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0112-0140_Rev _SoftMax Pro Software user guide.book

1. Read Type Endpoint Automix Off AutoCalibrate On Instrument Settings Specific to Absorbance Instruments Wavelengths One 405 nm Strips Read entire plate Instrument Settings Specific to Fluorescence Instruments Wavelengths One Excitation 485 Emission 538 Auto Cutoff Sensitivity 6 readings Automatic PMT Assay Plate Type 96 Well Standard clrbtm Wells to Read Read entire plate AutoRead Off Additional Protocol Information Standards in ug ml 0 0 1 0 25 0 5 0 75 1 0 and 1 4 each standard run in duplicate Standard Curve Fit Linear Blanks Plate blank run in duplicate SoftMax Pro Software User Guide 0112 0140 Rev A 89 90 6 Tutorials The following steps guide you through creating a protocol file for a quantitative protocol Reading an actual plate is not required as SoftMax Pro can generate simulated data STEP 3 DEFINE TEMPLATE The Template Editor is used to create a map of the contents of the microplate 1 10 1 12 Click the Template button in the Plate Section tool bar This opens the Template Editor showing all wells empty Click and drag the mouse to select the first two wells A1 and A2 Click the Group list on the Template Editor to see group selections Choose Blank The two selected wells in the template are now defined as the plate blank Select the two wells B1 and B2 and select Standards
2. 0112 0140 Rev A 31 32 4 Data Collection 4 2 6 4 2 7 4 2 8 abled manual cutoff selection When Auro Cutoffis enabled the instrument sets the cut offs based upon the wavelengths chosen for reading when Spectrum is selected as the read type however a manual setting for the emission monochromator is required with the default being no cutoff filter Determining a manual setting for a cutoff filter is based upon the known value of the Stokes shift which is the difference between the wavelengths of the excitation and emis sion maxima If the Stokes shift is small it may be advisable to choose an excitation wave length that is as far as possible from the emission maximum while still being capable of exciting the fluorophore Doing this causes less of the excited light to overlap the emission spectrum allowing better selection and quantitation of the emitted light See the individ ual instrument manuals for more information regarding cutoff filter settings SHAKE LMAX Il AND LMAX 11384 ONLY This function shakes the plate to mix the injected reagent with the solution in the micro plate You can specify a duration in seconds Shaking begins upon clicking the Read button INTEGRATION SPECTRAMAX L LMAX II AND LMAX 11384 ONLY Enter the amount of time to perform a single integration Dual Read type allows the choice of Special where you can specify a separate integration time for P and M injections For Kinet
3. LOCKING AND UNLOCKING SECTIONS Sections Notes Plate Group Graph CuvetteSet can be individually locked if the user has the necessary permissions enabled for their account Newly created sections are unlocked by default and can be locked and unlocked by clicking the lock unlock button on the right side of the tool bar for each section it is a toggle so the icon changes from locked to unlocked SoftMax Pro Software User Guide 0112 0140 Rev A 119 120 8 SoftMax Pro GxP Changing the lock setting for the Experiment changes the lock state of all sections within that Experiment Changing the lock setting for one or more sections within an Experiment causes the lock setting button for the Experiment to change to mixed reflecting the fact that the Experi ment contains both locked and unlocked sections Toggling the Experiment s lock setting button overrides any changes made to individual sections SoftMax Pro Software User Guide 0112 0140 Rev A 9 1 9 2 9 3 9 4 9 Robotics amp LIMS Integration INTRODUCTION In order to integrate Molecular Devices readers and SoftMax Pro software with robotic systems from other manufacturers or to automate the export of data from SoftMax Pro to a desired LIMS package SoftMax Pro supports a remote control interface Molecular Devices has tested the interface but does not provide technical support for specific inte gration needs Molecular Devices strongly re
4. 0112 0140 Rev A PathCheck Pathlength Correction SpectraMax Plus Plus384 190 340PC M2 M26 M5 MI J o cep ATA ERERUM AA a VR pe n 34 AUTOR lee eee ee be hae ua 39 Blanking Pre Read Plate s Cs odo Lex exe ox KK KK KK KK KK KK KK KK KK KK teg 40 AutoCalibrates cas Baca don ona Wa E RU bu Kel we Alaye Wa Wl o pic bs 40 Well Sean EGIGGE una octets oL les dab AA H bistir eaii 41 Assay Plate pie 42 Strips Wells to Rd reos eta v 43 Compound Source Flex Only sou eicd v oes ee KK KK KK KK KK KK KK KK KK K 43 Compound Transfer Flex Only c go so is dew KK KK KK KK KK KK KK KK KK KK KK 43 Trit rare Flex Only excretion As gee e J 44 Pipette Tips Layout Flex Only o WW rado KK KK KK KK KK KK KK 45 Compound amp Tip Columns Flex Only KK KK KK KK KK KK KK KK 45 Settling Time SpectraMax M5 and M5 Only KK RR KK KK 47 Speed Read Absorbance Only Endpoint Kinetic and Spectrum Scan Only 47 Column Wavelength Prot Lii eer EO kK KK KK E pte KK KK KK KK KK k 47 rie RGA SED DD cn gg tox reg Ca es 48 Template Editors Vp al Dti a e ph utes D n balan E n SOM K ey W Wale Kl a Ghote nt 48 Selecting Wells or Cuvettes in the Template Editor oooooooooomoo o 48 COI pd ou putet ta ii SAR berg CENE See ERU bord 49 Baranle oido Atout ua A Lea pt ashe asians EIU 51 SI Lo arri o en dne e els dotes d vs es vtt vet dou ede Gut esti ust ete 51 Clear ose ut AN SE DI AM P E MP LUE 51 zz St spa Mee 51 Blanking io
5. 090158 Append UserlD GxP ONLY Checking Append UserID adds the UserID for the user logged on to SoftMax Pro GxP to the Assigned Name or Protocol Name specified Append Date Checking Append Date adds the date to the Assigned Name or Protocol Name speci fied If Append Time is not also checked SoftMax Pro indexes the runs for that day start ing with 1 and increments with each successive run For example the first file AutoSaved on November 18th 2005 at 9 01am and 58 seconds would be named as Data 11 18 05 1 while the second file AutoSaved would be named Data 11 18 05 2 and so on Append Time Checking Append Time adds the time in the format hhmmss to the Assigned Name or Protocol Name specified For example the first file AutoSaved on November 18th 2005 at 9 01am and 58 seconds would be named as Data 090158 1 while the sec ond file AutoSaved would be named at the exact time the second read completed Data 090744 1 and so on Format Format options allow you to specify what type of file is AutoSaved Version 5 supports three file formats SoftMax Pro XML and Tab Delimited After each Plate section or CuvetteSet section is read the data is saved to a new file For protocols with more than one Plate section each subsequent AutoSave file contains all the data from the just completed plate read as well as all previous readings For example if you open a protocol containing two Plate section
6. 142 MinRFU 69 142 MinRLU 69 142 Multiple Plots 78 N NameCurSection 128 New NewNotes robotic command 128 NewPlate robotic command 128 Notes 128 Plate 128 New Columns 75 New Experiment 17 New Section 18 New Summary 19 Normal Display 142 Notes Section 19 O OD 142 Onset OD 142 Onset RFU 142 Onset RLU 142 Onset Time 67 142 OpenAssay Robotic Command 128 OpenCDrawer Robotic Command 128 OpenDrawer Robotic Command 129 Opening Files 106 OpenT Drawer Robotic Command 129 Optical Density 142 P Parallel Line Analysis 83 Parameters 80 PathCheck 143 Interfering Substances 38 Pathlength Correction 34 Photomultiplier Tube 143 Pipette Tips 143 Pipette Tips Layout 45 Plate Plate Type 143 Plate Editor 42 Plot X Dialog Box 77 PMT 143 Point to Point 143 Polarization 31 Port Speed 8 Pre Read Plate Blanking 40 Print 19 Printed Report 107 Programmer Notes 125 Protocol File 105 143 Protocols Menu 11 Q Quadratic 143 R Ranged Data Display 143 Raw Data 144 Read 15 27 Cuvette 57 Data Display During 59 Interval 144 Microplate 56 Mode 144 SoftMax Pro Software User Guide 0112 0140 Rev A Read Mode 30 Read Type 28 Reader Settings 7 Readings per Well 144 Recalculation Options 74 Reduction 20 Limits 144 Polarization Display 63 Raw Number 61 Reduced Data 144 Reduced Number 62 Reference 144 Reading 57 Ref 16 Remote Control 121 126 Rename Experiment 17 Reorder Ex
7. Interface to SoftMax Pro Remote Commands that Return Values 134 MEC Example Code iii KK oe KK KK d pana edid ipods kk kk d raf d 134 Appendix Glossary Telus d cese as doeet diy edet exte cob dx e eyed i We n n ya y ed 137 GON 6s scot ERA E Ira 147 SoftMax Pro Software User Guide 0112 0140 Rev A 1 1 1 Introduction SoftMax Pro software controls Molecular Devices spectrophotometers Absorbance Luminescence and Fluorescence microplate readers providing extensive data calculation and analysis capabilities under a GLP GMP work environment for pharmaceutical bio technology academic hospital and government customers Over 120 assay protocols are included to speed life science research and drug discovery assay development and screening Researchers can customize Experiment protocols ana lyze and display data and create meaningful reports The straightforward yet powerful programming capabilities of SoftMax Pro can further enhance any specialized data collec tion and analysis needs through custom assay development This industry leading software package is also widely integrated with industry leading robotics systems Optional Validation Tools are also available to reduce the time effort and cost of certifying and re validating MDC laboratory tools Two editions are available SoftMax Pro software and SoftMax Pro GxP Both support all MDC instruments including S ectraMax VersaMax VMax EMax Gemin
8. SoftMax Pro Software User Guide 0112 0140 Rev A 27 28 4 Data Collection 4 2 2 4 2 3 INSTRUMENT SETTINGS VARY FOR DIFFERENT MODELS Only the settings relevant to the instrument connected to the computer or chosen in Edit gt Preferences are available in the Plate gt Settings dialog box However this guide lists many of the common settings across instrument models READ TYPE The Read type specifies the spatial temporal and chromatic properties of the read Endpoint In an Endpoint read a reading of each microplate well is taken at a single or at multiple wavelengths Depending on the read type selected values can be reported as optical density 96 Trans mittance if this is selected in the Reduction dialog box relative fluorescence units RFU or relative luminescence units RLU For multiple reads per well see Section 5 3 2 Kinetic Reads and Section 4 2 16 Well Scan Editor LMax II and LMax 11384 and SpectraMax L Only Endpoint on an LMax II or SpectraMax L instrument line means gt A single integration of samples in 96 or 384 well plates gt Two injections if desired in selected wells of a 96 or 384 well plate only one injection is possible in 384 well plates with the LMax II instrument line A pre read of the microplate can be made before injection of buffer or reagent or reading of the samples Normal pre read uses the same integration time as sample read while Special pre read all
9. box This checkbox is shown only when two or more wavelengths are present in the reading Enabling this checkbox causes SoftMax Pro to use quadratic interpolation to calculate the interpolated values This results in the loss of one point at each end of the plot Prior to beginning the calculation SoftMax Pro determines the starting point by averaging the initial data points of the separate wavelengths For example if the first data points of the two wavelengths have time values of 0 3 and 0 5 seconds SoftMax Pro averages these and uses 0 4 seconds as its initial point for interpolation DATA MODE TRANSMITTANCE ABSORBANCE For Absorbance reads only you can choose whether to display absorbance data as 96 Transmittance or Absorbance OD Separate mathematical calculations are used for handling OD and T calculations for Pre read plate blanking PathCheck and Reference because OD calculations are per formed on a linear scale whereas 96T calculations are performed on a logarithmic scale However SoftMax Pro does not perform other calculations differently for absorbance and 96 T modes Because of this Molecular Devices recommends that T be used only for raw OD raw OD with Pre read plate blank subtraction or Path Check corrected raw OD readings Use caution when using 96T on reduced numbers or any readings that apply other calculations since the data may not be calculated correctly CUSTOM REDUCTION FORMULAS If the predefined reduction
10. commercially available microplates are of such high quality low and uniform background that pre reading is not necessary In fact it is less desirable because if there is a speck of dirt or a dust particle in any well the erroneous value is subtracted from the sample OD in the subsequent read PathCheck and Interfering Substances Any material that absorbs in the 900 nm 1000 nm spectral region could interfere with PathCheck measurements Fortunately there are few materials that do interfere at the concentrations typically used SoftMax Pro Software User Guide 0112 0140 Rev A 4 2 Instrument Settings 4 2 13 Turbidity is the most common interference if you can detect any turbidity in your sam ple you should not use PathCheck Turbidity elevates the 900 nm measurement more than the 1000 nm measurement and causes an erroneously low estimate of pathlength Using Cuvette Reference does not reliably correct for turbidity Samples that are highly colored in the upper visible spectrum may have absorbance extending into the near infrared NIR and can interfere with PathCheck Examples include Lowry assays molybdate based assays and samples containing hemoglobins or porphyrins In general if the sample is distinctly red or purple you should check for interference before using PathCheck To determine possible color interference do the following 1 Measure the OD at 900 nm and 1000 nm both measured with air reference 2
11. gt Open and navigate to the Tutorial folder 2 Open the file named Tutorial2 pda The data in this file was generated using the parameters you have just created in this protocol file 3 The display for each well that is defined by a group boundary shows a raw value with a reduced number 4 Thewavelength reduction formula is shown at the bottom of the display Wavelength Combination Lm1 The Kinetic reduction formula is shown at the top of the Plate section Max Min Click the Display button in the Plate section tool bar to open the Display dialog box Click the Reduced button oN o Gc Choose Grayscale from the drop down menu The dialog box updates to show the low and high limits for grayscale display values which are automatically set to the minimum and maximum data values 9 Click OK to dose the dialog box The data display in the Plate section updates to show the data represented by a heat map STEP 8 DATA ANALYSIS GROUP SECTIONS SoftMax Pro creates group sections automatically when you define groups in the Template Editor The content of Group sections depends on the wells that are assigned to the groups in the Template 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 Scroll down and click the small triangle on the tool bar of the Data group to open it and make it active T
12. to three decimal places The number of decimal places is config urable in the Calculation dialog box STEP 11 PRINT A REPORT 1 SoftMax Pro has very flexible report printing The default setting in SoftMax Pro is for all Experiment sections to be included in the printed report You can exclude sections from printing as described in Chapter 7 File Management amp Printing 2 Choose File gt Print to print the whole Experiment 3 Select the appropriate settings for your printer and then click Print or OK to print the Experiment report Congratulations You have completed this tutorial SoftMax Pro Software User Guide 0112 0140 Rev A 103 6 Tutorials 104 SoftMax Pro Software User Guide 0112 0140 Rev A 7 1 7 File Management amp Printing FILE CREATION AND MANAGEMENT SoftMax Pro uses two basic file types data files and protocol files DATA FILES Data files contain the raw data collected by your reader along with any data analysis you may have completed SoftMax Pro automatically creates data files based on the capabilities of the connected reader or if a reader is not present based on the reader type specified in Edit gt Preferences PROTOCOL FILES Protocol files are Experiment template files that contain microplate well layout assign ments and all other Reader configuration data but no microplate data A large number of predefined protocols are installed with SoftMax Pro Savin
13. 23 Trituration mixes the contents of the wells in either or both the compound source and assay plates by withdrawing and re pipetting from the wells You can set the volume of fluid to be withdrawn from the well and the number of times cycles it is pipetted back into that well For assay plates you must enter a value for the height at which the transfer occurs The height must take into account the amount of fluid currently in the well in order for the pipette to engage the fluid and allow the desired mixing The tip must be in the fluid in order to triturate PIPETTE TIPS LAYOUT FLEX ONLY Use the Pipette Tips Layout to specify where the pipette tips are physically loaded in the FlexStation instrument Molecular Devices recommends that you use a full rack of tips each time you perform a transfer of fluid for Flex reads since this prevents potentially serious errors from occurring when partial tip racks are used For example if you should mistakenly enable a pipetting function from a tip that is not present your samples will not receive the intended com pound If you use a partial rack of tips ensure that the layout described in Pipette Tips Layout matches the tips inserted in the tip drawer If you change the Assay Plate Type setting after making selections in the Pipette Tips Lay out tab for example from a 96 well plate to a 384 well plate the selection of pipette tips is reset to all tips The Assay Plate setting takes precedenc
14. 9 File Management amp Printing 105 File Protection 113 Filters 7 Fixed Weighting 83 Flex 29 139 FLIPR 139 Fluorescence 139 140 Polarization 30 Read Mode 30 Fluorescence Polarization 30 SoftMax Pro Software User Guide 0112 0140 Rev A Fluorophore 140 Font Face 19 Font Size 19 Font Style 19 Formulas Copying 76 Four Parameter Logistic 140 G Gain 140 G Factor 140 Good Curve Fit 84 Graph Options Dialog Box 78 Graph Options 24 Graph Section 24 Grating Factor 140 Grayscale Data Display 140 Group 140 Group Settings 23 Section 22 Adding or Editing a Summary in 76 Group Settings 49 Group Associated Blanks 54 GxP Admin 115 GxP Menu 118 H Hide 77 Replicates 77 Hierarchical Calculation 65 IC50 80 Import ImportTemplate Robotic Command 127 Information 110 Templates 111 Incubator 16 140 Injection and Delay 32 Injection Wells 33 Installation 3 Instrument Icon 141 Instrument Settings 15 Integration 32 Interleaved Display 141 Interpolate Raw Data 72 J Judging a Good Curve Fit 84 K Kinetic Well Graphs 63 L Lag Time 69 141 Lambda at Maximum 70 Lambda at Minimum 70 Large Display 141 Linear 141 Locating Protocol and Data Files 106 Log Logit 141 Luminescence 30 142 M Mask 21 65 142 Maximum 142 Time at 145 MaxOD 69 142 MaxRFU 69 SoftMax Pro Software User Guide 0112 0140 Rev A 149 MaxRLU 69 142 MEC C Commands 134 Minimum 142 MinOD 69
15. Assign button A total of eight samples will be assigned to columns in this template Continue to highlight subsequent columns of wells and enter the following dilution factors Column A3 through H4 0 05 Column A5 through H5 0 5 Column A6 through H6 1 Column A7 through H7 5 Column A8 through H9 10 Column A10 through H10 100 The template is now complete Click OK to accept the entries and close the Template Editor The outlines of the groups are now shown in the Plate section An associated Group section is created each time a group is created in a template so we now have a Group section called Data STEP 4 SET REDUCTION PARAMETERS 1 Click the Reduction button in the Plate section tool bar to open the Reduction dialog box The default setting for Wavelength Combination Lm1 is fine For Kinetic Reduction change the setting to Max Min SoftMax Pro Software User Guide 0112 0140 Rev A 6 2 Tutorial 2 EC 50 Protocol Tutorial Flex only 6 2 5 6 2 6 6 2 7 6 7 Set Limits as follows Min RFU 0 Max RFU 50000 Lag Time 0 End Time 200 Baseline Options Zero Baseline 3 Pts Smoothing Moving Average 1 point no smoothing Click OK to close the dialog box STEP 5 SET DISPLAY PARAMETERS 1 2 4 Click the Display button to open the Display dialog box The default display for Flex readings is raw RFU This tutorial example shows a reduced number so check the With reduced nu
16. If the command is the Read remote command the status can be periodically checked by sending ReturnStatus remote command To parse the response from SoftMax Pro JAAA FARRER EE KEE RR ERKEK RR AA IN kek ok Sise RK AAA LAA KAA SEEN ALAR IA OnCopyData Message handler for WM_COPYDATA 1 Receive data from SoftMax Pro 2 Retrieve the response 3 If the response is for ReturnStatus parse it accordingly FEAR AAR A AOKI SESI AA IOI IR ACE ION AK AAR AALS ARAN AAI BOOL CMySampleClass OnCopyData CWnd pWnd COPYDATASTRUCT pCopyDataStruct CString aStr char temp char pCopyDataStruct gt lpData aStr SetString temp pCopyDataStruct gt cbData 1 aStr n if aStr Find ReturnStatus 1 SoftMax Pro Software User Guide 0112 0140 Rev A 135 9 Robotics amp LIMS Integration CheckInstrumentState aStr Wor do your own parsing return TRUE To add a short pause between remote commands const DWORD kMaxTimeOut 1000 1 second for inti 0 i lt noOfCmdsToSend i SendCmdToSMP cmdStr Put 1 second buffer DWORD start GetTickCount while GetTickCount start lt kMaxTimeOut 136 SoftMax Pro Software User Guide 0112 0140 Rev A A 1 A Appendix GLOSSARY OF TERMS Transmittance T Transmittance is the ratio of transmitted light to the incident light for absorbance read ings T Iy l T 1007 where Ip is incident light
17. Pro Software User Guide 0112 0140 Rev A 4 4 Reading a Microplate or Cuvette 4 4 2 DATA COLLECTION FROM A CUVETTE Two types of data collection are possible using a cuvette a reference and a sample reading You can start a reading at any time after defining instrument settings It is not necessary to define groups and assign cuvettes within the Template Editor first but you can use the Template Editor to create the appropriate number of cuvettes in the section Alternatively it is easy to create a small number of cuvettes one at a time with the Cuvette gt New Cuvette command Instrument settings must be the same for all cuvettes in a CuvetteSet section Sample Reading You can highlight any cuvette in the CuvetteSet section and choose to read it The Read button changes to Stop allowing you to terminate a reading if desired If you select more than one cuvette the software starts with the leftmost selected cuvette that does not contain sample read data CuvetteSet sections have a limit of 96 cuvettes If this limit is reached create a new Cuvet teSet section Reference Reading As in a spectrophotometer taking a reference reading can be done either on air or using a cuvette containing the buffer of your sample The reference may be read before or after reading samples If no reference has been taken in a CuvetteSet section before samples were read and you then read a reference the following occurs gt The r
