapter 3 SOFTmax PRO for Lmax Overview Window Size You may change the size of the window shown on the Macintosh by dragging the size box at the bottom right corner of the window in any direction You can expand the window to the full size of the screen by clicking the zoom box in the upper right corner of the window If you extend the bottom edge of the window beyond the last section or the right edge beyond the edge of the sections the intervening space will be gray To resize the window again drag the size box to a different position or click the zoom box again Clicking the zoom box will restore the previous window size NO If you move the SOFTmax PRO for Lmax window such that the title bar is under the status bar you will have to hide the status bar before you can move the window to see the title bar again nx LAS nuunuu w wwu BEEEEBE You may change the size of the window shown in Windows by dragging the side bottom or corner of the window when the cursor changes into a dou ble headed arrow You can expand the window to the full size of the screen by clicking the Maximize button in the upper right corner of the window If you extend the bottom edge o
260. ternal to SOFTmax PRO for Lmax If you autosave as a text file the SOFTmax PRO for Lmax version of the data will not be saved automatically and you will not be able to automatically re open the data file in SOFTmax PRO for Lmax If you choose to save to this format you will need to save your data manu ally using the Save or Save As commands in the File menu if you also want to save a SOFTmax PRO for Lmax file If you attempt to quit SOFTmax PRO for Lmax before saving a file containing data that has not yet been saved a message box will appear asking if you want to save the file You can assign any name to a file you save manually If your protocol contains more than one experiment section when you Autosave to SOFTmax PRO for Lmax file format each experi ment will be saved to a separate data file When Autosave is enabled the box below the Autosave option becomes active allowing you to choose the folder within which data files will be saved and the type of file to be saved You may choose to autosave as a SOFTmax PRO for Lmax file default or as a text file and when saving as a text file you may choose to append to the same file SOFTmax PRO for Lmax User s Manual Chapter 2 Installation M COS OF Tmas PROB for max 1 0 Figure 2 7 Autosave Information Windows The default folder for saving files is the SOFTmax PRO for Lmax folder To save data files in a different folder click the button A dialog box will
261. text format 6 13 Group menu 3 31 Group section 5 22 menu commands buttons 3 15 Group Settings command 3 32 Group Settings dialog box assign area 4 21 sample area 4 20 sample descriptor 4 17 Group associated blanks 1 11 4 25 4 26 H Help 1 7 1 8 formula 1 7 search function Windows 1 9 Hide command 3 32 5 23 Hide Formulas command 5 24 Hide Replicates command 3 32 Highlight 1 15 I Icon instrument 2 10 Importing data 6 20 Incubator command 3 27 Incubator button 3 6 Installation 2 7 Instrument settings 3 19 4 4 7 6 mode 4 9 summary 4 4 Instrument Setup command 3 27 SOFTmax PRO for Lmax User s Manual K Kinetics 4 10 4 11 5 18 display defaults 5 5 interval 4 10 limits 5 18 negative OD values 5 18 reduction 5 18 5 19 run time 4 10 Kinetics limits 5 18 L Lag Time 5 19 Large Display 1 16 Lid display 3 6 Limits data display 5 9 MaxRLU 5 18 Linear curve fit 1 16 5 35 Log Log curve fit 5 37 Log Logit curve fit 1 16 5 39 Long kinetics 1 4 Luminescence 1 17 M Macintosh computer 2 5 software 2 7 Mask 5 15 button 5 15 wells 5 15 Mask command 3 29 5 15 Maximize button 1 17 MaxRLU 1 17 5 18 Memory insufficient Macintosh 8 11 Menu Assay 3 28 bar 3 4 Experiment 3 26 File 3 22 Graph 3 30 Group 3 31 Notes 3 30 Plate 3 28 View 3 26 Message boxes 8 3 Minimize command 3 26 Minimize button 1 17 MinRLU 1 17 5 19 Modem port 2 11 Molecular Devic
262. the icon also has the words No port selected through it along SOFTmax PRO for Lmax with an X open the Preferences and select the proper communica window has an X through tions port setting should agree with the physical port to which the it cable from the instrument is connected to the computer 3 Quit SOFTmax PRO for Lmax turn off power to both the computer and the instrument Check that cable connections between the instrument and computer are secure Turn both machines on again and restart SOFTmax PRO for Lmax If an X still appears over the icon it may be that you are using the wrong type of cable See Communication 1 following for more information The Macintosh computer has You can purchase a serial switch that will allow you to choose between only one serial port and can t communication with the Lmax or the printer See Communication 2 connect to an Lmax and following for more information printer at the same time Communication 1 Instrument Icon Shown with an X If the instrument icon shown in the upper left corner of the SOFTmax PRO for Lmax window has an X through it check whether the Lmax is turned on If it is then check the SOFTmax PRO for Lmax Preferences settings see Setting Preferences below to ensure that the proper communications port modem or printer for the Macintosh COMI or COM 2 for the PC is selected This set ting should agree with the physical port to which