18. Reference with Path Check is different from a reference reading of a cuvette in a CuvetteSet section by clicking the Ref button in the CuvetteSet section tool bar The cuvette reference used for PathCheck calculations measurements at 900 nm and 1000 nm does not produce data that can be viewed in a CuvetteSet section and is used only with data in microplates not cuvettes Once you have read a plate with PathCheck turned on PathCheck information is stored permanently in the data file You have the option of applying or not applying PathCheck to the absorbance values as you choose If you did not have PathCheck turned on during the plate read you cannot apply PathCheck after the read PathCheck on SpectraMax 190 and 340PC PathCheck on the SpectraMax190 and 340PC uses the water constant only Be aware that if your sample matrix contains an organic solvent such as ethanol or methanol the esti mated pathlengths will be lower than the true values and PathCheck normalized values will be higher than the corresponding 1 cm values see discussion above gt Open Plate gt Settings and select Patb Check gt Check the PathCheck option gt Ifyou have determined a plate background constant for your microplate enter it in the designated field see Use Plate Background Constant on page 38 Once you have read a plate with PathCheck turned on PathCheck information is stored permanently in the data file You have the option of applyi
19. Removing a Password 5 idad fade phen deg Pp a ekla a ak ORAE IQ ERES Suspending a Password ui audaciae Ene KK KK KK KK aC Ace rara ce dard SoftMax Pro GxP Solution OVERVIEW ces Vas da fete od tl ie io inl cbr dud ea e Debbie d How the Programs Work Together 223 e KK o KK KK KK KK KK KK KK KK KK KK KK GxP en GU JJN EMIIZMMIMMJMJMMDMMAMM MN N NTMMDMDMIDNEMDMN HR DDB NIA Using SoftMaz Pro GIxD ous kaye Ra as Mace da hale a e w k l User Log On edere mt cere beste e e ec mi U UZaoggadE N TM PGS aute Cola Most obe acre ud ehe IA GxP Menus aie A VADER ee a E RP Vg PER be rte Ran Statemellts cod edet A i eg edat ee reet ene Locking and Unlocking Sections su exar vx sho a a KK KK KK KK KK Robotics amp LIMS Integration Intfoduction 3 eo ER Ted a Seow Eee aces Sample Source Code and Applications iaa d Ee KK KK KK KK KK PEOPLE Remote Control AAA REFS DUE A MEE SS Send Receive Remote CommandSs JW kk kk kK KK KK KK KK KK KK KK KK KK KK KK The WM SETTEXT Approach tic de sk Aka ke dl e The WM_COPYDATA Approach 5o a voted kK KK KK KK KK KK KK ada SoftMax Pro Software User Guide 0112 0140 Rev A vi Summarizing Remote Commanding KK KK KK KK KK KK KK EVER 124 Programmer Notes esee n 125 Remote Control in SoftMax Pro GxP kk kk kk KK KK KK KK es 126 SoftMax Pro Commands daxa iee cee adi es 126 Visual Basic Excel Macro Example J KK KK KK KK KK KK KK KK KK KK ERR 132 MEC C
20. SoftMax Pro software DATA DISPLAY THE DISPLAY DIALOG BOX At any time in an active Plate section you can click the Display button or select Plate gt Display to change how the data is presented Choices available in this dialog box depend on whether a section is active which instru ment is chosen in the Preferences or is connected to the computer and which read type you have chosen for example the choice for Plots is not shown if Endpoint is selected Raw Selecting Raw displays the default data type for the selected read type 5 Endpoint Raw absorbance fluorescence or luminescence values 5 Kinetic The change in raw OD RFU RLU values over time displayed as a plot 5 Spectrum Raw OD RFU RLU values for the range of wavelengths displayed as a plot 5 Well Scan Raw OD RFU RLU values as shades of gray gt Flex Raw RFU RLU values over time displayed as a plot SoftMax Pro Software User Guide 0112 0140 Rev A 61 62 5 Data Analysis Reduced The reduced number is a combination of plate blank subtraction in the Plate section wavelength reduction and if applicable a Spectrum Well Scan Kinetic or Flex reduction The reduced number is reported in the Group section when a template has been defined Number To view by reduced number alone select Number To see a reduced number as well as another type of display check With reduced num ber Threshold Threshold shows the raw data as a plus for val
21. and the Instrument 4 Compound Source 43 138 Compound Transfer 43 Computer System Requirements 3 Connect Instrument 3 Copy Formulas 76 Summaries 76 Template 21 Copy and Paste Column 75 Copy and Paste Data 21 Create Multiple Plots 78 Summary 76 Create New Files 106 Cross Platform Files 106 SoftMax Pro Software User Guide 0112 0140 Rev A 147 Curve Fit 79 Cubic Spline 138 Exponential 139 Five Parameter Logistic 80 Four Parameter Logistic 140 Judging a Good Curve Fit 84 Linear 141 Log Logit 141 Minimum Number of Standards 82 Options 83 Parallel Line Analysis 83 Quadratic 143 Semi Log 144 Weighting 83 Custom Reduction Formulas 72 Cutoff 31 138 Cuvette Reading 57 CuvetteSet Section 23 138 D Data Analysis 61 Data Display During a Reading 59 Data Reduction 65 Default Protocol 105 Delete Experiment 17 Delete Group 23 Delete Section 18 Display Dialog Box 61 Interleaved 141 Normal 142 Dual Read 29 Duplicate Experiment 17 Duplicate Group 23 Duplicate Section 18 E EC50 80 Edit Plate Type 42 Edit Summary 19 20 76 Emission Spectrum Scan 139 End Wavelength 70 End Time 69 139 Endpoint 28 Enter Registration Information 4 Error Bars 79 E Statements 118 Excitation Spectrum Scan 139 Experiment 16 Exponential 139 Export Format 8 Graph 24 113 Information 107 Template 111 ExportAs Robotic Command 127 F Fast Kinetic 29 File Locating Protocol and Data Files 106 Management 105 Prefix
22. as well as the usual plate reading parameters Dual Read SpectraMax L and LMax 11384 only Also an Endpoint reading Dual Read is designed to perform two separate integra tions one after each of two possible injections in any or all wells of a 96 well plate or 384 well plate LMax 11384 can inject twice in 96 well plates only Integration times for both readings are the same unless otherwise specified You can program a delay after each injection and you can read all or only some of the wells in the plate Pre reading a microplate is not possible with this read type Default values reported are relative luminescence units RLU The default reduced value is Lm1 Fast Kinetic SpectraMax L LMax II and LMax 11384 Only Fast Kinetic performs repeated readings of one or more wells of a 96 or 384 well microplate up to a 100 point maximum integration All readings of a single well are made before the next well is read Pre reading a microplate is not possible in Fast Kinetic reads One or two injections can be made in each well of a 96 well microplate and 384 well plate for the SpectraMax L at the start of the reading Integration time can be set from 0 1 to 100 seconds Default data reductions are VMax per Sec VMax Time to VMax or Onset Time SoftMax Pro Software User Guide 0112 0140 Rev A 29 30 4 Data Collection 4 2 4 READ MODE The Read mode is the form of detection used by the instrument All read ty
23. calculate this reduction SoftMax Pro determines the Kinetic point within the reduction limits that has the maximum signal level OD or 96T and divides it by 2 to get the 1 2 Maximum value Then it finds the time value at the 1 2 Maximum Slope This setting determines the slope of the combined plot i e the slope of the line using linear regression after the wavelength combination reduction It is calculated using all visible time points in the reduction window Slope is the same as VMax Rate when VMax Rate is set to the same number of points as the run but is different if you have modified VMax Points Area Under Curve This reduction estimates the area under the curve as defined by the data plots 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 coordinates of the data points The areas defined by each of the trapezoids are then computed and summed Kinetic Limits Limits define the data that are viewed and included in data reduction If you alter a limit to show less data you can always display the excluded data again by changing the limit Unless you have selected the Absolute Values option the display of OD RFU RLU values is relative to the first point measured for each well e the first point is always set to 0 0 and all other data are shifted accordingly The Absolute Values option allows you to see the unshifted data see below
24. certain instruments when reading using Fluorescence and Time Resolved Fluores cence you must also choose whether to read excitation at a fixed wavelength and scan emission wavelengths or vice versa within the range of the instrument 250 nm 850 nm A procedure to optimize excitation and emission wavelengths for a given proto col is outlined in the instrument manuals for the readers that support this function Please see those manuals for more information For Endpoint or Kinetic Readings gt Choose the number of wavelengths to read 5 Specify the wavelengths from the entries in the lists or type any wavelength within the available range of the instrument over an existing setting The lists show only common monochromator settings For Spectrum Readings Choose a Start and a Stop wavelength and a wavelength step for each increment between reads The minimum selectable increment is 1 nm Cutoffs The choices in this portion of the Wavelengths setting depend upon the selections for read type Endpoint Kinetic Spectrum or Well Scan and read mode Fluorescence or Time Resolved Fluorescence The term cutoff refers to filters used to block unwanted residual excitation light and minimize background interference No emission cutoff filter is used when Luminescence is selected With other read types choices are to enable or disable Auto Cutoff with specific filter settings available if it is dis SoftMax Pro Software User Guide
25. command can be sent to log on the user through the robotic interface SOFTMAX PRO COMMANDS SoftMax Pro commands are single words with no spaces If parameters are required with a command a colon is used between the command and the parameters Commands may be sent using either the WM SETTEXT or WM COPYDATA approach Completion messages or data are returned directly when commands are sent using the WM COPYDATA method Completion messages or data are placed in the system clipboard when commands are sent using the WM SETTEXT method AppendCurSectionTitle Append text to the title of the current Plate section CDrawer FlexStation only This command returns the status Open or Closed of the compound or reagent plate drawer Close Closes the current document If the data has not been saved the document is closed anyway with no user warning CloseCDrawer FlexStation only Tell SoftMax Pro to close the compound drawer CloseDrawer Tell SoftMax Pro to close the instrument drawer CloseTDrawer FlexStation only Tell SoftMax Pro to close the tips drawer Copy This is equivalent to pressing CTRL C or selecting Edit gt Copy If a Plate section has just been read or is the only selected section the plate data is copied to the clipboard The data copied follows the display settings for the current plate normally Raw Data SoftMax Pro Software User Guide 0112 0140 Rev A 9 6 SoftMax Pro Commands Oth
26. command or click the Graph button on the tool bar The data in the enlarged Well Graph update to show new data points as they are received from the instrument Other choices and settings within SoftMax Pro affect the way in which data is displayed the type of reduction specific time settings and so on See Chapter 5 for more on data analysis SoftMax Pro Software User Guide 0112 0140 Rev A 59 60 4 Data Collection SoftMax Pro Software User Guide 0112 0140 Rev A 5 1 5 2 5 2 1 5 Data Analysis INTRODUCTION Any SoftMax Pro data file has a minimum of one Plate section or one CuvetteSet section Most files also have an assigned plate template and associated Group Graph and Notes sections However depending upon the protocol and how you want to report the data only some of these sections may be present in every file Some simple protocols such as reading the absorbance of proteins at 280 nm in a cuvette or in a microplate with PathCheck allow you to calculate concentration in the Plate or CuvetteSet sections In such an example Group and Graph sections are necessary only if you wish to display the data in tables or graphically Many common protocols consist of data acquired in a Plate section or CuvetteSet section Standards and unknowns are defined in a template Unknowns are then interpolated from a standard curve The following diagram shows the relationship between the different sections in
27. for the graph Existing plots can be edited by highlighting the plot name and clicking the Edit button which opens the Plot dialog box containing all the information for the selected plot Creating Multiple Plots You can 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 multiple dilution series of an unknown to graph together a series of patient samples or to plot several controls that have been run over time This example shows how three groups all of which are Standards can be plotted on the same graph 1 Groups are created in the Template Editor For this example four groups are created three for standards Standard1 Standard2 and Standard3 and one for Unknowns Unknownl 2 The associated Group sections are created using the Standards column format that by default generates columns for concentration and OD RFU RLU values 3 Create a new Graph section with Experiment gt New Graph Create a new plot named Standard 1 Click OK to open the Graph Options dialog box 4 Select Standard for the group to be plotted the concentration of Standard1 for the X axis and values of Standardl for the Y axis 5 Add a second plot to this graph by clicking New in the Graph Options dialog box to open the Plot 2 dialog box Change the second plot name to Standard 2 choose Standard2 for the group and change the X and Y axes to show con
28. for both raw and reduced views of the Well Graph SoftMax Pro Software User Guide 0112 0140 Rev A 5 3 Data Reduction 5 2 3 5 3 Graph Options Opens the Well Graph Options dialog box which allows you to enable or disable con nected points or plotted symbols on the graph X Axis Settings Y Axis Settings Opens the Well Graph Axis Settings dialog allowing you to set Auto range parameters choose the minimum and maximum values for the selected axis cause the axis to be auto ranged add or remove gridlines and add or remove axis tick marks Only the Well Graph being viewed is affected by changes in these dialog boxes MASKING WELLS OR CUVETTES Masking is the process of excluding outliers in certain wells or cuvettes from data reduc tion calculations To mask wells or cuvettes 1 Select the wells or cuvettes to be masked 2 Click the Mask button or select Plate gt Mask or Cuvette gt Mask Masking can be used as a what if tool 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 suppresses the blanking function while unmasking them enables it again DATA REDUCTION Because this chapter deals with more than one instrument model not all of the informa tion found here may apply to your instrument The reduction process in SoftMax Pro is based on formulas that reduce the raw data to show a single number for each w
29. for plates that work best with custom settings For both 96 and 384 well plates the Standard plate should be used unless the microplate you are using is listed For Luminescence reads white opaque microplates yield optimum results for most appli cations The light reflection with white microplates provides a high signal to noise ratio with very low crosstalk from adjacent plate positions Clear bottom microplates also give low crosstalk values although the signal efficiency is reduced in comparison to white microplates Transparent sample plates cannot be used with the LMax II series instru ments due to extremely high crosstalk The reading efficiency and the signal to noise ratio are lower in black plates because of the decreased light reflection so black plates are not usually recommended for luminescence Edit Plate Type Gemini SpectraMax M2 M25 M5 M5 FlexStation Only You can customize the plate type with Plate Edit Plate Type The Plate Type Editor includes five fields for entering in millimeters gt The distance from the left edge of the plate to the center of well A1 5 The distance from the top edge of the plate to the center of well A1 gt The horizontal distance between wells SoftMax Pro Software User Guide 0112 0140 Rev A 4 2 Instrument Settings 4 2 18 4 2 19 4 2 20 gt The vertical distance between wells gt The well diameter The distance from the left edge of the plate to t
30. formulas do not meet your needs you can create one or more different reduction formulas for any read type Endpoint Kinetic Spectrum or Well Scan Choosing Custom from any of the menus or dialog boxes displays a Formula button with which you can open a Formula dialog box SoftMax Pro Software User Guide 0112 0140 Rev A 5 3 Data Reduction Examples of some formulas that can be used when combining multiple wavelengths in a Formula dialog box are given in the table below Table 5 1 Wavelength Reduction Formula Examples 2 3 4 to 61 Lm1 Lm2 Lm1 Lm2 Lm3 Lm1 Lm2 Lmn Lm1 Lm2 Lm1 Lm3 Lm2 Lm3 Lm1 Lm6 Lm2 Lm5 Lm3 Lm4 ILm1 Lm2 ILm1 Lm3 ILm1 Lmn ILm1 Lm2 ILm1 Lm3 ILm1 Lmn Log10 Lm1 Lm2 Log10 Lm1 Lm3 Log10 Lm1 Lmn Pathlength Pathlength IPathlength ILmx constant For example Lm1 1 44 for quantitating a polyclonal antibody by measuring the absorbance at A280 with PathCheck on Average allvalues Averages together the optical densities for multiple readings at the same wavelength for example if you read the well six times at 280 nm Min allvalues Reports the minimum OD RFU RLU recorded for multiple wavelength readings in each well Max lallvalues Reports the maximum OD RFU RLU recorded for multiple wavelength readings in each well If lLmz lt A makeerr Reports low for any well w
31. from the Group list The selected wells B1 and B2 now show the default first sample name for the Standards group St01 The area in the top center of the Template Editor is now active showing the default values for concentration and units In this tutorial the concentration for the first Standard should be set to 0 pg ml If a value other than 0 is shown in the Concentration field highlight the existing value and enter 0 Click the Assign button or press either the Tab or Return key on the keyboard to assign this concentration to the selected wells You can check the concentrations you have assigned to the template by pressing and holding the Ctrl Shift keys Select wells C1 and C2 Standards is still showing as the default group name but a new sample descriptor is created automatically St02 so all you need to do is assign a concentration value of 0 1 pg ml to these wells After entering the value click the Assign button A total of seven standards will be assigned in duplicate in this template Select wells D1 D2 through G1 G2 and click the Series button In the Series dialog box leave the default values for the sample descriptor Std03 starting from the top and the concentration in pg ml Enter 0 25 for the starting value choose the Step by operator and enter 0 25 for the increment of the series Click OK These choices assign concentrations of 0 25 0 5 0 75 and 1 0 pg ml respectively to the selected wells which you
32. have not been added to the User Accounts file may use SoftMax Pro GxP as a Guest if the administrator has enabled Guest access to the User Accounts file using GxP Admin Guests can open view and print any existing Experiment data files or proto cols They are not able to modify or save changes to any existing Experiment data file or protocol FILES Files created in SoftMax Pro GxP use the file name extensions epr for protocol files and eda for data files and can be opened only by SoftMax Pro GxP Files saved from other editions of SoftMax Pro ppr pda dat pro etc can be opened by the SoftMax Pro GxP but have no associated audit trail SoftMax Pro Software User Guide 0112 0140 Rev A 117 118 8 SoftMax Pro GxP 8 3 3 8 3 4 GXP MENU The GxP menu in SoftMax Pro GxP provides access to most of the security and GxP management features in SoftMax Pro GxP The available commands depend on the user s permissions Reset User Accounts Displays a dialog box in which the user or administrator links to an existing User Accounts file The link can be made using a direct file path or a TCP IP connection see the GxP Admin User Guide for more information on configuring connections to User Accounts files Account Info Displays the permissions available for the currently logged on user Log On Prompts a user to enter a user ID and password All users must log on if they want to read new microplates or perform any d
33. is selected It is not possible to copy and paste the data from selected wells only It is possible however to copy individual cuvettes or groups of cuvettes and paste these into other CuvetteSet sections If you are copying cuvette data and the target CuvetteSet section does not contain the appropriate cuvettes for the data that is to be pasted new cuvettes are created automatically Note that pasting cuvette data for only some cuvettes clears the existing data from all other cuvettes in that section 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 are updated to match Copy and Paste Template There are two ways to copy and paste a template depending on whether or not you want to keep the same group names SoftMax Pro Software User Guide 0112 0140 Rev A 21 22 3 SoftMax Pro Interface 3 6 5 Keep Group Names Plate Copy Template copies the template from the active Plate section to the Clip board so that it can be pasted into a different Plate section using Plate Paste Tem plate either in the same or in a different Experiment or into a Plate section in another SoftMax Pro file Pasting a template into an Experiment where the groups from the source template do not exist creates the groups Existing groups of the same name are not changed by the pasting process For example if the source Experiment contai