263. the pro cess greatly When recalculation has been suspended you may wish to see the results of the changes you have made but may still not wish to enable continuous recal culation Choosing Recalculate Now 3 CTRL from the Edit menu will cause SOFTmax PRO for Lmax to examine all elements of the experiment and perform complete recalculation of the data Choosing this command does not enable continuous recalculation but simply causes the program to perform recalculation once To return to continuous recalculation choose Suspend Recalculation again The checkmark next to the command will disappear indicating that the function is no longer in use AMOH Recalculate Now 3 CTRL can be used to see intermediate results during long kinetic readings At any time you can choose Suspend Recalculation to delay calculations until a later time Displaying Data in Group Graph and Notes Sections 5 22 Group Sections The manner in which data is displayed in a Group section depends on the type of reading that was performed endpoint dual read or kinetic but can be customized by choices made in the data display reduction settings and the Edit menu e Resizing hiding columns e Adding deleting columns e Showing editing column formulas e Adding editing summaries e Changing text style Default displays for Group sections are shown automatically unless you choose a different display option The particular font to be u
264. the reduced data Show Raw Show Reduced button This button toggles the Well Graph between views of raw and reduced data When the Well Graph is opened the initial view depends on the Dis play settings for the Plate section If raw data is being displayed in the Plate the initial view in the Well Graph will be raw data and the button will be set to Show Reduced If reduced data is being displayed in the Plate section the initial view in the Well Graph will be reduced data and the button will be set to Show Raw E Scale to Data Scale to Limits button This button toggles between a view of the data within the reduction limits and a view of the entire data set Switching between these views helps to SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data visualize how the limit settings affect data reduction If raw data is being viewed in the Plate section the display in the Well Graph is set initially to show the plot scaled to the reduction limits If reduced data is being viewed in the Plate section the Well Graph display is set initially to show the plot scaled to the data If you are viewing raw data and click the Scale to Limits or Scale to Data button only the display of raw data in the Well Graph will be affected the button will not affect the view of reduced data The same is true if you click this button while viewing reduced data To produce comparable scaling of both raw an
265. the values used in calculations within SOFTmax PRO for Lmax While the raw values remain unchanged calculations will use reduced values if any and are therefore affected by settings chosen in the Reduction dialog box For more information see Data Reduc tion on page 5 16 SOFTmax PRO for Lmax User s Manual 8S Chapter 5 Displaying and Analyzing Data The Display Dialog Box Before or after a plate has been read you can click the Display button to ES Display Sar RE open the Display dialog box which allows you to change the way the data is presented Choices available in this dialog box depend on whether a Plate sec tion is currently active and which read mode you have chosen for example the choice for Plots is not shown if endpoint mode is selected Figure 5 2 shows the default Display dialog boxes for the different read modes The Display dialog box is divided into two parts left and right the default display Raw RLU is shown to the left of a dividing line the choices for reduced data displays are shown to the right of the dividing line Note data reduction itself is performed separately settings here simply determine how the reduced data will be displayed in the Plate section The dialog boxes below show the default display choices for the three modes raw RLU values numbers for endpoint raw RLU values numbers for dual read the change in raw RLU values over time displayed as a plot for both l
266. tic reading is reached reported in seconds Title Bar The horizontal bar at the top of a window that contains the title of the win dow or dialog box On many windows within the Windows environment the title bar also contains the Control menu box and Maximize and Minimize buttons On the Macintosh the title bar also contains the Close box and Zoom box Undo Cancels the previous operation if possible The paste function is enabled by choosing Undo from the File menu pressing 36V on the Macintosh or pressing CTRL V in Windows Vertical Display A display option for 384 well plates which shows the plate as two 8 x 12 arrays stacked vertically SOFTmax PRO for Lmax User s Manual 1 21 e Chapter 1 Welcome to SOFTmax PRO for Lmax Vmax Points The number of points in the kinetic curve included in each of a series of regressions to calculate the maximum rate Vmax or the time segment used to calculate the Time to Vmax Vmax units per sec The kinetic rate reported as signal sec RLU units per second It is calculated using a linear curve fit y Ax B A creeping iteration is performed using Vmax Points and the slope of the steepest line segment is reported as Vmax Rate M Zoom Box Macintosh A small box containing a smaller box at the right of the title bar You can click the Zoom box to return the window to its previous size 1 22 SOFTmax PRO for Lmax User s Manual Chapter 2 Installation Conn