34. name for the Controls group Co01 Do the same for wells D9 through F9 assigning the default second sample name Co02 to these wells The last items to be added to the template are samples with dilution To do this you need to create a new group Choose the wells of the template that will contain these samples A11 through G12 Click the Group menu and choose New In the Name field enter Sam w dil and choose Unknowns dilution for the Column Format Choose ug ml in the Sample Descriptor Units field Click OK The samples with dilution need to be changed to be part of a series however since we really want to include six different samples with replicates Highlight wells A11 through G12 again and click the Series button Leave the default direction from the top down and leave the default operator of divided by 1 Enter 10 for the starting value SoftMax Pro Software User Guide 0112 0140 Rev A 91 92 6 Tutorials 25 The template is now complete 26 Click OK to accept the entries and close the Template Editor The outlines of the groups you have defined in the Template Editor are now shown in the Plate section 27 An associated Group section is created each time a sample group is created in the template so we now have Group sections named Standards Unknowns Controls and Sam w dil STEP 4 SET REDUCTION PARAMETERS 1 4 Click the Reduction button in the Plate section tool bar to open the Reduction
35. regres sions to calculate the maximum rate VMax or the time segment used to calculate the Time to VMax Well Scan Some applications involve the detection of whole cells in large area tissue culture plates Well Scan reads can be used with such microplates to allow maximum surface area detec tion in whole cell protocols Since many cell lines tend to grow as clumps or in the corners of microplate wells you can choose from several patterns and define the number of points to be scanned to work best with your particular application SoftMax Pro Software User Guide 0112 0140 Rev A Symbols Baseline with Multiplier 71 A Absolute Values 69 Absorbance 30 137 Add Statements 118 Analyst 111 Anisotropy 31 Append Date 10 Time 10 Area Under Curve 68 70 137 Assay Plate Type 42 Assign Area 51 Author Name 137 AutoCalibrate 40 137 Automation 121 Automix 39 138 Autoprint 11 AutoRead 48 138 AutoSave 9 Autosize 77 B Background Constant Subtraction 37 Baseline Options 71 Baud 138 Blanking 53 Considerations 37 Group Associated Blanks 54 Plate Blanks 53 Pre Read Plate Blank 40 Index Bottom Read 138 C Calculation Hierarchical 65 Calibration 59 Change Section Order 107 Text Format 19 107 Close 126 Close Drawer 16 CloseCDrawer Remote Command 126 CloseDrawer Remote Command 126 CloseTDrawer Remote Command 126 127 Column Format 49 Column Wavelength Priority 47 Communication Between PC
36. source to each well cho sen to receive that transfer You can set up to 30 pL for a 384 well plate and up to 200 pL for a 96 well plate Time Point Determines the time after the start of the run when the fluid is scheduled to be dis pensed Minimum time This is the minimum time required before a pipetting event can occur The Time Point setting above cannot be set smaller than this value The minimum time value is cumulative it is not an interval between pipetting events The value is the minimum number of seconds of elapsed time from the begin ning of the read taking into account gt The mechanical speed of the pipetter head gt The time needed to aspirate and dispense fluids gt Trituration gt Automixing The minimum time for the second pipetting event depends on when the first pipetting event occurred The calculation for the second event starts at the end of the first event and adds to that the total time necessary to perform actions prior to aspirating new fluids from the compound plate and dispense them into the assay plate Compound Transfer settings work in conjunction with the Compound amp Tip Columns setting described below TRITURATE FLEX ONLY The available Triturate choices are derived from the number of compound transfers speci fied If no transfers are enabled no triturate settings are enabled SoftMax Pro Software User Guide 0112 0140 Rev A 4 2 Instrument Settings 4 2 22 4 2
37. tem SoftMax Pro Software User Guide 0112 0140 Rev A A 1 Glossary of Terms perature for the microplate chamber on instruments that have temperature control capability Instrument Icon Located in the Status Bar the instrument icon shows the status of the connection between the computer and the instrument If the icon has a through it the connection is not functioning or no instrument is connected if the instrument icon appears without a the connection is working properly Interleaved Display A display choice in which 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 This display is most useful when the 384 well plate comprises four daughter plates of 96 wells each Kinetic During Kinetic readings data is collected over time with multiple readings made at regu lar intervals measured in seconds The values calculated based on raw Kinetic data are VMax VMax per Sec Time to VMax and Onset Time Kinetic readings can be single or multiple wavelength readings up to six if using a SpectraMax instrument other than the Gemini or Gemini XS which have a maximum of four or the FlexStation which also has four Lag Time Lag time in a Kinetic protocol is the period of very slow growth microorganisms or the rate of reaction that may precede the rapid linear phase
38. that 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 useful for applications including coagulation chemistry where the changing concentration of the reagents does not change VMax but rather changes 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 reach a specified OD or RFU RLU onset OD RFU FLU This elapsed time data is useful for cascade reactions including clot formation endotoxin testing for example and clot lysis applications where the change in SoftMax Pro Software User Guide 0112 0140 Rev A 67 68 5 Data Analysis reagent concentration does not affect the maximum optical density change but changes the time required for the reaction to reach completion Time at Minimum This setting reports the time at the minimum OD RFU RLU or T that falls within the reduction limits Time at Maximum This setting reports the time at the maximum OD RFU RLU or 96T that falls within the reduction limits Time at Y Maximum This setting reports the time at the half of the maximum OD RFU RLU or T that falls within the reduction limits To
39. the Assay Plate Type COMPOUND TRANSFER FLEX ONLY Use the settings in this section to specify the volumes and rates of compound transfers Assay Plate Fluid The entry in the Znitial Volume field should be equal to the largest initial volume for any well in the plate SoftMax Pro assumes all wells hold the same initial volume This setting is used to compute the total volume in each well after all fluids have been dispensed to check for potential fluid overflow Transfers You may enable up to three compound transfers during a reading or choose none When transfers are enabled numbered transfer buttons are displayed to enable you to set the parameters for that transfer SoftMax Pro Software User Guide 0112 0140 Rev A 43 44 4 Data Collection 4 2 21 Pipette Height Determines the amount of fluid in microliters measured from the bottom of the microplate well above which the tip of the pipette remains You can set up to 130 pL for a 384 well plate and up to 300 pL for a 96 well plate Note that subsequent transfers require that you calculate the amount of fluid added Rate from 1 to 8 Determines the rate at which the fluid is pipetted into the well A setting of 1 is equal to 26 microliters per second and each subsequent number increases in increments of 26 microliters A setting of 3 to 4 may help to minimize cell damage and splashing Volume Determines the volume of material that is pipetted from the
40. the Template button in a Plate section SoftMax Pro Software User Guide 0112 0140 Rev A 5 4 Group and Graph Sections Group sections can also be created by duplicating existing Group sections within the Experiment file itself which is often an easier way to replicate Groups if many custom col umns or Summary objects have been added Reduced numbers from the Plate section are displayed in the Values column of the Group section tables by default Custom reduction formulas require the use of accessors and operators that are understood by SoftMax Pro For a list of these accessors and operators see the Formula Reference Guide Adding Columns Click the New Column button or use Group New Column to add a column to the active Group table 5 If no columns are highlighted the column is added at the end of the group 5 If you select a column first the new column is added to the right of the highlighted column New columns can contain references to other columns either in the current section or in a different one For example if you wanted to subtract the mean values in one group from those in another you could create a new column in either group to do this If the two groups were 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 sub tract the mean values in Group 2 from those in Group 1 The column formula
41. the instrument via the clipboard Wait for a message for up to mcsngMsg Timeout seconds before giving up Dim sngStart As Single Dim Clipboard As DataObject Set Clipboard New DataObject Clipboard Clear clear the clipboard for the data that we are about to get SendMsgToPRO strMessage sngStart Timer get the time that we started looking at clipboard wait for an answer for up to mcsngMsgTimeout seconds Do While Timer lt sngStart mcsngMsgTimeout GetMsgFromPro Clipboard GetText get text from the clipboard Clipboard GetFromClipboard Retrieve text from clipboard GetMsgFromPro Clipboard GetText 1 If Len GetMsgFromPro 0 Then if we got message back from the clipboard then exit with it in hand Clipboard Clear Exit Do End If DoEvents Yield to other processes while we wait SoftMax Pro Software User Guide 0112 0140 Rev A 133 134 9 Robotics amp LIMS Integration 9 8 9 8 1 make sure that we didn t just span midnight with our timer check If Timer sngStart Then Current timer is less than start time so reset the starting time In the worst case this routine might take twice as long to timeout if the message fetch spanned midnight This is unlikely and even if it does occur it doesn t hurt anything sngStart Timer End If Loop End Function MFC C INTERFACE TO SOFTMAX PRO REMOTE COMMANDS THAT RETURN VALUES Some SoftMax Pro software IPC commands return val
42. the top of the Experiment data file Statements Menu Once a statement has been created the Statements menu is displayed when the statement is active it is similar to the other section menus such as Plate Notes etc The Statements menu allows the logged in user to create or modify statements in the current Experiment data file Add Statement Allows the user to create a new statement if that permission is available to the user Edit Statement Opens the Electronic Statement Setup dialog box allowing the current user to change the title and text of the active statement Remove All Signatures If a statement has been signed a user with this permission can remove all signatures from the active statement When all signatures have been removed the file is editable again Copy Statement Copies the contents of the statement onto the clipboard Paste Statement Inserts the clipboard contents into a Statement section of the current Experiment Sign Statement Allows the user to add their electronic signature to a Statement section Once a Statement is signed the Experiment is locked and cannot be edited unless signatures are removed Multiple signatures may appear on one Statement Signatures are saved with data files but are not saved with protocols Add A Note Allows the user to add a note to the statement prior to signing it Agree Prompts the user for a password and signs the document with the user name date and time
43. the top right of the Status Bar While password protection is not available in SoftMax Pro GxP both editions support password protected files generated by older versions of SoftMax Pro SoftMax Pro provides no means of overriding password protection If you are concerned that you might forget your password store it in a safe place for future reference SoftMax Pro Software User Guide 0112 0140 Rev A 113 7 File Management amp Printing 7 4 1 SETTING A PASSWORD On a non password protected file 1 Choose the File Set Password command 2 Enteran author name and password 3 Click OK Passwords can be from 1 to 8 characters long and can include all characters available from the keyboard The Author Name is saved and printed with the data 7 4 23 CHANGING A PASSWORD On a password protected file 1 Choose the File Change Password command 2 Enter the old password and a new password 3 Click OK 7 4 8 REMOVING A PASSWORD On a password protected file 1 Choose the File Change Password command Check Remove Password Enter the old password Click OK A 0 N 7 4 4 SUSPENDING A PASSWORD If you do not remove password protection prior to trying to change a password protected file a dialog box is displayed in which you have to enter the password to continue Enabling the Suspend Password option stops this repeat prompting 114 SoftMax Pro Software User Guide 0112 0140 Rev A 8 1 8
44. 1 2 If this section is closed click the triangle indicator on the left side of the tool bar to open it The graph contains a single plot 3 The graph is configured to show Concentration on the X axis and 96 Activation on the Y axis A Parameter curve fit has been applied To apply a different fit select it from the Fit list in the Graph 1 section tool bar 4 To display the data in a bar graph or another format open the Graph gt Graph Options dialog box or click the Graph Options button in the Graph 1 section tool bar 5 This dialog box allows you to rename the graph choose the type of graph that is displayed and to format the text used in the graph You can add or remove plots click the New or Delete and you can edit the properties of existing plots 6 Change the type of graph to Cluster bar and click OK Step 10 Data Analysis Summary Formulas 5 Two Summaries are displayed at the bottom of the Data group section The second is Summary 2 EC 5096 0 044 Click this Summary to highlight it gt When selected the formula used to create this Summary is displayed in the Data group section tool bar Summary 2 InterpX Plot 1 Graph 1 50 5 Formulas can be simple or quite complex and are composed of elements that perform mathematical operations statistical analysis functions or refer to other elements con tained within SoftMax Pro Complete information about the possible choices for creat ing formulas can be found i
45. 1 Create a new file using the File gt New menu command 2 Modify all preferences and settings as necessary 3 Add any desired text information in Notes sections 4 Customize the columns formulas and reductions to suit your research needs 5 When the file is to your liking select the Protocols gt Save As Default Protocol menu item to save this as your new default protocol that will be displayed every time SoftMax Pro software is launched It is not mandatory to create a default protocol because the software detects the instru ment connected to the computer and automatically creates a new file based on instru ment For example if connected to a Fluorescence instrument with a cuvette port the software displays fluorescence units on both a Plate section and a CuvetteSet section if connected to an Absorbance instrument with no cuvette port the software displays absor bance units on a Plate section only In addition to these sections a blank Notes section is created Finally you can change your default protocol at any time To remove the default protocol used simply delete the file Default Protocol from Program Files Molecular Devices SofiMax Pro v5 2 SoftMax Pro Software User Guide 0112 0140 Rev A 25 26 3 SoftMax Pro Interface SoftMax Pro Software User Guide 0112 0140 Rev A 4 1 4 2 4 2 1 4 Data Collection INTRODUCTION TO DATA COLLECTION A typical process for preparing collec
46. 12 0140 Rev A 15 16 3 SoftMax Pro Interface 3 4 3 3 4 4 3 4 5 3 5 3 5 1 The next unread Plate or CuvetteSet section gt The first plate or cuvette if all have been read Menus are disabled while reading is in process CLOSE DRAWER OPEN DRAWER Use these commands to close and open the instrument drawer so that a plate can be inserted or removed INCUBATOR Control Incubator allows you to turn the incubator on or off When on you can enter a setting to regulate the temperature in the microplate chamber This command is available for all instruments except Emax VMax UVMax and LMax II REF Control gt Ref causes the SpectraMax M2 M2 M5 M5 Plus or Plus384 to read the cuvette in the cuvette port and applies the reading as a reference to all cuvettes in the active CuvetteSet section This command is available only when a CuvetteSet section is active and a SpectraMax M2 M2 M5 M55 Plus or Plus384 is connected or chosen in Edit gt Preferences EXPERIMENTS SoftMax Pro collects data from one or more microplates or cuvettes and stores it in a sin gle data file using the same or different instrument settings for different microplates or cuvettes For example microplates containing different samples can be read using the same or different modes all within the same Experiment Each SoftMax Pro file contains at least one Experiment Within an Experiment are one or more sections Experi
47. 140 Rev A 2 3 Connecting the Instrument via USB 2 3 1 2 3 2 Please refer to the instrument manuals for additional instructions related to the USB only instruments from Molecular Devices the SpectraMax L Luminometer and StakMax plate handling system SINGLE PORT ADAPTERS Single port adapters are very straightforward 1 Connect the MDC serial cable to the USB to serial adapter 2 Connect the USB to serial adapter to an open USB port on your computer 3 Turn your MDC microplate instrument on by pressing the black toggle to the position 4 Launch SoftMax Pro software and select the Edit gt Preferences menu item 5 Select the appropriate serial port most often COMI from the Serial Port pull down menu 6 You should now see a three dimensional image of your instrument in the top left corner of SoftMax Pro software immediately beneath the File menu 7 Confirm that your instrument is communicating properly by clicking on the instrument image Your specific reader should be automatically selected in the Reader pull down menu MULTIPLE PORT ADAPTERS Before attempting to use a multi port adapter be sure you have downloaded and installed the very latest software driver from the manufacturer s website If you are using a two port adapter from Keyspan such as their USA 28x Twin Serial Adapter you MUST be sure to connect your MDC microplate reader to PORT 1 Ports are usually identified on the side or top of the adapter it
48. 2 5 2 5 1 SETTING PREFERENCES Before using SoftMax Pro we recommend checking the global application settings in the Edit gt Preferences dialog box Here you will find 5 Reader settings such as the port used for the connection to the instrument supported readers and filter settings for use with Series I microplate readers gt Automatic file saving and printing options 5 File export settings when data is exported using the File Import Export menu item READER SETTINGS Reader When a reader is connected and is powered on the Reader list automatically reports the instrument connected to the computer If a reader is not connected to the computer the Reader list displays the model of the last reader connected A protocol file can be written if an instrument is neither connected to the computer nor selected in the preferences Choosing an instrument can be useful however since the options available in the Plate gt Settings dialog box depend on the choice of instrument here For example with a SpectraMax you can configure up to six wavelengths whereas you cannot do this with a VMax Filters Filter settings apply only to EMax VMax UVMax and ThermoMax instruments Check that the filter wavelength displayed for each position matches the wavelength of the filter physically installed in each position of the filter wheel If the settings in this dia log box are not correct subsequent readings will generate incorre
49. 404 501 and foreign patents and pending U S and foreign patents SpectraMax M2 and use thereof is covered by issued U S patent nos 5 112 134 5 766 875 5 959 738 6 188 476 6 232 608 6 236 456 6 313 471 6 316 774 6 320 662 6 339 472 6 404 501 6 496 260 and foreign patents and pending U S and foreign patents Automix is covered by U S Patent Number 5 112 134 PathCheck is covered by U S Patent Number 5 959 738 WASTE text engine c 1993 1998 Marco Piovanelli 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 Questions Phone 1 800 635 5577 1 408 747 1700 Fax 1 408 747 3603 Web www moleculardevices com SoftMax Pro Software User Guide 0112 0140 Rev A Introduction Major Functions of SoftMax Pros comica rra lInsttumeht Control gt lt dx pa ERES iia Data Collection and Display 0 0 0 cee eee eee Data Reduction and Plotting eg arta bog es eR Immediate Results Reporting and Analysis Two Available Editions Standard and GxP LLuuue Installation amp Setup Installations lu
50. 5 or M5 SoftMax Pro can collect data from one or more microplates or cuvettes and store it in a single data file using the same or different instrument settings for different microplates or cuvettes For example microplates containing different samples can be read using the same or different modes all within the same Experiment In addition to displaying data collected from a Molecular Devices microplate system instrument you can import display and analyze data collected from either a FLIPR or Analyst instrument DATA REDUCTION AND PLOTTING You can manipulate or reduce the raw data using dozens of built in formulas or define your own analysis structure to quickly and easily summarize the raw data More than one reduction can be shown and results from different microplates and cuvettes can be com pared within the same Experiment IMMEDIATE RESULTS REPORTING AND ANALYSIS Once you have defined instrument settings and have customized a SoftMax Pro software data file with assay information reduction settings custom columns in Group sections and summary objects you can Save As a Protocol file type to create an assay template that can then be used and distributed throughout a department or company for highly repeatable data collection and analysis that is completed the second the plate read has completed TWO AVAILABLE EDITIONS STANDARD AND GXP Two editions of SoftMax Pro are available Standard and GxP SoftMax Pro softw
51. AutoSave directory which is set in the Edit gt Preferences dialog Data files can also be saved to subdirectories of the Autosave directory SoftMax Pro appends pda to any file name passed in to it If a file with the same name already exists SoftMax Pro automatically overwrites the file with no warning If the subdirectory does not exist the file is not saved Example SaveAs myFile SaveAs mySubDirectory myFile SelectAll This is equivalent to pressing CTRL A or choosing Edit gt Select All SelectAll can be used to select data in group tables for copying not needed for copying plate data SelectNextPlateSection Selects next Plate section from the current Plate section SelectSect Select a section by name SelectSect can be useful for multi plate protocols or for selecting Group tables for copying In version 5 and above this command can also be used to spec SoftMax Pro Software User Guide 0112 0140 Rev A 9 6 SoftMax Pro Commands ify sections in different Experiments If no Experiment is specified SelectSect defaults to the active Experiment Example SelectSect Plate 0023499 SelectSect Group 1 Experiment 2 SelectSectNum X Select a section by its order in the document SelectSectNum can be useful for multi plate protocols or for selecting Group tables for copying Example SelectSectNum 3 SetFolderAs folderpath Set the current protocol directory You must specify the root directory Exampl
52. D I ege reu fis deu cuo EE EE Selecting an Experiment S aces a ee tbe a sen bcp Cede a Ken lla dra ica ue J Manipulating Experiments W dtd X EC dnd CEU Robe el D KK a E de SS D DD a nu n mnmnpereemm o Active Section Menus Wk kk kk kk kK KK KK KK KK kk K kk kk kK kk kk kk kk k Manipulating Sections aed cea de ddr a E ne a lk dd kay a ak le e NAAA pp paa Plate Section au 35 te cae shot ze Por es ym Group Section ci E N A N Nen RES eo aad whe boas e ls da CuvetteSet Section iss e ooo per dp were we eae IN NNN echo TD NECEM Creating a Default Protocolo iud dato oce t Ee KK KK Re lobe KK KK KK KK KK kK AR Data Collection Introduction to Data Collection lee Instrument Settings kk kk kk kk KK KK KK KK kK KK KK kK KK kk kK s Instrument Operation oi esta orb donee tools Instrument Settings Vary for Different Models 0ooooococcoooomoooo Best Typen Sae S ad d sec La DEL ava Mosca baze dad Read Modelado e Opa UR Wavelengths MTM MM Shake LMax II and LMax 11384 Only iiie e Euren pe Re Pere KK Integration SpectraMax L LMax II and LMax 11384 Only Injection and Delay SpectraMax L and LMax 11384 Only Injection Wells SpectraMax L and LMax 11384 Only ooooooo oooooo Sensitivity Gemini SpectraMax L M2 M2 M5 M5 FlexStation Fluorescence Luminescence Only KK KK KK KK eese SoftMax Pro Software User Guide
53. Editor to create a Plate Blank 1 Open the Template Editor for the Plate section 2 Select the wells to be used for the Plate Blank 3 Select Blank from the Group list Unlike pre read plate blanks in a plate blank group the average value of all the wells in the Blank group is subtracted from each individual well value on the microplate If you have an instrument that supports PathCheck the Plate Blank is subtracted after pathlength correction Use Plate Blanks when all samples on the plate have been prepared in the same way and therefore can be corrected from a single blank reading Plate Blank subtraction can be turned off in the Reduction dialog box so that the data can be reviewed with or without plate blank subtraction For Kinetic and Spectrum data the blank value is averaged and subtracted from each point in the read SoftMax Pro Software User Guide 0112 0140 Rev A 53 54 4 Data Collection CuvetteSet Sections To perform a similar function to Plate Blanking for CuvetteSet sections you can assign cuvettes to a Blank group in the Template Editor of the CuvetteSet section and create a Template Blank 1 Open the Template Editor for the CuvetteSet section 2 Select the cuvettes to be used for the Template Blank 3 Select Blank from the Group list The average value of all the cuvettes in a Template Blank group is subtracted from individual cuvette readings in the same CuvetteSet section For Kinetic and Spectr
54. Graph Sections uta e kk kk KK KK KK kk kk kk kk kk kk kk k 74 Group Sections esie ene tod EO Ai e EROR ek r Sa EA Ren 74 Graph Section osha ver bad e dp uoo dud pr e OR Ee ERE a 77 Tutorials Tutorial 1 Quantitative Endpoint Protocol Tutorial KK RR KK KK 88 Step 1 Create a New Data File ot or KK KK KK KK KK KK KK KK KK KK KK ate 88 Step 2 Define Instrument Settings 2 Lost Peres KK KK KK KK KK KK KK KK K KK se 89 Step 3 Define Template ti oon a acr d n a e a aces adi rey cud 90 Step 4 Set Reduction Parameters ci Ak rev pes KK KK KK KK KK KK KK KK KK KK 92 Step 5 Set Display Parameters 0 alias KK KK KK KK KK KK KK KK KK KK KK KK des 92 Step or Save the Protocol ze nei eee aah E 92 Step 7 Read tie Dea spy ku kl Wla wa pora Ese teal ls baena 93 Step 8 Data Analysis Group Sections leeren 93 Step 9 Data Analysis Standard Curves 5s Lum dert S es KK KK KK KK dx 96 Tutorial 2 EC 50 Protocol Tutorial Flex al KK KK RR KK KK 96 Step be Create a New Data Files y k Pee ba en l Wl va hd Pale cal 97 Step 2 Define Instrument Settings 52e kk KK KK KK KK KK KK KK KK KK K KK aea 97 Step 3 Define Template aat Xwene e yk E ben yk ea Weed bek Tee 98 Step 4 Set Reduction Parameters 0 eee cece cence 98 Step 5 Set Display Parameters 254 tase kK See KK KK KK KK KK KK KK KK KK ey 99 Step 6 Save the Protocol J kk kK KK KK KK KK KK KK KK Now KK KK KK KK k 99 Step 7 Read the Plate i i A yey sel ayn c
55. Guide 0112 0140 Rev A 122 9 Robotics amp LIMS Integration 9 4 1 THE WM SETTEXT APPROACH Call the Win32 RegisterWindowMessage function to register a unique message tag Soft Max Pro uses this tag information when the command is received to distinguish between a Windows generated message and an external command The tag should be a non zero value UINT tag RegisterWindowMessage SOFTMaxPROMsg All messages must be sent to the SoftMax Pro main window Note that if SoftMax Pro is not currently running this returns a null value requiring your program to discontinue until SoftMax Pro is restarted HWND hwnd FindWindow SOFTMaxPROMainWnd SoftMax Pro If the target SoftMax Pro is the GxP version the Window name should be SoftMax Pro GxP HWND hwnd FindWindow SOFTMaxPRORemote SoftMax Pro GxP A complete list of remote commands can be found in SoftMax Pro Commands below Do not forget that the tag value from RegisterWindowMessage above is sent with the command string See Microsoft documentation regarding the SendMessage function for more information SendMessage hwnd WM_SETTEXT tag LPARAM remoteCommandString For example to send SoftMax Pro a Read command SendMessage hwnd WM_SETTEXT tag LPARAM Read If return data is expected such as with remote commands ReturnStatus ReturnTiming and ReturnData the data is returned to the clipboard The advantage of this a
56. Guide 0112 0140 Rev A 4 3 Template Edftor 4 3 8 COPYING AND PASTING TEMPLATES To copy a template 1 Activate the source template by clicking in the Plate section containing it 2 Choose the Plate gt Copy Template command 3 Activate the destination Plate section by clicking it 4 Choose the Plate Paste Template command Copying and Pasting within the Same Experiment Section When copying and pasting within the same Experiment section the group names and sample names of the pasted template are identical to those of the source template In effect the destination template is 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 Changes made to the template on the destination plate are also made on the source plate Data from the two Plate sections are analyzed together Copying and Pasting between Different Experiment Sections When copying and pasting between different Experiment sections the group names and sample names of the pasted template are identical to those of the source template but since they are in different Experiments the full name is different for example group experiment 2 instead of group experiment 1 Changes made to the template of the destination Plate section do not affect the source section Data from the two Plate sections are not analyzed together Copying and Pasting Tem
57. Max Pro main window If SoftMax Pro is not cur rently running this returns a null value thus requiring your program to discontinue until SoftMax Pro is restarted HWND hwnd FindWindow SOFTMaxPROMainWnd SoftMax Pro If the target SoftMax Pro is the GxP version the Window name should be SoftMax Pro GxP HWND hwnd FindWindow SOFTMaxPRORemote SoftMax Pro GxP Get your window handle This is the window to receive the return message HWND MyWnd GetSafeHwnd Assign the command to send Place the command in heap space which appears to be nec essary with older Windows operating systems char cmdStr Read Example remote command to send char msgStr strdup cmdStr String duplication copy command to heap space Create the Win32 COPYDATASTRUCT message that is sent See Microsoft documenta tion regarding this structure for details COPYDATASTRUCT cd This struct is defined in Microsoft include file SoftMax Pro Software User Guide 0112 0140 Rev A 124 9 Robotics amp LIMS Integration 9 4 3 cd dwData DWORD MyWnd Handle to a window receiving return information cd cbData strlen msgStr 1 Length of message string adding 1 for null terminator cd IpData msgStr String pointer to remote command Finally send the message to SoftMax Pro A complete of list of remote commands can be found in SoftMax Pro Com
58. Negative Kinetic values decrease with time and limits should be set accordingly below 0 to view negative Kinetic data SoftMax Pro Software User Guide 0112 0140 Rev A 5 3 Data Reduction 5 3 3 MaxOD RFU RLU The limit for the maximum value to report Any values from the reading that are above this limit are excluded from data reduction The default for MaxOD is 1 0 while it is 20 000 for RFU RLU The MaxOD RFU RLU reduction parameter can be used to exclude the non linear portion 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 MaxOD RFU RLU allows the use of the maximum number of linear points to calculate the slope of the line and thus to determine the rate for each well MinOD RFU RLU The limit for the minimum value to report Any values from the reading that are under this limit are not shown and are excluded from data reduction The default is 0 OD or RFU 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 but does not prevent data from being collected Kinetic plots do not display the data collected prior to the Lag Time The defau
59. New Summary dialog box is placed at the bottom of the Group section as follows Name Description Formula Result You can check the Hide Name option to display the Summary as Description Formula Result The name assigned to a Summary is important since you can reference the Summary by name in other formulas building one upon another Move Summaries horizontally by dragging them left or right If you do this while holding down the Ctrl key the Summary snaps in line with a column Copy and Paste 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 recreating it Summaries can also be copied and pasted within SoftMax Pro After pasting both formulas and Summaries can be edited Contiguous column formulas may be copied and pasted together Multiple Summaries may also be copied and pasted together if the Shift key is held down while selecting the Summaries To copy a formula or Summary 1 Highlight the formula or Summary 2 Select Edit gt Copy 8 C CTRL C 3 Activate the destination if necessary with a formula access the dialog box into which it will be pasted 4 Select Edit gt Paste 8 V CTRL V SoftMax Pro Software User Guide 0112 0140 Rev A 5 4 Group and Graph Sections 5 4 2 Autosize Use Group gt Aut
60. New command opens a new Experiment file named Untitled based on the default protocol or the instrument selected in the Edit gt Preferences dialog box OPENING FILES To open a supported protocol or data file use the File gt Open command SoftMax Pro allows you to open files from prior versions of the program However once you save a file it is readable only by the current version If you need to continue using a data file with an older version of SoftMax Pro do not overwrite the original when re saving it While SoftMax Pro software opens both SoftMax and SoftMax Pro data files two features are not forwards compatible sample blanks and Auto range limits in Graph sections The data for sample blanks obtained using SoftMax software is read when these files are opened in SoftMax Pro but no calculations are performed automatically using this data Auto range limits are not available when SoftMax files are opened using SoftMax Pro so limits for the display of information in graphs must be re entered manually Cross Platform Files SoftMax Pro files created by either Windows or Mac users can be opened on either plat form The Mac honors SoftMax Pro 3 letter file extensions as well File Type File Extension Mac Document Type SoftMax Pro Protocol File ppr sPFd SoftMax Pro Data File pda SPFd SoftMax Protocol File pro sPFd SoftMax Data File dat SPFd SAVING FILES MANUALLY Use the File gt Save command t
61. OK to save these settings From the Preferences dialog click Edit after selecting an AutoSave instance to modify the AutoSave settings Location You can control where files are AutoSaved by modifying the options listed in the Location section The default location set in SoftMax Pro is an invalid dummy path that should be deleted before saving another location To save data files to an assigned folder click Assigned Folder and choose a different folder If the AutoSave function reports 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 a newer version Name You can control the automatic naming of all AutoSaved files by modifying the options listed in the Name section Protocol s Name If selected each AutoSaved file begins with the name of the protocol used to generate the data that has been AutoSaved SoftMax Pro Software User Guide 0112 0140 Rev A 10 2 Installation amp Setup Assigned Name If selected any text in this field is automatically inserted at the beginning of each AutoSaved file name Files are also numbered sequentially for example Data 1 Data 2 etc If the Assigned Name field is left blank the date and time of each plate read is used as the file name for each AutoSaved file For example the first file AutoSaved on November 18th 2005 at 9 01am and 58 seconds would be AutoSaved as 11 18 05
62. Reduction settings shown to the right of or below the data display depending on the plate type and printer settings gt Fluid transfer and checksum status for the FlexStation instrument If the plate grid in the Plate section is colored then a template has been defined for the Plate section Each group defined in the template has a different color the icon of the cor responding group table has the same color If your experiment requires multiple plates you can create as many Plate sections as needed Template The Template button or Plate gt Template opens the Template Editor which allows you to describe the samples in the wells providing the link between the raw data and analysis groups You can create new groups in the template or assign wells to already established groups A group is a set of associated samples that has been created in the Template Some group types such as Standard or Unknown can be created as part of the default protocol and you can create others as required When you create a group and assign samples to it a cor responding group table is automatically created Each Plate section in your Experiment requires a template to be created to identify groups within that plate However two or more Plate sections may use the same template or the same groups may be used in different templates Reduction The Reduction button or Plate gt Reduction opens the Reduction dialog box which is used to speci
63. SOFTMAX PRO SOFTWARE Version 5 for Mac and Windows User Guide Molecular Devices Molecular Devices Corporation 1311 Orleans Drive Sunnyvale California 94089 Part 0112 0140 Rev A SoftMax Pro Software User Guide 0112 0140 Rev A Molecular Devices Corporation SoftMax Pro Software Version 5 for Mac and Windows User Guide Copyright O Copyright 2007 Molecular Devices Corporation All rights reserved No part of this publication may be reproduced 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 1311 Orleans Drive Sunnyvale California 94089 United States of America Trademarks SoftMax FlexStation FLIPR SoftMax SpectraMax Analyst and VMax are registered trademarks and Automix EMax PathCheck SpectraPlate SpectraStrip ThermoMax UVMax VersaMax ScreenStation ScreenPlay and Acquest are trademarks of Molecular Devices Corporation These trademarks are not to be used in any type of promotion or advertising without written permission from Molecular Devices Corporation All other trademarks are the property of their respective owners SoftMax Pro and use thereof is covered by issued U S patent nos 5 766 875 5 959 738 6 188 476 6 232 608 6 236 456 6 320 662 6 339 472 6
64. Set the Serial Port xo None 3 Select the other instrument from the Reader list Failure to do so could cause instrument errors when you attempt to read To achieve the shortest possible interval for Kinetic readings choose wavelengths in ascending order The minimum interval for Flex reads is dependent on the Compound Transfer settings and the number of wells to be read per column PATHCHECK PATHLENGTH CORRECTION SPECTRAMAX PLUS PLUS384 190 340PC M2 M2 M5 M5 The Beer Lambert law states that absorbance is proportional to the distance that light travels through the sample A ebc where A is the absorbance is the molar absorbtivity of the sample 6 is the pathlength and c is the concentration of the sample In short the longer the pathlength the higher the absorbance Microplate readers use a vertical light path so the distance of the light through the sample depends on the volume This variable pathlength makes it difficult to perform extinction based assays and also makes it confusing to compare results between microplate readers and spectrophotometers The standard pathlength of a cuvette is the conventional basis for quantifying the unique absorbtivity properties of compounds in solution Quantitative analyses can be performed on the basis of extinction coefficients without standard curves e g NADH based SoftMax Pro Software User Guide 0112 0140 Rev A 4 2 Instrument Settings enzyme assays Wh
65. Setting Preferences 2 5 3 column but the wells are read sequentially the reaction advances in the other wells while the first is being read Consequently the data in the other wells must be interpolated against the amount of time that has elapsed between injection and reading in order to compare data from one well to another and to compare data from one run to another Once exported well interpolated data cannot be re imported into SoftMax Pro This interpolation is different to the interpolation option in the Plate gt Reduction dialog box 384 to 4 x 96 time format This is used when you are using 384 well plates and conducting extended Kinetic reads with more than 255 time points This option enables you to work around architectural issues in Microsoft Excel that prevent the import of data files with more than 255 col umns SAVE DATA AFTER READ AUTOSAVE When this option is enabled the collected data is saved automatically to a user defined location immediately after each plate read is completed This greatly reduces the likeli hood of lost data particularly when AutoSave is set to save files to corporate network vol umes that are backed up on a regular basis You can add as many AutoSave instances as desired each with its own settings After click ing Add you are presented with the Add AutoSave Entry dialog that allows you to specify the location file naming conventions and file format Once specified simply click
66. SoftMax Pro GxP SOLUTION OVERVIEW Molecular Devices provides a fully integrated hardware and software solution for adher ence to Good Manufacturing Practices GMP Good Laboratory Practices GLP and many other regulatory compliance requirements including 21 CFR Part 11 This solution consists of gt An MDC microplate reader gt SoftMax Pro GxP software for data collection and analysis 5 GxP Admin software for user and permission management gt MDC File Server optional for TCP IP based file serving of one or more multiple User Accounts file GxP Admin manages all SoftMax Pro GxP users and user permissions and creates and stores this information in a proprietary User Accounts edb file SoftMax Pro GxP isa version of SoftMax Pro that includes features to provide regulatory compliance with 21 CFR Part 11 requirements SoftMax Pro connects to and controls all MDC microplate readers and it analyzes all col lected microplate and cuvette data SoftMax Pro GxP is a special software release that includes full user and permission control and audit trail generation in addition to all the data collection and analysis features found in the Standard edition With the audit trail features in SoftMax Pro GxP all Experiments are fully traceable and can be completely reconstructed Before SoftMax Pro GxP can be launched and used a User Accounts file must be created by the administrator using GxP Admin software The User Accounts file rec
67. Subtract the 900 nm value from the 1000 nm value 3 Do the same for pure water If the delta OD for the sample differs significantly from the delta OD for water then it is advisable not to use the PathCheck feature Use of Cuvette Reference on SpectraMax Plus Plus384 M2 M2 M5 M5 does not correct for the interference with the current calculation scheme in SoftMax Pro Currently Cuvette Reference involves a single auto mated read at 900 nm and 1000 nm and the automated calculations in SoftMax Pro do not compensate for color or solvent interference However you could correct for such interference by taking two cuvette measurements and using a different set of calculations For further information contact Molecular Devices Technical Support Organic solvents could interfere with PathCheck if they have absorbance in the region of the NIR water peak Solvents such as ethanol and methanol do not absorb in the NIR region so they do not interfere except for causing a decrease in the water absorbance to the extent of their presence in the solution Their passive interference can be avoided by using the Cuvette Reference If however the solvent absorbs between 900 and 1000 nm the interference would be similar to the interference of highly colored samples described above If you are considering adding an organic solvent other than ethanol or methanol you are advised to run a Spectrum scan between 900 nm and 1000 nm to determine if the solvent w
68. TEP 1 CREATE A NEW DATA FILE 1 Double click the SoftMax Pro icon to start the program 2 Choose the File gt Open command and open the file Tutoriall pda If you are using an Absorbance instrument the CuvetteSet 1 section can be deleted as the use of a cuvette is not covered in this tutorial To delete this section 1 Click the title bar of the CuvetteSet 1 section 2 Select Edit gt Delete CuvetteSet 1 The Experiment should now have a Notes section Introduction Plate section Plate 1 a Graph section Graph 1 and two other group sections Controls and Unknowns that will be used in this tutorial SoftMax Pro Software User Guide 0112 0140 Rev A 6 1 Tutorial 1 Quantitative Endpoint Protocol Tutorial 6 1 2 STEP 2 DEFINE INSTRUMENT SETTINGS 1 Click the Plate 1 section to make it active and then click the Settings button in the tool bar or select Plate gt Settings At the top of the dialog box Endpoint is selected by default as the read type no change is required Clicking the setting name bold heading on the left side of the Instrument Settings dialog box shows the choices available for that setting in the right pane allowing you to make changes Following are the details about the quantitative protocol settings we will use to create a protocol file using SoftMax Pro Select Fluorescence as the read type and make sure all other settings match those in the table Table 6 1 Example Settings