267. tings for a Dual Reading 0 0 eee eee 4 10 Instrument Settings for a Long Kinetics Reading 0 0 000004 4 10 Instrument Settings for a Fast Kinetics Reading 0 0 0000 4 11 Wate Type DOLI e eo euadere autos vuv eq v RES NCRR TU EATER AER ERESER SS ER 4 12 Partial Plate Read Wells to Read Dialog Box with Wells B2 through F9 Selected 0 0 eee eee eee eee tenet ete neee 4 12 Template Editor Dialog BOX 4 2 uut ttu mmo tom to Ro or o ga Gun 4 13 The Appearance of the Template Editor Tool Bar under Difterent Circums EO EI CDSU a o aue d acu ud e aoa v EE 6 dus das d 4 15 The Group Drop Down List seeeeeeeeee RII 4 16 Group Settings Dialog Box Using Default Settings 0 00 4 16 SOFTmax PRO for Lmax User s Manual ix Figure 4 15 Figure 4 16 Figure 4 17 Figure 4 18 Figure 4 19 Figure 4 20 Figure 4 21 Figure 4 22 Figure 4 23 Figure 4 24 Figure 4 25 Figure 4 26 Figure 4 27 Figure 4 28 Figure 4 29 Figure 4 30 Figure 5 1 Figure 5 2 Figure 5 3 Figure 5 4 Figure 5 5 Figure 5 6 Figure 5 7 Figure 5 8 Figure 5 9 Figure 5 10 Figure 5 11 Figure 5 12 Figure 5 13 Figure 5 14 Figure 5 15 Figure 5 16 Figure 5 17 Figure 5 18 Figure 5 19 Figure 5 20 Figure 5 21 Figure 5 22 Figure 5 23 Figure 5 24 Figure 5 25 Figure 5 26 Figure 5 27 Figure 5 28 Figure 5 29 Figure 5 30 Figure 5 31 Figure 5 32 Figure 5 33 Group Settings
268. tion for Standards lt lt 254 zebeepr REED RReREE RERO dens 7 22 A Portion of the Standard Group Section with Formulas Shown by Pressing the CTRL Shift Keys ctr bedrmR RR Re RR ERR 7 23 Standards Group Section with Formula Shown for Highlighted Column MeanValt 22222122 odwiedeesivdededuiedadsdsiedetedsdadededs 7 23 Sizing the Concentration Column Wider 0 c eee eee eee eens 7 24 Columns Before and After Autosize sees 7 25 Calculation Dialog POX rm 7 25 New MaxValue Column Appended to Standards Section 7 26 Summaries in the Standards Section 00 cece eee eee 7 27 Calculation Dialog Box from Summary in Standards Section 7 27 Standard Section Before and After Hide Replicates 0000 7 28 Standard Curve Graph Section Linear Fit Applied 0 4 7 29 Standard Curve Graph Section with 4 Parameter Fit Applied 7 30 Graph Options Dialog Box seeeeeeeeeee e enn ees 7 30 Print Dialog Box for Macintosh 1 1 0 0 00 eee cece eens 7 31 Print Dialog Box for Windows Epson Stylus 800 Shown 7 32 Example of an Alert Message 0 0 ccc cece teen ens 8 3 8 Pin Din Connection to Macintosh Computer 0 000 e cece eee eee 8 8 8 Pin DIN to 8 Pin DIN Serial Cable 0 0 0 eee 8 8 25 Pin DIN to 25 Pin or 9 Pin Serial Cable 0 0 00 eee ee eee 8 9 About This Macintosh Dialo
269. tion is Vmax units per sec Other reductions avail able from the pop up menu are Vmax Time to Vmax Onset Time Time at Minimum Time at Maximum Time at 1 2 Maximum Slope Area Under Curve or you can choose Custom and create a different reduction formula SOFTmax PRO for Lmax User s Manual 5 19 Chapter 5 Displaying and Analyzing Data BO 5 20 Vmax and Vmax units per sec The maximum slope of the kinetic display of relative luminescence units ver sus time The default value displayed is in units second where units are rela tive luminescence units RLU The rate is calculated with the use of Vmax Points see below which determines the number of contiguous points over which Vmax is calculated The number of Vmax Points defaults to the maxi mum taken during the reading you can change this number in the Reduction dialog box if desired NORY Vmax Vmax sec 60 000 A linear regression is performed to determine the slope of the line using the equation y m X B where m is the slope of the line The reaction rate is reported in units sec Time to Vmax This is an alternative method for analyzing non linear kinetic reactions which reports the elapsed time until the maximum reaction rate is reached rather than reporting the maximum rate itself Used in conjunction with Vmax Points Time to Vmax is the time to the midpoint of the line defined by Vmax Points and used to calculate Vmax This elapsed time data is usefu
270. tion is the same as e number Accessora Example AntiLog 1 2 718 e Plate Reduction Accessors e Referencing objects by AntiLogiO number name Returns the AntiLog base 105 of a number or list of numbers Statistical Functions This is the same as 10 number Text Functions Example AntiLog 13210 Figure 1 2 Formula Help Window SOFTmax PRO for Lmax User s Manual 1 7 Chapter 1 Welcome to SOFTmax PRO for Lmax BO H Windows On Line Help You can access on line help within SOFTmax PRO for Lmax for Windows by selecting the Help menu in the menu bar Three types of help are immediately available you can view the contents of the help file seek specific help about formulas or get general information about how to use the help function w www ununun anunuuuu WEEE Help 4 Click here Contents for the Help Menu Index or press ALT H Using Help SOFTmax PRO for Lmax Help Options About S0FT mas PAO for Lmax Figure 1 3 Help Menu for SOFTmax PRO for Lmax for Windows Choosing Contents by clicking on the menu item with the mouse or pressing ALT H C brings up the SOFTmax PRO for Lmax help Contents window showing the general organization of the help that is available Help Softmax Pro Help 4 Window a File Edit Bookmark Help oo Menu Softmax Pro for Lmax Help The SOF Tmax Pro for Lmaxz Window Contents of the Reading a Microplate Help File Sections Templates Status