69. The Sample area contains the Sample list and sample descriptor values Sample List The Sample list reports the sample name assigned to the currently selected wells 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 sam ple names would be Sta01 Sta02 Sta03 and so on SoftMax Pro automatically increments sample names for a group as you successively assign samples to columns for example However you can type a sample name directly into the Sample field Sample Descriptor Field Use the Sample Descriptor field to enter sample information such as the sample dilution or concentration The Sample Descriptor field is displayed only if the selected group has associated descriptor information ASSIGN Click this button to apply a sample name to selected wells CLEAR Clears the group assignment of the selected wells SERIES This button is used to define several samples as a series allowing you to easily enter incre mental sample descriptors for example dilutions or concentrations and sample names to the template as long as the increment can be expressed as a mathematical operation Replicate Samples and Series You can assign the same group or sample definition same sample name and description within a group to multiple wells to create replicates For example you might want to read standards in re
70. a compound 1 Highlight one or more compound cells 2 Select one of the items from the Compound list The wells shown in this tab setting convey graphically the volumes of liquid in the assay plate for each pipetting event Using the color associated with each event the compound settings display the dispensed compound volume as a percentage of the total assay well volume An initial volume entered on the Compound Transfer tab is shown as a gray fill As the liquids are cumulative the first event s volume is shown above the initial liquid volume if any the second event s volume is shown above the first and the third event s volume above the second To deselect a tip or compound assignment 1 Select the appropriate cells 2 Select Backspace on a Windows PC or Delete on a Mac To change an assignment 1 Select the wells 2 Choose new values or type a value on the keyboard Note If you have multiple wells selected and you type a value that value is shown in the first selected well and subsequent wells increment to the next higher value Again this does NOT take into account the actual pipette tips shown to be available in the Pipette Tips Layout selection SoftMax Pro automatically enters information in the Compound and Tip Columns tab using a best fit algorithm that uses the number of transfers the Wells to Read section and the Pipette Tips Layout selected area The algorithm assumes gt All columns in the Wells to Read
71. a standard curve It is designed to be used by both Absorbance and Fluores cence instruments gt Tutorial 2 leads you through the steps required to create and run an EC50 protocol using a graph of concentration vs RFU on a FlexStation instrument using a Flex read The source files for these tutorials are installed with SoftMax Pro software and can be found in Program Files Molecular Devices SofiMax Pro v5 2 Tutorials If you are new to SoftMax Pro we suggest that you read and follow the steps in one of the tutorials before using SoftMax Pro for the first time since it will help you learn the basic features quickly More information about any of the operations performed in this tutorial can be found in Chapters 4 and 5 Hard and Soft Parameters The only settings that must be defined before reading a microplate are the instrument set tings in Plate Settings These settings tell the instrument the conditions that should be in place in order to collect data They include the read type Endpoint Kinetic Spectrum Well Scan or Flex the number of wavelengths specific wavelength settings 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 and data reduction settings These param eters can be defined prior to reading a microp
72. ad of the 4 parameter logistic equation when you need to fit to a non symmetrical sigmoidal shape The non symmetry is typically seen in the rate of change of the slope of the curve as it approaches the asymptote With 4 parameter fits the rate of change of the slope is the same at the lower asymptote as at the upper asymptote With 5 parameter fits the upper and lower portions of the curve can have very different shapes The minimization method for the 4 parameter and 5 parameter equations is based on the Levenberg Marquardt Method Discussion of these methods 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 rela tionship between each pair of data points The line segment 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 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 SoftMax Pro Software User Guide 0112 0140 Rev A 81 82 5 Data Analysis The equations are computed wi
73. alculate Absorbance or Transmittance of sample read in the cuvette port Semi Log Curve Fit The semi log function fits the best straight line to a set of data for log X plotted against Y The resulting curve displayed will be a straight line with the X axis drawn in logarith mic scale Series The Series function allows you to work with groups of wells in the Template so that the standard value or dilution factor increases or decreases in specified steps The name can be incremented as well Slope A reduction option that determines the slope of the combined plot e the slope of the line using linear regression after the wavelength combination reduction This reduction SoftMax Pro Software User Guide 0112 0140 Rev A A 1 Glossary of Terms uses all time points visible in the reduction window Slope is the same as VMax Rate when VMax Rate is set to the same number of points as the run z the default Slope is not the same as VMax Rate if you have modified the number of VMax Points Spectrum A read type that allows you collect absorbance or fluorescence data across a spectrum of wavelengths This data is displayed as OD RFU or RLU versus wavelength The default reduction displays the wavelength that corresponds to the maximum signal Standard Curve A mathematically idealized representation of the relationship between the concentration of a standard calibrator X value and the OD RFU RLU Y value The standa
74. an inappropriate fit For best results make sure that the logistic fit is appropri ate for your assay and that the entire range of the assay is represented in your standards SoftMax Pro Software User Guide 0112 0140 Rev A 5 4 Group and Graph Sections If your standards do not clearly define both a high and low asymptote the spline fit may be more appropriate for your assay Parameter Settings 4 or 5 Parameter Curve Fit Weighting Cu ve fit weighting is useful when uncertainties in the data are not random and may be related to the time the concentration or the number of data points for example In such cases you can up weight or down weight points on the curve using a weighted sum of squared errors 2 WSSE Xwi Yovo for weights w observed values y and function values j By default SoftMax Pro does not apply weighting but you may choose a variable weight for each point and enter it as a function in the Parameter Settings dialog box The formula must be an array of numbers that has the same number of points as the curve For example you could use a formula Weight Group 1 to use a column of weights from the Group section Group 1 Different weight values result in different Chi Squared values for each curve fit To access the Chi Squared values of a particular curve fit use the accessor ChiSquared Plot 1 Graph 1 Manually Set Parameters SoftMax Pro offers the option of manually assigning individual fi
75. and I is transmitted light Absorbance A Absorbance is the amount of light absorbed by a solution To measure absorbance accu rately it is necessary to eliminate light scatter In the absence of turbidity absorbance optical density A log o where Ip is incident light and I is transmitted light In this manual we use the terms absorbance and optical density interchangeably Area Under Curve Reduction formula for a Kinetic or Spectrum scan that determines the area under the Kinetic or Spectrum plot or under a plot in a Graph section Author Name The Author Name is entered from the password dialog box at the time when file shielding is turned on The Author Name is different from the User Name in that it may be the name of the author of a protocol file that is later used to read data AutoCalibrate For Absorbance instruments an air reference reading is taken before runs and sometimes between Kinetic readings during a run as determined by the type of instrument and read settings For Fluorescence instruments the measurement occurs before reads and some times between Kinetic reads if the interval is long enough SoftMax Pro Software User Guide 0112 0140 Rev A 137 138 A Appendix Calibration data is stored in memory and is used by SoftMax Pro until the instrument is powered down or the wavelength is changed AutoCalibration can be turned off Automix I he Automix function determines how often if a
76. are present in a reading and you choose to combine these wavelengths while Zero Baseline is selected the zero baseline applies only after the wavelength combination Baseline with Multiplier This option makes Flex data look more like FLIPR data FLIPR data can be imported into SoftMax Pro and this option modifies the plot and reduction calcula tions to make Flex data comparable to the FLIPR data This method averages the first number of points you specify the upper box divides the raw data by this average value and then multiplies by 100 96 The Multiplier field allows you to enter a number from 0 0001 to 9999 that is used to multiply the Baseline value SoftMax Pro Software User Guide 0112 0140 Rev A 71 72 5 Data Analysis 5 3 6 5 3 7 This calculation is performed only on the reduced data and only after any wavelength combinations are calculated To see the modified plot choose Reduced Plot in the Display dialog box or click Show Reduced in a zoomed well Graph window Interpolate Raw Data For Flex reads each data value is logged with its own read time which can make it difficult to perform wavelength combination calculations correctly For example to take a ratio of two different wavelengths in a single well it is necessary to take a ratio Lm1 Lm2 of values that have been read at two different points in time To solve this problem select the nterpolate Raw Data checkbox in the Reduction dialog
77. are provides instrument control and analysis functions for all Spectra Max FlexStation Gemini and earlier instruments as well as import and analysis of data files from FLIPR and Analyst instruments SoftMax Pro GxB together with a Molecular Devices plate reader and Molecular Devices GxP Admin software provides a fully integrated hardware and software solution for adherence to Good Manufacturing Practices GMP Good Laboratory Practices GLP and many other regulatory compliance requirements including 21 CFR Part 11 SoftMax Pro Software User Guide 0112 0140 Rev A 2 1 2 Installation amp Setup INSTALLATION INSTALLING AN INSTRUMENT Please consult your instrument manual for instructions on connecting your instrument to a computer COMPUTER SYSTEM REQUIREMENTS Recommended computer hardware and operating system software specifications that will ensure proper operation of SoftMax Pro can be found online at http www moleculardevices com downloads SMPv5x_SysReqs pdf Molecular Devices recommends that you increase the amount of RAM in your computer to a minimum of 2 GB in the following cases gt If you plan to use SoftMax Pro with a FlexStation instrument gt Ifyou plan on using your microplate reader with the StakMax integrated plate han dling system gt If you use large more than 40 Plate sections per file or complicated 384 well ADME protocols etc files gt If you plan to use robotic applicat
78. ase be sure to obtain the latest software driver from the manufacturer before connecting to your instrument LAUNCHING SOFTMAX PRO SOFTWARE After the instrument is powered on and has completed its start up sequence double click the SoftMax Pro icon on your desktop to start the program ENTERING REGISTRATION INFORMATION The first time you run SoftMax Pro after installation a first launch wizard will guide you through the setup process In particular it requires you to enter your name company and the serial numbers of SoftMax Pro and the connected instrument The software serial number can be found in the SoftMax Pro software box and the instru ment serial number is located on a label affixed to the back of the instrument You will also be prompted to register your software with Molecular Devices over an inter net connection Registration of SoftMax Pro activates the product warranty and entitles you to receive software updates when available CONNECTING THE INSTRUMENT VIA USB Many Windows and Mac based computers no longer have a standard RS232 serial port If you have a computer without a standard serial port you will need to use a USB to serial adapter These adapters are available at many computer supply stores as well as through online merchants Molecular Devices recommends serial adapters made by Keyspan www key span com as they have been proven repeatedly in field operations SoftMax Pro Software User Guide 0112 0
79. ata analysis Log Off Terminates the user s connection to SoftMax Pro GxP Change Password Allows the currently logged on user to change his her password Add Note to Audit Trail Opens a dialog box in which the user can securely append a note to the permanent audit trail for the current file Show Audit Trail Opens a file showing a list of all user activities recorded for the current file STATEMENTS Within SoftMax Pro GxP a user with sufficient permissions can add a Statements section to an Experiment data file Electronic statements provide a means of designating that an Experiment has been approved reviewed or verified Users with appropriate permissions can also apply electronic signatures to these statements Once a statement has been signed the file cannot be modified Electronic statements are saved with Experiment data files and protocol files but signa tures are not saved with protocol files Only one electronic statement section is allowed per file and is always the first section displayed for each Experiment data file Add Statements Experiment New Statements allows a user to enter a title for each statement or comment and to enter a brief note for all other users who may be using or accessing a particular Experiment data file SoftMax Pro Software User Guide 0112 0140 Rev A 8 3 Using SoftMax Pro GxP 8 3 5 After the first statement has been created it appears as the Statements section at
80. ax Pro sends a completion message once the command has been executed An asynchro nous window message is returned as a window callback typically an OnCopyData han dler inside the Win32 COPYDATASTRUCT types data packet For example if the Read command is sent an OKRead text string is returned after the read is com pleted gt The user does not wants to receive a completion message Use SendMessage combined with WM SETTEXT for messages that do not require a completion message The advantage of this approach is that it may be universally applied to Visual C and Visual Basic modules Excel macros etc Remote commands that DO return data There are three remote commands that return data ReturnStatus Return Timing and ReturnData If the caller has enabled queue operation the command ReturnStatus is handled immedi ately and not queued as are other remote commands Data is returned in one of two ways gt After sending the remote command using the WM_COPYDATA message the caller can look for return data through an asynchronous Windows message inside a Win32 COPYDATASTRUCT type data packet An asynchronous window message is returned as a window callback typically an OnCopyData handler gt After sending the remote command using the WM_SETTEXT message the return data is available immediately on the clipboard The advantage of this approach is that it may be universally applied to Visual C and Visual Basic modules Excel macro
81. be exported with the Plots gt Export Graph command COPYING AND PASTING Plate section data CuvetteSet section data and Notes section text can be copied from external programs and pasted into a Plate section CuvetteSet section or Notes section For 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 of Edit Preferences Pre read data cannot be pasted into a Plate section After data is pasted into a Plate section the information to the right of the display shows imported data If you have created a template for a Plate section prior to pasting data numbers are shown only in the groups you have created in the template Data in other wells is not reported but is available if you alter the template Clearing the template or assigning groups to all wells in the plate will show the complete data set FILE PROTECTION Password Protection avoids changes being made to a protocol or data file by persons who do not know the password and provides an electronic signature with the file gt When a protocol file is password protected you may read data into the file but may not change it in any other way without entering a password gt When a data file is password protected you may view the file but are unable to make changes without entering a password When a file has been password protected an icon showing a lock appears at
82. before SoftMax Pro GxP software can be used This can be completed by each end user by the administrator or by IT personnel 5 After each Windows log on account is linked to a User Accounts file users can launch SoftMax Pro GxP software log on and use the software to collect and analyze micro plate data gt If enabled by the administrator Guest users can open view and print Experiment data files GXP ADMIN FEATURES GxP Admin supports the following major functions 5 Template Management Create templates or groups of SoftMax Pro GxP permissions This makes managing large numbers of users easy as the administrator can use tem plates to quickly and easily assign sets of permissions to new users gt User Management Add new users modify user permissions and inactivate users Once created users cannot be deleted 5 File Reporting Export user information and permissions to a text file View the audit trail of administrator activities 5 License Management Add new site license information which is used by GxP Admin to control the number of users registered to use Molecular Devices software programs SoftMax Pro Software User Guide 0112 0140 Rev A 8 3 Using SoftMax Pro GxP 8 3 8 3 1 8 3 2 gt Password Aging Users can be required to change their password on a regular basis users are prevented from re using their previous password 5 Idle Timeout Automatically log users out of SoftMax Pro af
83. bers of points are included in density settings SoftMax Pro Software User Guide 0112 0140 Rev A 41 42 4 Data Collection 4 2 17 Four scanning patterns are available a horizontal line a vertical line a cross which is a combination of a horizontal and vertical line and a grid pattern The density setting determines gt The number of points read in the line patterns gt The maximum number of horizontal and vertical points included in the grid pattern With a grid pattern all points that land within 2 8 mm of an edge are excluded The minimum number of points is 3 for most plate sizes while the maximum number of points depends on the well diameter for 384 well plates only one read per well is allowed The SoftMax Pro export format supports Well Scans The data from each position is out put in subsequent rows or plates starting with the top left point and proceeding with hor izontal priority ASSAY 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 microplate to be read Changing the Plate Type within a Plate section causes well assignments stored in the pre vious template to be discarded The groups created previously remain however so you can select new wells and apply existing groups to these wells Plate Types The Plate Type list includes entries for microplates with standard dimensions and addi tional entries
84. can check by holding down Ctrl Shift Select wells H1 and H2 A new sample descriptor is created automatically St07 so all you need to do is assign a concentration value of 1 4 pg ml to these wells After entering the value click Assign or press the Tab or Return key SoftMax Pro Software User Guide 0112 0140 Rev A 6 1 Tutorial 1 Quantitative Endpoint Protocol Tutorial 13 14 15 16 17 18 19 20 21 22 23 24 This tutorial example uses seven separate unknown samples with each unknown being placed in four wells The unknown samples should be entered on the template as follows Un01 wells AS through D5 n02 wells A6 through D6 n03 wells EG through H6 n04 wells A7 through D7 n05 wells E7 through H7 n06 wells A8 through D8 n07 wells E8 through H8 U U U U U U Start by highlighting wells A5 through D5 Select Unknowns from the Group menu The selected wells will now show the default first sample name for the Samples group Un01 Continue highlighting the wells as described above assigning new unknown sample descriptors to each subsequent group This tutorial example uses two controls with each control being placed in three wells The controls should be entered on the template as follows Co01 wells A9 through C9 Co02 wells D9 through F9 Start by highlighting wells A9 through C9 Select Controls from the Group menu The selected wells now show the default first sample
85. centration and values respectively for Standard2 6 Repeat the process for a third plot labeled Standard 3 SoftMax Pro Software User Guide 0112 0140 Rev A 5 4 Group and Graph Sections 7 Now that all plot information is complete click OK in the Graph Options dialog box to close it and the resulting graph shows the three plots created 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 Y axis Error bars are lines that extend beyond a plotted value in either or both directions and graphically represent some amount of error in plot ted data Curve Fitting When you first create a graph of the data it does not have a fit associated with it You can fit any plot to one of ten curve fitting functions Linear Semi Log Log Log Quadratic 4 Parameter logistic 5 Parameter logistic Log Logit Point to Point Exponential and Cubic Spline These selections are shown in the Fit list in the Graph section tool bar All plots on a graph must have the same type of fit Typically a standard curve refers to the curve fitted to the plot of concentration versus mean value for the Standard group Once you have selected a fit type SoftMax Pro determines the parameter values that best fit the data The function with these parameters is drawn on the graph Ideally the type of fit used should be determined by the underlying chemistry of th
86. ch they appear in an Experiment The time in the Delay field specifies the delay time between plate reads TEMPLATE EDITOR The Template Editor is used to describe the location of samples in a microplate or cuvette and thus provides the link between raw data and analysis groups Each template is composed of samples and groups gt A set of one or more replicate wells makes up a sample gt A set of related samples forms a group For example you may have a group named Standards that consists of seven samples named STDO1 STDO2 STD07 and a group named Unknowns that consists of five samples names UNK01 UNKO2 UNKOS Each sample would be typically applied to a column or some other subset of wells on a microplate or to one or more cuvettes Each well designated as being part of a group has associated with it a group name a sam 8 8P group group ple name or replicate ID a sample descriptor optional and a column format for the calculations and data reported in the associated Group section Samples and groups may exist across multiple Plate sections as well as within a single Plate section Deleting the wells assigned to a group from the template does not delete the group To delete a group name in the Template Editor you must delete the corresponding Group section from the Experiment SELECTING WELLS OR CUVETTES IN THE TEMPLATE EDITOR Open the Template Editor for a Plate section with the Plate gt Templat
87. ckground Constants To determine Plate Background Constants 1 Filla clean microplate with water 2 Readat the wavelengths you will be reading your samples 3 The average OD value is the Plate Background Constant Enter it in the designated field under Plate gt Settings gt Path Check 4 If you intend to read your samples at more than one wavelength there should be a corresponding number of Background Constants It is important that you put water in the wells and not read a dry plate for the Back ground Constant Dry plates have a slightly higher OD value than a water filled plate because of differences in refractive indices Using a dry plate results in PathCheck normalized values that are lower than 1 cm cuvette values Omitting the Background Constant results in PathCheck normalized values that are higher than 1 cm cuvette values Use Pre Read This method is less desirable and less convenient than the first two When you fill the microplate with water and pre read the plate SoftMax Pro stores the pre read values Then you empty the microplate and add your samples and read again SoftMax Pro subtracts the pre read values on a well by well basis A1 from Al B1 from B1 etc Molecular Devices enabled the Pre read feature in earlier versions of SoftMax Pro because the early UV transparent microplates had high and variable background OD values Use of Pre read dramatically improved the quality of the data at that time Now however
88. column SPEED READ ABSORBANCE ONLY ENDPOINT KINETIC AND SPECTRUM SCAN ONLY Speed Read uses fewer flashes per read in order to decrease read time Speed Read turns off AutoCalibration and uses the air calibration values stored in the instrument As a result Speed Read may not provide as accurate an absorbance measure ment of each wavelength in the scan as with the normal read COLUMN WAVELENGTH PRIORITY Choosing column or wavelength priority determines the order in which a plate is read in multi wavelength readings 5 In column priority an instrument reads the entire microplate or chosen number of strips at the first wavelength and then goes back and reads the microplate at the sec ond and subsequent wavelengths 5 In wavelength priority the instrument reads all wavelengths for the first column of wells in the microplate and then reads all wavelengths for the second column erc For most instruments there is a choice between column or wavelength priority when doing Endpoint or Kinetic readings for all read modes If Column Wavelength priority choices are not offered the following priorities are used by default gt Endpoint Kinetic column priority 5 Spectrum Well Scan Fast Kinetic Flex wavelength priority SoftMax Pro Software User Guide 0112 0140 Rev A 48 4 Data Collection 4 2 27 AUTOREAD 4 3 4 3 1 AutoRead enables the automatic reading of all Plate sections in the order in whi
89. commends that customers consult a third party automation company if internal software resources or expertise are not available All leading automation vendors have integrated SoftMax Pro software and Molecular Devices instruments with their robotics systems A latest list of approved robotics part ners can be found on the Molecular Devices website If you are a robotics system vendor or an individual integrator and you need more infor mation please contact your local Molecular Devices sales representative or send an email with the subject SoftMax Pro amp Robotics describing your interest to support moldev com SAMPLE SOURCE CODE AND APPLICATIONS Automation sample source code and applications are included as a part of the SoftMax Pro installation package The sample source code is available as Visual Basic Visual C or Excel Macro projects and can be found in the Automation Examples folder in the Soft Max Pro application directory REMOTE CONTROL SoftMax Pro software can be controlled by other Windows applications with simple ASCII string based messages Using the Microsoft Windows Win32 SendMessage func tion SoftMax Pro remote command strings such as ReturnStatus Read or SaveAs myFile can be sent as a Windows message to SoftMax Pro for remote process execution SEND RECEIVE REMOTE COMMANDS The user can choose between sending remote commands using Windows message WM_SETTEXT or WM_COPYDATA SoftMax Pro Software User
90. created by duplicating existing Group sections SoftMax Pro Software User Guide 0112 0140 Rev A 3 6 Sections 3 6 6 Group sections are divided into gt Tool bar gt The body of the Group table gt Summaries Group Settings Use the Group gt Group Settings dialog box to edit the Group name descriptors units and format The Column Format field in the Group Settings dialog specifies the default columns for the data calculated and reported in the active group Delete Group You can delete a selected group with the Edit gt Delete Group Name command Deleting a Group section automatically deletes references to that group from the Tem plate Duplicate Group You can duplicate a selected group with the Edit gt Duplicate Group Name command Group section tables can also be duplicated When a Group table is duplicated its name is added to the Template Editor s list of group names but the group is not assigned to any wells CUVETTESET SECTION CuvetteSet sections are used to collect data from the cuvette port of the SpectraMax M2 M2 M5 M55 Plus or Plus384 to define an analysis template and to define data display and data reduction The Template Editor in the CuvetteSet section is used to describe the contents of the Cuvette CuvetteSet sections are divided into gt Tool bar gt Data display can be shown in three different ways three samples per row one sample per row and in a grid
91. ct data because SoftMax Pro has no way to verify the identity of the filters in the filter wheel The SpectraMax 250 accepts two optional filters but the wavelengths for these filters are entered using the instrument s front panel For specific information regarding how to install optional filters in the SpectraMax 250 refer to the instrument manual Serial Port Check that the setting for the serial port matches the physical port used to connect the computer and the instrument If you are using a Keyspan serial adapter with a Mac computer set the port to Printer None Select None to use the serial ports on your computer for other purposes such as a modem connection a network connection or printing SoftMax Pro Software User Guide 0112 0140 Rev A 2 Installation amp Setup 2 5 2 When None is selected the Status Bar in SoftMax Pro shows an instrument icon with a through it along with the words No port selected You cannot communicate with your instrument Port Speed When a FlexStation instrument is selected as the reader a Port Speed option is available SoftMax Pro automatically detects the baud rate of the instrument to which the computer is connected and establishes a communication rate accordingly This option allows you to view the baud rate that is being used and to manually set a different rate This should not be done unless a serial communications problem occurs MANUAL EXPORT FORMAT Data collec
92. d Fluorescence read mode displays Inte gration Delay and Integration next to the read type selection instead of Start and End Fluorescence Polarization RFUs By using a fluorescent dye to label a small molecule its binding to a large molecule can be monitored through its speed of rotation Fluorescence Polarization mode returns two sets of data one for fluorescence intensity parallel P to the excitation plane and the other for fluorescence intensity perpendicular S to the excitation plane These S and P values are used to calculate the Polarization mP and Anisotropy r values in SoftMax Pro Although the Raw S amp P value is the true SoftMax Pro Software User Guide 0112 0140 Rev A 4 2 Instrument Settings 4 2 5 raw data returned from the instrument the calculated Polarization mP and Anisotropy r values are treated as the raw data and these values become the basis for further reduc tion calculations You can choose to display any of these data types on the Plate section When Raw S amp P is displayed the mP value is by default used for all reduction calcula tions When exporting the displayed raw values are exported whether mP r or Raw S amp P Polarization mP is calculated as follows _ parallel G x perpendicular PEOD parallel G x perpendicular Anisotropy r is calculated as follows pi parallel G x perpendicular parallel 2G x perpendicular WAVELENGTHS With
93. d for each well is the excitation wavelength of max imum fluorescence Exponential Curve Fit The exponential function used to generate this curve fit is y A B 1 exp x C Flex A read type using fluorescence that is available with the FlexStation instrument You can specify compound additions and trituration for individual wells Data is collected over time with readings taken at regular intervals Flex runs are measured in seconds and frac tions of seconds Flex readings can be single wavelength or multiple wavelength up to four FLIPR Molecular Devices FLIPR Fluorometric Imaging Plate Reader system was developed to perform cell based high throughput screening HTS assays Fluorescence The light emitted by certain substances resulting from the absorption of incident radia tion To measure fluorescence accurately it is necessary to reduce light scatter SoftMax Pro Software User Guide 0112 0140 Rev A 139 140 A Appendix The governing equation for fluorescence is Fluorescence extinction coefficient concentration quantum yield excitation inten sity pathlength emission collection efficiency Fluorescence Polarization By using a fluorescent dye to label a small molecule its binding to another molecule of equal or greater size can be monitored through its speed of rotation SpectraMax M5 and MS Fluorescence Polarization read mode returns three different raw data types Raw S a
94. dates to show the data represented by a heat map STEP 8 DATA ANALYSIS GROUP SECTIONS SoftMax Pro creates Group sections automatically when you define groups in the Tem plate Editor The content of Group sections depends on the wells that are assigned to the groups in the Template 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 Scroll down until the Standards group is visible The columns in this group are set up as follows from left to right 5 Sample the sample names assigned for the Standards group gt Concentration the concentrations assigned to each sample in the template 5 BackCalcConc Returns the curve fit associated with the plot of Standards from the Standard Curve against the Values column SoftMax Pro Software User Guide 0112 0140 Rev A 93 94 6 Tutorials gt Values raw OD values obtained for each sample gt MeanValue the mean of data from the Values column for each sample gt StdDev standard deviation for each sample value gt CV coefficient of variation for each sample value In this part of this tutorial you will view formulas associated with columns and Summa ries resize individual columns Autosize all the columns add a column and hide repli cates Viewing Formulas 5 To view the formulas associated with all columns in the Standards g
95. dialog box The default reduction setting for a single measurement wavelength Lml is fine For Absorbance instruments make sure that Absorbance is chosen as the Data Mode and that Use plate blank is not checked Click OK to close the dialog box STEP 5 SET DISPLAY PARAMETERS 1 2 3 Click the Display button to open the Display dialog box The default display for Endpoint readings is raw OD for Absorbance instruments and raw RFU for Fluorescence instruments It is not necessary for the purposes of this tutorial to show a reduced number so make sure the check boxes next to the options under Show are not checked If you decide later that you would like to view the reduced data you can change the display options after the reading since this does not affect the actual data only how it appears on the screen and printout Click OK to close the dialog box STEP 6 SAVE THE PROTOCOL 1 2 3 Choose the File gt Save As command On the Mac select the popup menu command SoftMax Pro Protocol On Windows select Pro Protocol Files ppr from the Save as type list and give the file the name Endpoint Tutorial in the File name field Click Save to save a copy of the protocol file to the hard disk Close Tutoriall pda You are asked whether or not to save changes say Dont Save Use File gt Open to open the ppr file you just created This creates an untitled data file Congratulations You have just created a Sof
96. e SetFolderAs c SoftMaxPRO Basic Protocols SetPort X Tell SoftMax Pro to set a serial COM port SetTemp XX X Send a command to set the current instrument incubator temperature Setting temperature to zero turns off the incubator Shake On Off This command if set to On starts shaking the microplate tray until the Shake Off com mand is sent By default the shake will stop after 30 seconds in most instruments Example Shake On Stop Send SoftMax Pro a command to stop reading If queue operation is enabled this com mand is not queued the queue is cleared and the command is processed immediately This is similar to clicking the Stop button on the SoftMax Pro tool bar except the Stop but ton does not affect remote command processing or its queue This command should be sent before any Close or Quit command if there is a possibility that the instrument is still reading If the data has not been saved the application quits with no user warning TDrawer FlexStation only This command returns the status Open or Closed of the tip rack drawer SoftMax Pro Software User Guide 0112 0140 Rev A 131 9 Robotics amp LIMS Integration TrayLock On Off When sent before a read this command keeps the microplate tray locked inside the instrument after a read if the parameter is set to On If set to Off the tray comes out Example TrayLock On Version Returns the version of the active So
97. e another after both chambers have reached equilibrium During warm up however you may notice a discrepancy in temperatures which is normal READ If a Plate or CuvetteSet section is active or if only a single Plate or CuvetteSet section has been created in the Experiment section clicking the Read button starts the reading This reading is based on the settings chosen in the Control Instrument Settings dialog box If more than one Plate or CuvetteSet section has been created in the Experiment section and no Plate or CuvetteSet section is currently active clicking the Read button opens a dialog box asking you to select the plate or cuvette to read If AutoRead is selected in the Instrument Settings all of the plates cuvettes will be read sequentially REFERENCE BUTTON This feature is active only when a CuvetteSet is selected Use the Reference button to mea sure a reagent blank The Reference value is stored and subtracted automatically from the Read values The reference reading may be taken before or after the sample reading This setting applies only to the SpectraMax M2 M2 M5 MS5S Plus and Plus384 STAKMAX BUTTON Displays the StakMax software interface allowing you to control the StakMax microplate handling system The StakMax software interface accessed through SoftMax Pro is the only method of interacting with the StakMax instrument When the StakMax software is launched it automatically connects to the StakMax inst
98. e assay and could be set before data is read When a fit is performed the parameter values are displayed in the legend at the bottom of the graph The correlation coefficient describes how well a change in x values correlates with a change in the y values The R2 value should only be used for linear curve fits 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 Linear 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 Semi Log The semi log function fits the best straight line to a set of data for log x plotted 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 log x SoftMax Pro Software User Guide 0112 0140 Rev A 80 5 Data Analysis where A is the y intercept of the line and B is the slope log in this equation is the common or base 10 logarithm 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 th
99. e command or the Template button the same applies to CuvetteSet sections gt Select one or more wells by clicking and dragging on the grid gt Non contiguous selections can be made by Shift clicking individual wells gt Clicking and dragging row or column labels selects entire rows or columns gt Non contiguous row or column selections can be made by 3 clicking Mac or Ctrl clicking Windows row or column labels gt Double clicking anywhere within a group selects the entire group gt Clicking in the empty border around the Template deselects all wells SoftMax Pro Software User Guide 0112 0140 Rev A 4 3 Template Edftor 4 3 2 GROUP Use the groups in the Group list to assign groups to well selections on the plate grid T he list contains two default entries Blank and New and any user defined groups gt Blank Creates a plate blank in the active section The name blank is reserved for use by SoftMax Pro and should not be used when creating custom groups 5 New Opens the Group Settings dialog box in which you can create a new group Group Settings The Group Settings dialog box allows you to define the name for a group of related sam ples a descriptor associated with the samples and the initial column format for the data calculated and reported in the associated Group section Whenever a new group is created whether or not wells are selected in the template a Group section is created in yo
100. e data points It measures the degree to which the points fall on the calculated 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 inclusive 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 perfect fit you can simply apply one of the other types of curves SoftMax Pro Software User Guide 0112 0140 Rev A 5 4 Group and Graph Sections For non linear curve fits SoftMax Pro 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 SoftMax Pro Software User Guide 0112 0140 Rev A 85 86 5 Data Analysis SoftMax Pro Software User Guide 0112 0140 Rev A 6 Tutorials The hands on tutorials included here are designed to take you through the basic features of SoftMax Pro gt Tutorial 1 leads you through the steps required to create and run a quantitative proto col using
101. e logarithm of the dose on the X axis The resulting display is a straight line with both axes drawn in loga rithmic scale The equation for the log log fit is log10 y A B log10 x where A is the log10 Y intercept of the line for log10 x 0 and B is the slope log in this equation is the common or base 10 logarithm Quadratic The quadratic function fits the best parabola to the data The parabola is a curved line based on the equation y A Bx Cx 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 mea sure of the curvature of the parabola The quadratic fit is most appropriate when the standard curve has a tendency to curve up or down 4 Parameter Logistic 5 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 4 parameter or 5 parameter fit Both logis tic and log logit fits are based on the equation 42 up x o where D is the Y value corresponding to the asymptote 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 corresponding to the mid point between A and D commonly called the IC50 or EC50 The coefficient B describes how rapidly the curve makes its transition from the asymptotes in
102. e over all other fluidics module set tings COMPOUND amp TIP COLUMNS FLEX ONLY When one or more compound transfers are enabled use these settings to choose the tips and compounds that are used for transfers This tab displays 5 One two or three transfers matching the setting made in the Compound Transfer tab gt The Tips Column list contains tip numbers matching the selected columns in the Pipette Tips tab 5 The Compound Column list contains numbers from 1 through the number of columns of the compound plate or the number of troughs selected in the Compound Source tab A Tips T or Compound C row has up to 12 columns to represent the number of col umns in a 96 well assay plate The actual number of columns and their placement further depends on the number of columns and their locations as selected in the Wells to Read tab To assign a tip to an assay plate well 1 Highlight one or more tip cells SoftMax Pro Software User Guide 0112 0140 Rev A 45 46 4 Data Collection 2 Select one of the items available in the Tips list Choosing Fill fills the selected cells with the tip numbers that correspond to the same column numbers tip 3 with column 3 Note Fill does not take into account the actual tips that are available if less than a full rack is used the choices in the Fill list represent the actual tip numbers that are specified as being available in the Pipette Tips Layout tab Similarly to assign
103. each plate is read The report contains information from each section that is designated to be included in the report For more information on reports and including or excluding sections see Section 7 2 Printing THE PROTOCOLS MENU The Protocols menu is a dynamically generated menu listing all top level folders and the protocol files they contain When SoftMax Pro software is installed several folders each containing predefined protocol files for use with SoftMax Pro software are automatically created within the Program Files Molecular Devices SoftMax Pro folder To change the folder used to generate the Protocols menu choose Protocols gt Set Folder and navigate to the desired folder This allows you to display a custom set of protocols that your company may be using instead of the set that ships with SoftMax Pro software Additionally custom folders can be created within Program Files Molecular Devices Sofi Max Pro Once a protocol file ppr or epr file is placed in this custom folder both the folder name and the protocol name are listed in the Protocols menu Some protocols will not be appropriate for your particular instrument they might have reductions that are appropriate only for Absorbance or Fluorescence instruments for example so please check the Revision Notes section located at the top of each MDC authored protocol file for reader suitability before using All protocol files can be opened run and cust