271. tiple contiguous wells can be selected by dragging across them Non contiguous selections can be made by holding down the Shift key and clicking individual wells Clicking a row label will select the entire row clicking a column label will select the entire column Contiguous columns or rows 1 12 or A H for a 96 well plate for example can also be selected by dragging Non contiguous columns or rows can be selected by holding the 3 command key on the Macintosh or the CTRL key in Windows and clicking in labels Double clicking anywhere within a group will select the entire group To deselect all wells quickly click in the empty border around the Template e When you leave the Template Editor by clicking the OK button the Plate section will show the boundaries of the groups created in the tem plate Group labels and sample descriptors will not be visible you can see them by holding down the Ctrl Shift keys When you release the key s the original view of the Plate section will return 4 14 SOFTmax PRO for Lmax User s Manual BO Chapter 4 Reading Microplates The Template Editor Tool Bar The tool bar will appear differently depending upon what type of group is selected in the Group drop down list The following illustration shows how the tool bar will appear under various circumstances Edit Sample Uno1 Group Unknowns um v Clear A group is selected that does not contain sample descriptor info
272. tration vs values a Pici 5 pecami t Concentration vs aues E Pore peci Concentration 3s sake Phi OHONMOR nalis Concenrsdon vie values Plott COMOROS Conceriralion vs vasusz Figure 5 27 Graph Displaying Eight Plots You can add as many plots to a graph as you wish and these can be of the same or different types of groups If you create a graph and later wish to add one or more plots to it click the Graph Options button on the Graph section tool bar and then click the New button This will cause the Plots dia log box to reappear allowing you to add or modify the plot information SOFTmax PRO for Lmax User s Manual 5 33 Chapter 5 Displaying and Analyzing Data BO Error Bars When data is displayed as a scatter or bar graph you can choose to display error bars for the plot of the data for the X and or Y axis Error bars are lines that extend beyond a plotted value in either or both directions and graphi cally represent some amount of error in plotted data Renilla Masa 1000 7 LL LJLLLIIIILLI 1001 i i i r Seah ale mm szixim oils m pr aiunt a 141 i ee alle ae Pr 1r 7 pcr cw T I PrI 7 j Ll p E I ki dja k za alsia skis sk b xax s m ber Hia s A SS EM NUS DIG RR UR UR REG IG RR UR A I iei ELE re Wis m d bgla m i g rm ee a p 3401 i LFU E rr d a i bole biki iak E Buil anie a RLU o F Error Bars 1 10 100
273. ts connected Cluster Bar or Stack Bar and you can set the height of the graph to be something other than the default percentage 10076 The font used for the title legend and or axes can be changed and or modified in size and style Plots can be added deleted or edited Editing plots allows you to change the plot name its source from which experiment and group as well Section Mame New Window 3 30 SOFTmax PRO for Lmax User s Manual Chapter 3 SOFTmax PRO for Lmax Overview as the items to be plotted for example concentration vs values Button Equivalent in the Graph section X axis Sets the X axis parameters tick marks scale label gridlines and autorange Button Equivalent in the Graph section Y axis Sets the Y axis parameters tick marks scale label gridlines and autorange Button Equivalent in the Graph section Section Name Allows you to change the name of the Graph section Mouse Equivalent double click the name of the Graph section Standard Curve to the right of the Graph section icon Export Graph Creates a file containing a picture of the graph with a filename of your choice On the Macintosh the graphic is in PICT format in Windows the graphic is in EMF Enhanced MetaFile format New Window Opens the active section in a separate window Mouse Equivalent double click the icon in the Graph section Group Menu Group Create Calumn Create Su
274. tus Bar contains the Lmax icon the current temperature within the microplate chamber and buttons allowing you to activate the Read function the incubator etc Template Editor A representation of the microplate shown as a grid of wells that can be used to designate the location of blanks standards controls unknowns empty wells or to assign wells to other groups you create The template is a map of the microplate It tells the software what is in the microplate and where it is located Threshold Data Display Data is displayed as plus for values above asterisk for values within and minus for values below user defined limits Time at Half Maximum Reduction for a kinetic run that reports the time at half the maximum signal within the reduction limits as follows SOFTmax PRO for Lmax first deter mines the kinetic point within the reduction limits that has the maximum signal level It then scans the kinetic plot from left to right until it finds two points that have signals bracketing one half of that signal value A linear interpolation between these two points is used to estimate the Time at Half Maximum Time at Maximum Reduction for a kinetic run that reports the time at the maximum signal within the reduction limits lime at Minimum Reduction for a kinetic run that reports the time at the minimum signal within the reduction limits Time to Vmax The elapsed time until the maximum reaction rate of a kine
275. ultaneous use of the software on four 4 additional computers solely for the purpose of an alyzing already acquired data is expressly permitted The user is expressly prohibited from using the soft ware to control any other instrument computing hardware or peripherals other than MDC s MAXline Microplate Readers This license agreement does not grant the user title to the software or any copyrights of proprietary rights in the software The user may not sublicense rent lease modify translate decompile or disassemble the software for any purpose The user however does own the media on which the software is i pen Or subsequently recorded or fixed MDC retains title and ownership of the software recorded on the original disc and all subsequent copies of the software regardless of the form or media in or on which the original and other copies may exist This license is not a sale of the original software or any copy Limited Warranty MDC warrants that the disc on which the program is furnished is free of defects in materials and workman ship and will operate under normal use for a period of 90 days from the date of delivery Except as provided above the Software is provided as is without additional warranties of any kind either express or implied including but not limited to any implied warranty of merchantability or fitness for a particular purpose The entire risk as to the quality and performance of the Software is the User s MDC does
276. uplicate 6 24 Duplicate command 1 14 E Edit Column command 3 31 Edit Summary command 3 30 3 31 End Time 5 19 Endpoint 1 3 4 9 5 18 display defaults 5 5 reduction 5 18 Enlarged well graphs 5 12 Error bars 5 34 Error messages 8 4 pasted text 8 5 Experiment active 3 7 3 26 menu 3 26 section 3 7 Exponential curve fit 1 14 5 42 Export 6 20 command 3 22 6 16 data 6 20 format 2 12 Preferences setting 2 11 6 16 F Fast kinetics 1 4 File autosaving 6 9 creating 6 3 6 4 cross platform 6 6 data 6 4 6 5 management 6 3 6 4 menu 3 22 protocol 6 4 6 5 saving 6 9 7 18 saving manually 6 9 stationery 7 18 Format text 6 13 Formula 5 46 column 5 27 copy 6 23 custom 5 46 custom reduction 5 21 editing 5 24 help 1 7 plot 5 29 summary 5 27 Formula button 5 21 G Glossary of Terms 1 11 Graph 5 28 command 3 29 connect points 5 44 copying 6 21 curve fit 5 34 5 35 error bars 5 34 formulas 5 29 options 5 44 standard curve 7 29 summary 5 29 Graph menu 3 30 SOFTmax PRO for Lmax User s Manual e Graph section 3 8 3 17 5 28 5 35 curve fit 5 34 5 35 menu commands buttons 3 18 plots 5 35 Graph Settings 3 31 Graph Type command 3 30 5 44 Graphing wells 5 12 Gray Scale display 5 9 5 11 Group 3 8 3 15 7 21 boundaries 4 14 columns 7 22 adding 5 26 copy 6 23 formulas 5 24 5 27 paste 6 23 customizing 3 8 sample descriptor 4 22 sample name 4 22 series 4 23 Template Editor 4 13
277. urnData This command is used to ask SOFTmax PRO for Lmax to send back the data from the currently selected plate Generally this is used to send the data directly to the caller instead of using the clipboard In the Visual Basic or Excel environments this command functions like the SOFTmax PRO for Lmax Copy command if a plate is the currently selected section ReturnStatus This command sends back the current instrument status The status is an ASCII string that has information about the instrument type and version whether the instrument is busy or idle its current temperature and the state of its drawer The exact format of this response depends on the type of instru ment in use SOFTmax PRO for Lmax User s Manual A 5 Appendix A SOFTmax PRO for Lmax Remote Command Language a A 6 ReturnTiming This command sends back the number of seconds that it would take to read the current plate For multi plate documents the plate used is chosen in the same way as described in the Read command above SaveAs XXX Have SOFTmax PRO for Lmax save the current document file The file will be saved in the current SOFTmax PRO for Lmax Autosave directory which is set in the Preferences dialog accessed through the Edit menu Data files can also be saved to subdirectories of the Autosave directory Notes e SOFTmax PRO for Lmax will append lda to any filename passed in to it e If a file with the same name already exi
278. v ceu Se Ss EN Dp OE UNSER RES Soe eM 8 11 Chapter 8 Troubleshooting 8 2 SOFTmax PRO for Lmax User s Manual 2 Chapter 8 Troubleshooting Introduction When alerts message boxes or error boxes appear while you are using SOFT max PRO for Lmax they usually contain information describing the situation In some circumstances however the information may not be complete enough to indicate what you should do to remedy the problem At other times errors may occur that are not directly associated with SOFTmax PRO for Lmax but affect its operation The information in this chapter is provided to assist you if an error occurs for which you do not have a solution or if you don t know the source of a problem This chapter presents general information regarding problems you may encounter first followed by specific types of problems grouped by kind dis play issues printing errors etc Within each group is general information regarding such problems sometimes followed by a table containing specific problems and their suggested remedies If you encounter a problem during operation of SOFTmax PRO for Lmax first check to see if a table is associated with that type of problem and see if a remedy is listed there When remedies require more space than the table can allow a reference to a numbered text section will be listed in the table Refer to that text section by number beneath the table to learn more about solving the proble
279. ve Group Section 0 eee ete e 5 31 Graph Options Dialog Box 0 0 eee cette tenes 5 32 Graph Options Dialog Box with Second Plot Added for GA h 5 32 Graph Displaying Eight Plots 00 cece eee eens 5 33 Plot Showing Error Bars 1 1 2 ccc ccc e een ees 5 34 Gropa with NO Fil uu d anderem p EUROS Wr UR ER RUP RUNE RUE I US UR SUR RU n 5 35 Graph with Linear Fit simulated data 20 0 cece eee eee ee 5 36 Graph with Semi Log Fit simulated data 2 0 0 cece eee eee ees 5 37 Graph with Log Log Fit simulated data 0 0 cee eee eee eee eee 5 38 Graph with Quadratic Fit simulated data 2 0 0c cece eee eee eee 5 39 SOFTmax PRO for Lmax User s Manual Figure 5 34 Figure 5 35 Figure 5 36 Figure 5 37 Figure 5 38 Figure 5 39 Figure 5 40 Figure 5 41 Figure 5 42 Figure 6 1 Figure 6 2 Figure 6 3 Figure 6 4 Figure 6 5 Figure 6 6 Figure 6 7 Figure 6 8 Figure 6 9 Figure 6 10 Figure 6 11 Figure 6 12 Figure 6 13 Figure 6 14 Figure 6 15 Figure 6 16 Figure 6 17 Figure 6 18 Figure 7 1 Figure 7 2 Figure 7 3 Figure 7 4 Figure 7 5 Figure 7 6 Figure 7 7 Figure 7 8 Figure 7 9 Figure 7 10 Figure 7 11 Figure 7 12 Figure 7 13 Figure 7 14 Figure 7 15 Figure 7 16 Figure 7 17 Figure 7 18 Figure 7 19 Figure 7 20 Figure 7 21 Graph with 4 Parameter Fit simulated data sseelee lesse 5 40 Graph with Log Logit Fit