104. eference reading is applied to all cuvettes in the CuvetteSet section unless you have highlighted specific cuvettes see below 5 The time and date of the reading are displayed under each cuvette in the section three cuvettes per row and one cuvette per row displays only gt The reference is applied to any new cuvettes subsequently created in that section You can have more than one reference per CuvetteSet section If you have cuvettes to which you want to apply a different reference 1 Highlight the cuvettes in the CuvetteSet section 2 Place the appropriate reference in the cuvette port 3 Click the Ref button or select the Control Ref command 4 The time and date stamp of the reference for the selected cuvettes changes Any new cuvettes created in the CuvetteSet section after reading a second reference use the new reference reading SoftMax Pro Software User Guide 0112 0140 Rev A 57 4 Data Collection The reference read is automatically applied to the normal read The reference value is not displayed in SoftMax Pro If you wish to see the reference value you can choose one of the following procedures Procedure 1 1 Create a cuvette in the CuvetteSet section and Ref on air empty cuvette port 2 Read a cuvette containing the blanking solution If you want to have both reference and read values available do the following 1 Create a CuvetteSet with up to the maximum of 96 cuvettes 2 Define a template b
105. ell or cuvette Further analysis of this reduced number is done in Group and Graph sections SoftMax Pro performs calculations hierarchically when reducing raw information col lected from the instrument Calculations in the Plate section or CuvetteSet section are performed in the order shown below If an option has either not been selected in the Instrument Settings dialog box is not available for the instrument you are using or has not been defined in the template or Reduction dialog box SoftMax Pro continues with the next listed calculation Plate Section 1 Convert OD to T 2 Pre read plate blank or plate background constant subtraction or division for 96T 3 PathCheck pathlength correction if available different equations for OD and T 4 Plate blank subtraction 5 Wavelength reduction SoftMax Pro Software User Guide 0112 0140 Rev A 65 66 5 Data Analysis 5 3 1 5 3 2 6 Kinetic or Spectrum reduction 7 Group blank subtraction CuvetteSet Section 1 Convert OD to 96T 2 Reference is subtracted from the reading optical density mode or the read data is divided by the reference 96 T mode but see below 3 Template blank subtraction 4 Wavelength reduction 5 Kinetic or Spectrum reduction 6 Group blank subtraction ENDPOINT READS Wavelength Combination The default reductions for Endpoint readings are Lm1 Pathlength and Custom for all instruments when you read at a single wavele
106. en using a cuvette the pathlength is known and is independent of sample volume so absorbance is proportional to concentration In a microplate pathlength is dependent on the liquid volume so absorbance is propor tional to both the concentration and the pathlength of the sample Standard curves are often used to determine analyte concentrations in vertical beam photometry of unknowns yet errors can still arise from pipetting the samples and standards The Path Check feature automatically determines the pathlength of aqueous samples in the micro plate and normalizes the absorbance in each well to a pathlength of 1 cm This novel patented approach to correcting the microwell absorbance values is accurate to within 2 5 of the values obtained directly in a 1 cm cuvette vertical light path horizontal light path cuvette microplate wells Figure 4 1 Cuvette and Microwell light paths Reference measurements made by reading the cuvette or using factory stored values derived from deionized water can be used to normalize the OD data for microplate wells PathCheck is used to normalize the data acquired from absorbance endpoint microplate readings to a 1 cm pathlength correcting the OD for each well to the value expected if the sample were read in a 1 cm cuvette The SpectraMax Plus Plus384 M2 M2 M5 and M5 offer two methods of pathlength correction the first uses a cuvette reference and the second uses a water constant that is stored
107. ent in the file Graph Options Use the Graph Options button or Graph gt Graph Options to configure the style and con tent of a graph You can select from three types of graphs gt Scatter with symbols plotted and the points connected 5 Cluster Bar gt Stack Bar Select New to add a new plot to the graph from any of the Group sections in the Experi ment X Axis Use the X Axis button or Graph X Axis to edit X axis parameters tick marks scale label gridlines and auto range Y Axis Use the Y Axis button or Graph gt Y Axis to edit Y axis parameters tick marks scale label gridlines and auto range Export Graph Exports a graph to file On the Mac the graph is saved in PICT format on Windows it is saved in EMF Enhanced Metafile format SoftMax Pro Software User Guide 0112 0140 Rev A 3 7 Creating a Default Protocol 3 7 CREATING A DEFAULT PROTOCOL While earlier versions of SoftMax Pro software opened to a specific Tutorial template SoftMax Pro versions 4 8 and later open to a blank protocol that matches the instrument connected to the computer If no instrument is connected to the computer and if no default protocol file exists Soft Max Pro uses the instrument chosen in Edit gt Preferences to create a new protocol To use a customized protocol file as your default template every time SoftMax Pro is launched you must specifically save a file as your default protocol
108. er sections such as Groups can also be copied The SelectAll command should be used first Drawer This command returns the status Open or Closed of the assay plate drawer ExportAs a filepath Tell SoftMax Pro to export data in the standard SoftMax Pro format The ExportAs command without the a attribute overwrites any existing file without warning However you can choose to append export data to the end of an existing file by specifying the a attribute In addition this exported file can then be imported and all data can be viewed as a single document file Please note that when using the attribute a a single space is required between the attribute and the following directory and file string Example ExportAs c SoftMax Prolexportfile txt ExportAs a c SoftMax Prolexportfile txt GetAutosaveState Returns the AutoSave state Return value is one of following 1 AutoSave is turned on 2 AutoSave is turned off 3 AutoSave is enabled but the specified format is not SoftMax Pro file pda format GetNumPlateSections Returns the number of Plate sections in active document ImportTemplate filepath Have SoftMax Pro import a template file gt The file should comply with SoftMax Pro s template file format gt A Plate section should be selected prior to importing a template Logoff SoftMax Pro GxP only This logs off the currently logged on user in SoftMax Pro GxP Logon user_id password SoftMax Pro GxP
109. es from 0 through 9 based on user defined limits Values above the high limit are displayed as plus and values below the low limit are displayed as minus SoftMax Pro Software User Guide 0112 0140 Rev A 143 144 A Appendix Raw Data Signal reported from the instrument with no alteration This is reported as OD RFU or RLU depending on the instrument type Read Interval The interval of time between the start of one reading and the start of the next reading dur ing a Kinetic or Flex run Read Type The method used to read the microplate or cuvette Endpoint Kinetic Spectrum Well Scan or Flex Not all types are available with all instruments Read Mode The type of reading performed in the instrument Fluorescence Time Resolved Fluores cence Luminescence Absorbance or Fluorescence Polarization Readings per Well The number of times programmable by the user that readings are taken on a well in Flu orescence and Time Resolved Fluorescence modes This setting also determines the amount of time that data is collected in Luminescence mode Reduced Data Data reduction causes the raw signal values reported by the instrument to be calculated and displayed according to user defined formula settings Reduction Limits Sets limits on data to be included in reduction for Kinetic Spectrum Well Scan or Flex readings Reference A reading of buffer or water taken in the cuvette that is used as T to c
110. ese limits are not shown and are excluded from data reduction Minimum Reduction for a Spectrum scan that reports the minimum signal within the reduction parameters MinOD MinRFU or MinRLU The limit for the minimum value you want to use for display and analysis of Kinetic or Spectrum scan data Any values that are under this limit are not shown and are excluded from data reduction The default is 0 To display negative Kinetics the value should be set below 0 zero Normal Display The default display for 384 well plates in the Plate section Onset OD Onset RFU or Onset RLU The change in signal required to compute the onset time for Kinetic readings Onset Time The time it takes for a given increase in signal called the onset OD or RFU or RLU to occur Optical Density OD The amount of light passing through a sample to a detector relative to the total amount of light available Optical Density includes absorbance of the sample plus light scatter from turbidity SoftMax Pro Software User Guide 0112 0140 Rev A A 1 Glossary of Terms PathCheck Instrument Settings option within SoftMax Pro for the SpectraMax M2 M2 M5 M5 Plus Plus384 190 or 340PC that allows data collected in a Plate section to be normal ized to a 1 cm pathlength Photomultiplier Tube PMT A vacuum tube that detects light especially from dim sources through the use of photoe mission and successive instances of secondary emis
111. etic reductions are applied to the value at each time point after the wavelength combi nation formula is applied VMax per min and VMax per sec reductions are available for all instruments that are capable of Kinetics the default Kinetic reduction is VMax VMax VMax is the maximum slope of the Kinetic display of mOD min or RFU RLU per second VMax is calculated by measuring the slopes of a number of straight lines where VMax Points see below determines the number of contiguous points over which each straight line is defined VMax Points This setting defines the maximum size of the line segment used to determine the slope of the line used in calculating the rate of the reaction The default is the total number of points taken in the reading reading except on the FlexStation instrument where the default is 5 The first slope is calculated for a line drawn beginning at the first reading as defined by Lag Time described below and ending at a total number of readings equal to the VMax Points setting The second and any subsequent slopes are calculated beginning at the second time point and ending at a total number of readings equal to VMax Points The steepest positive or negative slope is reported as VMax If the data plot displays fewer time points data points than VMax Points all of the time points are used to determine the slope of the data Time to VMax This is an alternative method for analyzing non linear Kinetic reactions
112. ftMax Pro 9 7 VISUAL BASIC EXCEL MACRO EXAMPLE Declarations Private Declare Function Find Window Lib user32 Alias Find WindowA _ ByVal IpClassName As String ByVal IpWindowName As String As Long Private Declare Function SendMessage Lib user32 Alias SendMessageA _ ByVal hWnd As Long ByVal wMsg As Long ByVal wParam As Long _ ByVal IParam As String As Long Private Declare Function RegisterWindowMessage Lib user32 Alias _ RegisterWindowMessageA ByVal mgsName As String As Long Const WM SETTEXT 12 No of seconds that GetMsgFromPro will wait before timing out Const mcsngMsg Timeout As Single 600 Public Sub Start Start the main form MainForm Show End Sub Public Function SendMsgToPRO ByVal msgStr As String As Long Function to send windows command to SoftMaxPro application This is the primary method of communication between the two processes on the same PC Dim IVal As Long hWnd FindWindow SOFTMaxPROMainWnd SOFTmax Pro If hWnd 0 Then 132 SoftMax Pro Software User Guide 0112 0140 Rev A 9 7 Visual Basic Excel Macro Example Beep Else softmaxMsg RegisterWindowMessage SOFTMaxPROMsg If softmaxMsg 0 Then Beep Beep Else IVal SendMessage hWnd WM SETTEXT softmaxMsg msgStr End If End If End Function Public Function GetMsgFromPro ByVal strMessage As String As String First send a request message to the instrument and then get back a text message from
113. fy the reduction of the data collected from the microplate reader typically to a sin gle number per well SoftMax Pro Software User Guide 0112 0140 Rev A 3 6 Sections Options available in the Reduction dialog box depend on the type of reading performed Endpoint Kinetic Spectrum Well Scan or Flex and the instrument used to collect the data See Chapter 5 Data Analysis for more details on Data Reduction Graph The Graph button or Plate gt Graph creates a graph from the data in the selected wells in the active Plate section It is enabled both during data collection and after data has been acquired for a Kinetic Spectrum Well Scan or Flex reading Double clicking a well also creates a graph of the data in that well Graph is disabled if the instrument settings are for an Endpoint reading Mask The Mask button or Plate gt Mask allows you to remove selected wells from the data analysis Mask is a toggle so that selecting masked wells and choosing the Mask command unmasks them Copy and Paste Data Data can be copied from one Plate section to another or from one CuvetteSet section to another within SoftMax Pro 1 Make the source section active by clicking on it 2 Select Edit gt Copy 3 C Ctrl C 3 Create a new section if necessary or make the destination section active 4 Select Edit gt Paste V Ctrl V When copying Plate sections all wells are copied even if only a subset of wells
114. g a data file as a protocol file removes any data in the file leaving only the configura tion information Creating a Default Protocol If you frequently find yourself adding the same sections Graph Group etc to the major ity of your Experiment data files or if you want to customize the document window that is opened every time you launch SoftMax Pro e g to include Summaries or other infor mation in the Notes section you can create your own default protocol file Create the protocol by opening the document you want to be the default protocol and select Protocols Save As Default Protocol This file resides in the Protocols folder Whenever you launch SoftMax Pro or use the File New command the document cre ated will be based on your default protocol file Resetting the Default Protocol Folder The Protocols menu is a dynamically generated menu listing all the folders one level deep and protocol files in a selected folder To change the folder used to generate the menu choose the Protocols gt Set Folder com mand and navigate to the desired folder SoftMax Pro Software User Guide 0112 0140 Rev A 105 106 7 File Management amp Printing ORGANIZING PROTOCOL AND DATA FILES In a multi user environment it is usually helpful to gt Create a protocol directory for each SoftMax Pro user or gt Create separate subdirectories for different types of experiments CREATING NEW FILES The File gt
115. g the maximum value obtained for each sample SoftMax Pro Software User Guide 0112 0140 Rev A 6 1 Tutorial 1 Quantitative Endpoint Protocol Tutorial Viewing an If Statement in a Column Formula The Unknowns Group section contains a column called Outliers The formula for this column uses an If statement that uses data from another Group section to report when data points lie outside the range of the minimum and maximum Standard values An If statement in a formula defines a condition that if found to be true gives a certain result and if not true gives a different result The basic structure of an If statement is If condition result if true result if false The Outliers formula is as follows If Values gt MinStd Standards and Values lt MaxStd Standards Outlier This statement says that if the data shown in the Values column 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 report nothing signified by two quotation marks with nothing between them otherwise report the word Outlier The MinStd or MaxStd calculations are used in the Summaries at the bottom of the Stan dards section but the names are not displayed by default To display them 1 Double click the first Summary Smallest standard value to open the Group Edit Summary dialog box 2 Uncheck t
116. ghtly This view should assist you in determining the best limits for reduced data Show Raw Show Reduced Toggles the Well Graph between views of raw and reduced data When the Well Graph is opened the initial view depends on the Display settings for the Plate section 5 If raw data is being displayed in the Plate the initial view in the Well Graph is raw data and the button is set to Show Reduced gt If reduced data is being displayed in the Plate section the initial view in the Well Graph is reduced data and the button is set to Show Raw Scale to Data Scale to Limits 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 visualize how the limit settings affect data reduction 5 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 gt 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 is 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 and reduced displays this button must be set to either Scale to Limits or Scale to Data
117. h otherwise blank field SoftMax Pro Software User Guide 0112 0140 Rev A 7 3 Import Export cuvettes Field Field Entry Description 13 End wavelength Spectrum end wavelength otherwise blank field 14 Wavelength step Spectrum wavelength step otherwise blank field 15 of wavelengths of wavelengths in Endpoint Kinetic Well Scan Flex reads for Spectrum blank field 16 Read wavelengths For Absorbance Luminescence wavelengths read separated by a space for Spectrum blank field for Fluorescence emission wavelengths for Ex Em sweep blank field 17 First column read First column read 1 to max always 1 for cuvettes 18 Number of columns Last column read 1 to max always 12 for cuvettes 19 Number of wells or Number of wells in a plate 24 96 384 etc or number of cuvettes read for a CuvetteSet Luminescence After Field 19 the format splits depending on the read mode Fields 20 28 are not present for Absorbance or Transmittance but are included for Fluorescence and 20 Excitation wavelengths Excitation wavelengths separated by spaces For Luminescence or sweeps this is a blank field the emSweep excitation wavelength is in field 16 21 Cutoff option Manual or Automatic 22 Cutoff filters Cutoff filter wavelength values separated by spaces 23 Sweep wave option EmSweep or ExSweep or blank field 24 Sweep fi
118. he Hide Name option and click OK MinStd and MaxStd are now displayed next to their Summaries at the bottom of the group 3 The formulas for MinStd and MaxStd are Min MeanValue Max MeanValue The MeanValue column reports the average of the well values in the Values column the Standard replicates and these are used in the Outliers formula Such a formula might be useful in finding points that might be better masked and thus not used in the calculations Fora complete discussion of writing formulas in SoftMax Pro as well as detailed examples of formulas and customized protocols see the SoftMax Pro Formula Reference Guide Showing and Hiding Replicates 5 Use the Group Hide Replicates command to hide individual replicate values Where appropriate average values are shown instead gt Show replicates again with the Group gt Show All command SoftMax Pro Software User Guide 0112 0140 Rev A 95 96 6 Tutorials 6 2 STEP 9 DATA ANALYSIS STANDARD CURVE 1 Scroll down the SoftMax Pro window until you see the section tool bar for Graph 1 2 Click the indicator on the left side of the tool bar to open it 3 This graph is set to show concentration on the X axis and mean value on the Y axis 4 You can edit graph properties in the Graph gt Graph Options dialog box Step 10 Print a Report 1 SoftMax Pro has very flexible report printing The default setting in SoftMax Pro is for all Experiment sections to be incl
119. he center of well A1 must also include an offset to accommodate the angle of the optics Appropriate measurements should be taken in the lab to determine the correct settings for these fields Plate information is saved to the file Custom Plates in the SoftMax Pro folder Because this is specific to the computer on which it is saved when a file with a custom plate is opened on another computer the plate type is changed to the standard plate type STRIPS WELLS TO READ You can choose to read a subset of wells or columns of wells in the microplate Partial plate reading is available for all read types although it is most useful for fast Kinetic anal yses The time required for Kinetic readings can be significantly reduced using this setting since the instrument does not have to read the entire plate To enable a partial plate read highlight the columns to be read Columns do not need to start with 1 but must be contiguous If you enable a partial plate reading only the wells to be read are visible in the data dis play indicating that no data will be collected for the other wells all wells are present in the Template Editor however COMPOUND SOURCE FLEX ONLY Select the plate used to store fluid compounds that are withdrawn and then injected into plate wells during the run The type of plate used should match the actual plate used in the Compound Source drawer and it should have the same number of wells as the plate selected in
120. he columns in this group are set up as follows from left to right 5 Sample the sample names assigned for the Data group gt Wells the wells that are assigned to the individual sample names these correspond to the individual wells in the template gt Concentration the concentration assigned to each sample in the template 5 WellValues raw RFU values obtained for each sample gt MeanValue the mean of data from the Values column for each sample gt Activation sample activation relative to selected control group SoftMax Pro Software User Guide 0112 0140 Rev A 6 2 Tutorial 2 EC 50 Protocol Tutorial Flex only gt CV coefficient of variation percent for each sample value Viewing Formulas 5 To view the formulas associated with all columns in the Data group section hold down Ctrl Shift while the group is active or select Group gt Show Formulas A row of formu las is displayed beneath the column titles 5 If formulas are long they may not fit in the columns To see the complete formula for a particular column click once on a column and the formula is reported in the tool bar 5 To modify or change a formula for a selected column select the Group gt Edit Column command or dick the Formula button on the section tool bar Let s look more closely at the formula for 96 Activation gt Double click the Activation column The Calculation dialog box is displayed show ing the formula used to deri