280. ve the Edit menu will show Delete Plate 1 Using the Macintosh you can also delete the active section by pressing 3 within Windows you can press CTRL Take care when deleting sections that they do not contain information you wish to save You may also delete multiple sections simultaneously by making them active the Delete com mand in the Edit menu will change to show Delete Selection NOH Deleting a Group section automatically deletes references to that group from the Template See Chapter 4 Reading Microplates for more information Section tool bars contain buttons and pull down menus Buttons in the tool bar are only enabled when that section is active the section does not need to be open Menu equivalents exist for most tool bar functions Figure 3 4 shows the active tool bar from a Graph section 7 Graphet Fit Linear v Figure 3 4 Tool Bar from an Active Graph Section If you scroll the window vertically so that part of an open active section is hidden its tool bar sticks to the top of the window See Figure 3 5 This fea ture prevents the active tool bar from becoming unavailable if you are view ing the bottom of a section that is longer than the size of the window When the next section scrolls to the top of the window its tool bar replaces the pre vious one but is not active even if the section is open If you scroll back so that even a small part of the previous section is visible its tool bar returns
281. w Selections in this menu allow you to position the windows or icons on your desktop in different ways Apple Displays the SOFTmax PRO for Lmax version registration informa tion ROM version if available from the instrument and copyright informa tion Additionally a number of buttons are available in different sections of the SOFTmax PRO for Lmax experiment and section tool bars Clicking these but tons accomplishes many of the same actions as choosing menu items SOFTmax PRO for Lmax User s Manual Chapter 3 SOFTmax PRO for Lmax Overview Keyboard Shortcuts Eert netmei set Ellipsis indicates that choosing this command will open a dialog box Menu Title Dimmed Sa SOFT max PRO Command Figure 3 14 SOFTmax PRO for Lmax Menus Windows version SOFTmax PRO for Lmax User s Manual 3 21 Chapter 3 SOFTmax PRO for Lmax Overview BQ Apple Menu About OFI max PRO File Menu File New Chrl h Open triti Close Crl at Save Litres 5 ave As Import E port Set Password Print Setup Print Notesi Print Ctrl P Exit 3 22 Ctrl U About SOFTmax PRO for Lmax Macintosh only Displays the SOFTmax PRO for Lmax version registration information ROM version if available from the instrument and copyright information If an instrument is connected to the Macintosh running SOFT max PRO for Lmax the instrument type will also be displayed You will n
282. wed and included in data reduction but do not affect data collection If you alter a limit to show less data you can always display the hidden data again by changing the limit MaxRLU The limit for the maximum value you wish to report Any values from the reading that are above this limit will not be shown and will be excluded from data reduction The default is 20 000 for RLU The MaxRLU reduction parameter can be used to exclude the non linear por tion of the reaction from data analysis This type of data reduction is most useful in reactions where the initial portion of the data is linear You might also adjust the End Time setting to remove a non linear portion after a certain time point in the reaction Using MaxRLU allows the use of the maximum number of linear points to calculate the slope of the line and thus to deter mine the rate for each well SOFTmax PRO for Lmax User s Manual Chapter 5 Displaying and Analyzing Data MinRLU The limit for the minimum value you wish to report Any values from the reading that are under this limit will not be shown and will be excluded from data reduction The default is 0 RLU To display negative kinetics the value should be set below 0 zero Lag Time Specifies how many initial data points are excluded from the calculation of Vmax Rate Lag Time truncates the data used in the calculation It does not prevent data from being collected The kinetic plots will not display the d
283. which must be se lected individually If the text is broken up by summaries this com mand will select the largest block of text that it can bounded by summaries In a Plate section this command will select all wells if one or more wells in the grid array has already been selected If no wells have been selected this command will be dimmed In a Group section this command will select all columns in the group table but will not include any summaries at the bottom of the group table summaries must be selected individually Delete Section Selection 3 CTRL Deletes the currently active sec tion s or experiment s A warning dialog box will appear asking if you really want to delete the section s or experiment s Duplicate Section Experiment Creates new section s or experiment s that are exactly like the active section s or experiment s and positions the duplicated selection s after the currently active section s The names 3 24 SOFTmax PRO for Lmax User s Manual Chapter 3 SOFTmax PRO for Lmax Overview of the new section s or experiment s will have the word Copy ap pended to their name s For example if you duplicate a section named Plate 1 the duplicate section will be called Plate 1Copy Subsequent duplications will append an incremented number after the name e g Plate 1Copy 2 For more information about this command and what is included when a section is duplicated see Du
284. which will change the way the summary appears to show Description Formula Result NOH The name assigned to a summary is important since you can refer ence the summary by name in other formulas building one upon another The summary name can be referenced even if it is hidden If you wish to see the formulas associated with summaries without calling up the Calculation dialog box for each one you can choose Show Formulas from the Group menu To see these formulas only briefly hold down the shift and control Ctrl keys while that Group section is active The summaries will expand to show the complete formula along with the title They will resume their previous appearance when you release the option key SOFTmax PRO for Lmax User s Manual 5 27 Chapter 5 Displaying and Analyzing Data BO 5 28 Summaries in Group sections can be moved horizontally if desired by drag ging them left or right When you position the pointer over a Group sum mary the cursor changes to show a double headed horizontal arrow Clicking once highlights the summary dragging and then releasing the summary leaves it in the position where the mouse button was released To have summaries line up with particular columns in the Group hold down the control Ctrl key while dragging the summary When you release the con trol Ctrl key the summary will snap back so that it begins flush to the left side of the column under which it was released Each sum
285. wish depending on the amount of memory available 8 12 SOFTmax PRO for Lmax User s Manual Appendix A SOFTmax PRO for Lmax Remote Command Language Haoa EE ISI ENTER TIT TT T dts Sn TTL TEL TIT es Se eS A 3 SOFTmax PRO for Lmax Responses 0000 0005 A 3 SOFTmax PRO for Lmax Commands eee A 4 Visual Basic Excel Macro Example A 7 MFC C Interface to SOFTmax PRO for Lmax Remote Commands that Return Values A 8 Appendix A SOFTmax PRO for Lmax Remote Command Language A 2 SOFTmax PRO for Lmax User s Manual 8S Appendix A SOFTmax PRO for Lmax Remote Command Language Introduction SOFTmax PRO for Lmax can be controlled by other Windows applications by means of a simple ASCII string based messaging system A command string such as Read or SaveAe myFile is sent to SOFTmax PRO for Lmax which then parses and executes the command Interprocess communication is implemented by using the Windows Send Message PostMessage system When SOFTmax PRO for Lmax starts up it uses RegisterWindowMessage to register a unique message tag Other applica tions can retrieve this message tag by using the same mechanism smaxMsg RegisterWindowMessage SOFTMaxPROMsq ASCII commands are sent to SOFTmax PRO for Lmax using the WM_SETTEXT message a special message used for interprocess Windows messages because it allows character strings from the address space of one p
286. x User s Manual Chapter 6 File Management and Printing 2 Display options chosen for the Plate section section Whether raw or reduced data will be exported depends upon the display options If raw data is displayed raw data will be exported if reduced data is displayed this reduced data will be exported Open the Plate section section from which you will be exporting data Click the Display button in the tool bar of the Plate section to access the Display dialog box Choose the elements of the display settings that match the data you would like to export raw or reduced data with or without number and so on When finished selecting display preferences click to close the dialog box Ras RLU T ith reduced number Figure 6 13 Display Dialog Box When exporting data as tab delimited ASCII text all reduced dis plays will be exported as numbers regardless of how the data is shown in SOFTmax PRO for Lmax Choosing Grayscale Ranged or Threshold will export the same information as the reduced number display Choosing with number will also not affect the exported data since only the raw number is exported when raw numbers are displayed and only the reduced number will be exported when reduced data is displayed 3 Choices made in the Export dialog box After choosing Export from the File menu a dialog box will appear asking you to choose which sections you wish to export You can choose to export Pl
287. xist with the instrument or the computer or a communica tion problem may exist between the instrument and the computer This error message means that no signal was recorded in a given well for an endpoint reading or at one or more timepoints for a given well for a kinetic run The instrument sent a placeholder NAN stands for Not A Number in lieu of a number and continued to collect data Depending on the type of assay s conducted it may be very easy or difficult to see which wells were not read During an endpoint reading it might be quite easy to spot a missed data point finding one during a long kinetics reading might be quite difficult however Follow the instructions below for the type of reading that was performed when the error occurred in order to assist you in finding lost data points Lost Data with an Endpoint Reading If you get such an error message during an endpoint reading try displaying raw signal and visibly inspect the plate data for missing read points Lost Data with a Kinetics Reading If the error occurs during a kinetics run access the Reduction dialog box click the Reduction button for the plate section that was read Choose Cus tom for the reduction and then click the formula button that appears zft to bring up the Calculation dialog box For the kinetic reduction enter the for mula numpointsread and then close the dialog box In the Plate section set the display click the Di
288. y Dialog Boxes 5 6 Display Dialog Box for Long Kinetics Threshold Display 5 10 Endpoint Data Displayed as Threshold with Reduced Number 5 10 Fast Kinetics Data Reduced Displayed as Ranged 00004 5 11 Endpoint Data Reduced Displayed as Gray Scale esses 5 11 Graph Options Dialog 5 13 Well Graph for a Single Fast Kinetics Microplate Well Raw Data 5 14 Well Graph for Three Fast Kinetic Wells of a Microplate Raw Data 5 14 Masked WCU m 5 15 Reduction Dialog Boxes for All Read Modes 0000 00 eee eee ee 5 17 Limits in Reduction Dialog Box 0 0 0 cece eee ee 5 19 Custom Selected Showing Formula Button Endpoint lusus 5 21 Calculation Dialog Box Endpoint 5 21 Cursor Positioned on a Line between Columns 0 000 000 0 ee 5 23 Dragging a Column Divider Line 0 0 cece eee eee 5 24 The Show Formulas Command in the Group Menu 204 5 25 Formulas Shown in Group Columns eeeeeeeeeeeeeeeeeees 5 25 Formula Button Activated and Column Formulas Shown 5 26 Column Formula Dialog DOX auam itum m ttt ci c rm aes 5 26 alc lation Dialog BOX wicceuctaxeineseau Arie Rr RUR E RERO RE 5 27 Graph Options Dialog Box 1 2 00 eee cen nent e ene ees 5 30 Standards and Unknowns Groups Created 1 0 0 0 0 0 c eese 5 31 GA nati
289. y been saved and no changes have been made Save As Saves a file with a new file name or into a new folder or subdirec tory See Chapter 6 Saving Files Manually for more information about using the Save As command for saving files Import Export Import brings tab delimited ASCII text file information into SOFTmax PRO for Lmax and Export creates a file of Plate section and or Group table section data All information from SOFTmax PRO for Lmax can be saved as tab delimited ASCII or text format which is suitable for importing into word processing or spreadsheet pro grams After choosing Import a dialog box will appear asking you to choose the file you wish to import Files to be imported must contain specific header information For more information on importing files into SOFTmax PRO for Lmax see Exporting Importing Information on page 6 16 SOFTmax PRO for Lmax User s Manual Chapter 3 SOFTmax PRO for Lmax Overview After choosing Export a dialog box will appear asking you to name the file and choose a location where the file should reside In addition the dialog box will ask you to choose which sections should be export ed you can choose to export all or some Plate or Group table sections The data that is exported out of the Plate section will be the same data currently displayed in that Plate section either raw or reduced data One additional parameter that affects export function is located
290. y default they put the data in the clipboard but optionally they will use the WM_COPYDATA mechanism to return data directly to the caller In order to have the SOFTmax PRO for Lmax response returned in this fash ion the commands must be sent using WM_COPYDATA so that SOFTmax PRO for Lmax knows what to send the response to SOFTmax PRO for Lmax will then reply with another WM_COPYDATA mes sage that contains the response MFC Example Code long cmdStr CString cmdStr smaxMsg RegisterWindowMessage 90FTMaxPROMsg cd dwData DWORD GetSafeHwnad pass our hwnd for return messages cd cbData cmdStr GetLength 1 add one for O ca pDatazcmdStr GetBuffer O SendMessage hwnd WM_COPYDATA smaxMsqg LPARAM amp cd Later O0FTmax PRO for Lmax will reply with a WM_COPYDATA message To get it in MFC create a routine like this POOL XXXXXXX OnCopyData CWPropWnd COPYDATASTRUCT pCopyDataStruct UINTmsg laa CString aStr msg lag RegisterWindowMessage SOFTMaxPROREeplyMag get tag if pCopyDataStruct gt dwData msglag aotrz pCopyDataStruct gt pData pCopyDataStruct gt cbData do whatever return TRUE else return FALSE message is for someone else A 8 SOFTmax PRO for Lmax User s Manual e Numerics 4 Parameter curve fit 5 39 A About SOFTmax PRO for Lmax 3 22 Absolute values 5 19 Active 1 11 3 7 3 8 3 9 3 20 3 22 3 23 3 24 3 26 3 30 Alerts 8 3 ANSI Cha
291. ymbols in the graph In the Plots field of this dialog box each of the plots that have been created for this Graph section are listed Choosing any of these plots provides an indi cation of the data to be used for the X and Y axis data points and error bars on the right side of this field Plots can be deleted by highlighting the plot name and clicking the Delete button Clicking on the New button opens the Plot X dialog box allowing you to create a new plot for the graph Exist ing plots can be edited by highlighting the plot name and clicking on the button which opens the Plot X dialog box containing all the informa tion for the selected plot When creating a new Graph section the Graph Options dialog box opens after pressing either or Cancel in the Plot X dialog The new Graph section is not created however until you press in the Graph Options dialog box SOFTmax PRO for Lmax User s Manual 5 29 Chapter 5 Displaying and Analyzing Data BO Name of the plot you can change this Chart options box Delete plot button New plot button Edit plot button Plot name and symbol Graph Options Fonts options Standard Curve Height pg onts m Sample Text h C2 Cluster bar Sample Text X and Y axis Phi is labels Pc Pitty io all Plot 1 B 4 Concentration Standards v MeanYaluem Standard m Errors Mo Erro X and Y error bar Y Errors Mo Error description C

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