121. he currently selected plate Generally this is used to send the data directly to the caller instead of using the clipboard If data is to be returned to the clipboard this command functions like the SoftMax Pro Edit gt Copy command when a plate is the currently selected section ReturnStatus This command returns the current instrument status This command can for example be sent to check whether the instrument is busy or reading or staying idle The status is an ASCII string that has gt Information about the instrument type and version gt Whether the instrument is busy or idle 5 The instrument s current temperature SoftMax Pro Software User Guide 0112 0140 Rev A 129 130 9 Robotics amp LIMS Integration gt The state of its drawer The exact format of this response depends on the type of instrument in use If SoftMax Pro failed to place data in the clipboard then no return status is available Example ReturnStatus tab cr ReaderType lt tab gt Version lt cr gt State lt tab gt Offline Busy or Idle Temperature lt tab gt XXX Drawer lt tab gt Open or Closed ReturnTiming This command sends back the number of seconds that it would take to read the current plate For multi plate documents the plate to be used is chosen in the same way as described in the Read command SaveAs filepath Tell SoftMax Pro to save the current document file The file is saved in the current Soft Max Pro
122. he data can be chosen for another plot within the same graph gt You can use this feature to plot Kinetic or Spectrum data in Graph sections For exam ple to plot Kinetic data from well A1 in a Graph section you can enter the formula AlLm1 for the Y Axis and the TimeRun accessor for the X axis 5 The entered formulas are 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 the dialog box Four sizes nine colors and nine styles are available from drop down menus SoftMax Pro Software User Guide 0112 0140 Rev A 7 78 5 Data Analysis The Graph Options Dialog Box The Graph gt Graph Options dialog box is displayed when you are editing or creating a graph It allows you to 5 Change the name and size of the Graph section 5 Select the type of graph to be displayed scatter cluster bar stack bar gt Change the fonts used on the title legend and axes gt Access the Plor dialog box for creating editing and deleting plots Each of the plots that is created for the Graph section is listed in the Plots field Choosing any of these plots provides an indication 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 the New button opens the Plot dialog box allowing you to create a new plot
123. he maximum signal level It then scans the 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 Maxi mum Time at Maximum Reduction for a Kinetic or Flex run that reports the time at the maximum signal within the reduction limits SoftMax Pro Software User Guide 0112 0140 Rev A 145 146 A Appendix Time at Minimum Reduction for a Kinetic or Flex 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 Kinetic or Flex reading is reached reported in seconds Triturate FlexStation only To mix a solution by means of aspirate and dispense cycles User Name The User Name is the name of the person who reads data into the shielded file The User name is entered after the Read button is pressed but before the reading begins VMax The rate reported as signal min milli OD RFU or RLU units per minute for a Kinetic or Flex reading 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 It may also be reported as units per second the default for Fluorescence and Luminescence modes VMax Points The number of points in the Kinetic or Flex curve included in each of a series of
124. he microplate drawer INSTRUMENT SETTINGS AND CONTROL In addition to the buttons on the Status Bar most commands for instrument control are in the Control menu INSTRUMENT SETTINGS Use Plate gt Settings to configure all parameters for a plate read such as the read type End point Kinetic Fast Kinetic Spectrum Well Scan or Flex read mode Absorbance Fluo rescence Luminescence Fluorescence Polarization Time Resolved Fluorescence wavelength settings Automix parameters pre read blanking strips PathCheck Auto Read timing for Kinetic runs Speed Read for Spectrum scans and plate type Each Plate or CuvetteSet section can have a different instrument setup The available options in Plate gt Settings depend on the type of instrument that is con nected to the computer or selected in Edit gt Preferences see Section 2 5 Setting Prefer ences Plate gt Settings is only available when a Plate section is selected in the data file READ In a single plate experiment Control gt Read reads the plate or cuvette immediately If no Plate section or CuvetteSet section is active the Read command displays a dialog box in which you select the Plate section or CuvetteSet section to read The default Plate section or CuvetteSet section selected by pressing the Return key is chosen on these criteria gt The selected Plate or CuvetteSet section if one is selected SoftMax Pro Software User Guide 01
125. i ThermoMax and FlexStation Data from Analyst and FLIPR systems can also be easily imported and analyzed SoftMax Pro GxP is a special software release that includes full user and permission con trol and audit trail generation in addition to all the data collection and analysis features found in the Standard edition With the audit trail features in SoftMax Pro GxP all Experiments are fully traceable and can be completely reconstructed SoftMax Pro 5 2 is available for PC compatible computers using Windows 2000 and XP and Mac computers using OSX 10 2 8 or greater MAJOR FUNCTIONS OF SOFTMAX PRO INSTRUMENT CONTROL SoftMax Pro allows you to set up and run a complete protocol for all instruments Instru ment settings can be saved as a protocol file and used repeatedly for reading different microplates or cuvettes All stand alone instrument functions can be controlled using the software In addition SoftMax Pro provides capabilities that are not available when using an instrument in stand alone mode such as user defined Kinetic run times read intervals Automix parameters etc SoftMax Pro Software User Guide 0112 0140 Rev A 1 Introduction 1 2 DATA COLLECTION AND DISPLAY SoftMax Pro collects and stores all raw data received from the instrument Data is dis played in a grid format that corresponds to the wells in a microplate all instruments or individual cuvettes using SpectraMax Plus Plus384 M2 M2 M
126. ic enter the total Run Time and the desired Interval between readings The resulting number of Kinetic reads is reported below the run time For Fast Kinetic enter the integration time followed by the number of times this integra tion should be repeated for each well before moving to the next well INJECTION AND DELAY SPECTRAMAX L AND LMAX 11384 ONLY The SpectraMax L and LMax 11384 are equipped with two injectors having variable vol umes One labeled M for measurement injects directly in the reading position and the other labeled P for pre injection injects into the preceding position High precision pumps are used for the injections ensuring both instantaneous sample mixing after injection and precise injection volumes The injection system can only be used with 96 and 384 well plates SoftMax Pro allows selection of one or both injectors for any well of a 96 well or 384 well plate the LMax 11384 can use both injectors in a 96 well plate only For the LMax 11384 you can define the injection volume and the post injection delay which is the time between the last injection and the start of the reading For the SpectraMax L you can define a large range of injection parameters SoftMax Pro Software User Guide 0112 0140 Rev A 4 2 Instrument Settings 4 2 9 4 2 10 INJECTION WELLS SPECTRAMAX L AND LMAX 11384 ONLY Use the Injection Wells options to define the wells to which P and M injections are applied To a
127. ighting 83 Visual Basic 132 VMax 67 146 VMax Points 67 146 W Weighting 83 Well Scan 29 146 Reduction 70 Wavelength Combination 70 Well Graphs 63 Well Scan Editor 41 Window Resizing 13 X XML 11 Z Zero Baseline 71 152 SoftMax Pro Software User Guide 0112 0140 Rev A
128. in the instrument The SpectraMax 190 and 340PC use the water constant only The actual pathlength Z of a solvent is found from the following equation Sample OD 1000 OD 900 d cm n SoftMax Pro Software User Guide 0112 0140 Rev A 35 36 4 Data Collection When a cuvette reference is used for pathlength correction the value of amp is obtained by taking OD measurements on the fluid in the cuvette at two wavelengths 1000 and 900 nm k Cuvette OD iooo ODo00 When the water constant is used for pathlength correction the value of k is obtained from the instrument This constant is saved in the instrument in the factory and may differ slightly from instrument to instrument Once the pathlength Z is found the following equation is used for the pathlength correc tion OD T OD sample cm d cm PathCheck on SpectraMax Plus Plus384 M2 M2e M5 and Mbe Cuvette Reference or Water Constant To enable PathCheck for Absorbance readings 1 Open Plate gt Settings and select Patb Check 2 Check the Path Check option 3 Select either Water Constant or Cuvette Reference see below 4 Ifyou have determined a plate background constant for your microplate enter it in the designated field see Use Plate Background Constant on page 38 The PathCheck measurement is based on the absorbance of water in the near infrared region between 900 nm and 1000 nm If the sample is completely aqueous has no tur bidity a
129. inimizing the weighted sum of squares error The possible fits evaluated by the software algorithms are constrained by assuming they are parallel functions related by a relative potency term The Graph section where PLA has been enabled then displays the curve fit parame ters relative potency relative to the selected standard curve the degrees of freedom from both the independent and constrained curve fits and the probabilities obtained from the F test and Chi squared test for parallelism if applicable New function accessors have been added to SoftMax Pro to quickly summarize this information in a Notes section Please see the SoftMax Pro Formula Reference Guide for additional details To use parallel line analysis with a standard curve 1 You need to have a Graph section containing at least two plots one a Standard and one a Test sample 2 Select 4 Parameter or 5 Parameter from the Fit list on the Graph section tool bar 3 Open the Parameter Settings dialog box 4 Check the Use Parallel Line Analysis check box and select your standard curve from the Standard Curve menu 5 Click OK Axis Settings Selecting Auto range draws the graph so that all data points are displayed by automatically adjusting the minimum and maximum range settings to encompass the data 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 calculated curve and th
130. initial volume 0 uL Triturate N a Pipette Tips Layout Full Rack Compound amp None Tip Columns Automix Off AutoCalibrate Once SoftMax Pro Software User Guide 0112 0140 Rev A 97 98 6 Tutorials 6 2 3 6 2 4 Final Check of Instrument Settings Click OK to accept the settings and close the dialog box STEP 3 DEFINE TEMPLATE 1 10 Click the Template button in the Plate section tool bar This opens the Template Editor showing all wells empty Select the first column of wells A1 through H1 Select New from the Group list to create a new group Enter Data in the Name field Select Unknowns Dilution from the Column Format list Select pg ml as the units for the dilution factor Click OK to close the dialog box The selected wells now show the default first sample name for the Data group Data01 The area in the top center of the Template Editor is now active showing the default values for Sample name and Dilution Factor In this tutorial the default value of 0 pg ml for the dilution factor is correct You can check the concentrations you have assigned to the template by pressing and holding the Ctrl Shift keys Click the Assign button or press the Return key on the keyboard to assign this dilution factor if this is not the case Select wells A2 through H2 Assign a dilution factor of 0 01 pg ml to these wells by entering 0 01 in the Dilution Factor field and clicking the
131. into volatile RAM when the instrument is turned on Every time SoftMax Pro Software User Guide 0112 0140 Rev A 4 2 Instrument Settings 4 2 16 you perform a plate or cuvette read unless you have turned off AutoCalibration new air calibration measurements are made at the wavelengths used for the read and the volatile RAM values are updated for those wavelengths If you are doing a Kinetic read an Auto Calibrate value is taken before each read If you read a plate or cuvette with AutoCalibration turned off the current air calibration values in volatile RAM are used without updating Turning off AutoCalibrate allows the instrument to complete readings more quickly and to begin the first reading of a Kinetic plate faster If Speed Read is turned on AutoCalibrate is automatically turned off Molecular Devices recommends that you periodically update the air calibration informa tion stored in the instruments NVRAM This is particularly important if you are using Speed Read reading cuvettes without a reference or running Kinetics in microplates with read intervals that are too short to allow AutoCalibration Note NVRAM air calibration update is not supported by VMax and Emax To update the instrument air calibration values use the Control gt Calibration command gt When you select the Calibrate Plate checkbox to update the plate air calibration values the instrument steps through each wavelength available to the instru
132. ions INSTALLING SOFTMAX PRO Follow these instructions to install SoftMax Pro 1 Insert the SoftMax Pro CD into the CD ROM drive 2 Windows Open My Computer and then double click the CD ROM drive icon to display its contents Mac Double click the CD icon to display its contents 3 Double dick the SoftMax Pro v5 2 Installer icon and follow the screen prompts UNINSTALLING SOFTMAX PRO Before uninstalling the program make sure to backup your data and any saved files to a folder outside of the SoftMax Pro folder because uninstalling deletes the entire SoftMax Pro directory SoftMax Pro Software User Guide 0112 0140 Rev A 2 Installation amp Setup 2 2 2 2 1 2 2 2 2 2 3 2 3 Windows To uninstall SoftMax Pro software click the Windows Start menu then Set tings then Control Panel Double click Add or Remove Programs then scroll down to find SofiMax Pro v5 2 Click this once to select it then click the Remove button This is the recommended method of removing SoftMax Pro software from a Windows based com puter since it also removes related information from the Windows Registry Mac To remove SoftMax Pro drag the SoftMax Pro folder to the Trash then empty the Trash RUNNING THE SOFTWARE FOR THE FIRST TIME COMMUNICATING WITH AN INSTRUMENT To ensure that SoftMax Pro can communicate with your instrument turn on the instru ment prior to starting SoftMax Pro If using a USB to serial adapter ple
133. ith an OD RFU RLU less than A 118 if 1Lmx B high for any OD RFU RLU greater than B and the OD RFU RLU of makeerr 117 any well that lies between A and B ILm1 For EMax VMax UVMax or ThermoMax instruments the choice available for reading at two wavelengths is designated 1 Ref While the instrument performs a reading at the two wavelengths you set one for the measurement and one for the reference data from these readings is automatically reduced to a single value in the display and you cannot view the data from the individual wavelengths Custom reduction formulas utilizing mathematical operators or terms can be used to obtain specific types of data The table below provides some examples of such formulas for Kinetic and Spectrum readings SoftMax Pro Software User Guide 0112 0140 Rev A 73 74 5 Data Analysis 5 3 8 5 4 5 4 1 Table 5 2 Kinetic and Spectrum Reduction Formula Examples Kinetic VMaxcorr combinedplot Reports VMax correlation coefficient for plots in all 96 IVMaxpoints readinterval wells VMax Delta combinedplot Reports the VMax Rate of the delta between each time IVMaxpoints readinterval point Spectrum or Kinetic Nthitem Lm1 X Reports the optical density at item X in the list of readings For example if you have a Kinetic run with 20 time points and X is 10 it reports the OD RFU RLU of the 10th time point Similarly if y
134. ive S evene ciet n a 99 Step 8 Data Analysis Group Sections v iss eei KK KK KK KK KK Ron d 100 Step 11 Printa Report 4x oos ec a ELA AW SA ELA AE EM WAW IE a 103 7 File Management amp Printing File Creation and Management WWW kk kk KK KK KK KK KK KK KK KK KK KK KK KK 105 Data Pl a HEE an En ERUNT E 105 Protocol Piles AV YE AE 105 Organizing Protocol and Data Files aiii ia KK KK KK KK KK 106 SoftMax Pro Software User Guide 0112 0140 Rev A Creating New Elles a LA bere tob pees V k Ede ie doe W he W A es Opening N Y A JM MJ J MJ Saving Files Manually gya u Kak a eka tee Ea N dya greed pb rur ra SAD padre Aah tas rt Printings cete ey eurer N N IM MM hM DM MA_U nAANND3 gt y_o 2a i__i iI on jjMJJA 8 NPE A Printing Reports escocia pr Ep uh RE e a EA aes Including or Excluding Sections i306 aria debebat KK KK KK KK Changing Section Order oys k sa nae la a pd Changing Text Fotindtzs 44 eiua sas ate KK KK KK KK KK KK KK KK kK KK kK ADA Lim port Export tinted nosis ln wh nln ahs eley baka whe lie ek wana edere ball Aporte eve pe Et yn dh De any eid eene tere us Importing and Exporting Templates isla o lie KK KK KK KK KK tios a Exporting Gr ph amp lt as i 2 a Copying and Pasting 2 abc P xul kk sad x File Protector ISI HE de Tot rte LE tee REC d UE del Settinp a Rss Word s rep red PESE MERI VES Q un CERRO Deni pol apod Changing a Password vi oed Eu ada
135. labeled and look very much like the table itself Following the data a blank line is included and then a line with a single termination field End Each subsequent data set in the file includes the lines described above a blank line and a line with the termination field IMPORT SoftMax Pro imports data in tab delimited ASCII text format allowing you to import data collected by Criterion Host Analyst AD HT Acquest Analyst Host Analyst GT ScreenPlay ScreenStation or FLIPR operating software FLIPR into SoftMax Pro Only data that can be displayed in Plate or CuvetteSet sections can be imported Import supports up to 384 well plates if you import 1536 well plate data only the top left quadrant of data is imported Import When data is imported a new Experiment is created in the open SoftMax Pro file for the imported data New Plate sections and CuvetteSet sections are created for each data set within the file Plate pre read and CuvetteSet reference data can also be imported but data for pathlength correction cannot SoftMax Pro Software User Guide 0112 0140 Rev A 7 3 Import Export 7 3 3 The time date stamp for a section containing imported data shows imported data rather than a date or time File gt Import Export gt Import Analyst allows you to import data files generated by Analyst instruments including Analyst AD HT GT Acquest and ScreenStation Files for import must be generated as verbo
136. lank in the CuvetteSet section 3 Place buffer in the cuvette and select Read 4 Click the Read button to read the remaining samples The optical density of the cuvette designated as the template blank is subtracted from the optical density of all the other cuvettes in the CuvetteSet section Procedure 2 1 Create a CuvetteSet with up to the maximum of 96 cuvettes 2 Reference and read the cuvette 3 Create a template with the appropriate groups 4 Create a column in each Group table with the formula wellprereadLm1 If you have a multiple wavelength reading and want to see the reference at each wave length create additional columns with the formula wellprereadLmx where x is 2 3 or 4 corresponding to the wavelength for which you want to see a reference The OD RFU RLU with the reference subtracted is displayed in the Values column The reference value for each sample is displayed in the custom column you have created SoftMax Pro Software User Guide 0112 0140 Rev A 4 4 Reading a Microplate or Cuvette 4 4 3 4 4 4 CALIBRATION For some instrument models Control gt Calibration performs an air reading at all available wavelengths and stores those values in the instrument When you select the Calibrate Plate checkbox to update the plate air calibration values the instrument steps through each wavelength one by one taking a reading through the air calibration hole at the edge of the plate holder drawer Th
137. late but they are applied only after the plate is read and can be changed at any time In these tutorials you will build a complete protocol based on the settings found in the Tutorial file which will include both hard and soft parameters You will save the com pleted protocol file prior to collecting data this file could then be used to generate future data files SoftMax Pro Software User Guide 0112 0140 Rev A 87 88 6 Tutorials 6 1 6 1 1 Before starting ensure that the instrument that is connected to the computer is chosen in the Edit gt Preferences dialog box 1 Choose your instrument from the Reader list if it is not already chosen 2 Click OK Note Some settings mentioned in the tutorials are not available for some instruments TUTORIAL 1 QUANTITATIVE ENDPOINT PROTOCOL TUTORIAL Tutorial 1 is designed for both Absorbance and Fluorescence instruments It assumes that you have read the previous chapters in the manual and have a basic understanding of the logic and operation of SoftMax Pro The following steps are required to create and run a quantitative protocol using a standard curve Create a new data file Define instrument settings Define template Set reduction parameters Set display parameters Save the protocol Read the plate start the simulator Data analysis Group sections o oco N O OC FP Y N Data analysis Standard curve 10 Print a report S
138. les in three different ways three samples per row one sample per row and in a grid of cells corresponding to a microplate format Data Mode Reduction option to view absorbance data as OD absorbance or Transmittance fluo rescence data as RFU relative fluorescence units or luminescence data as RLU relative luminescence units SoftMax Pro Software User Guide 0112 0140 Rev A A 1 Glossary of Terms Default Protocol A protocol file included with the SoftMax Pro program or created by a user that resides in the default folder The Default Protocol file defines the initial information that appears when you open SoftMax Pro or when you create a new file from within the program Both Mac and Windows versions of this file are called Default Protocol and both versions append the 3 character ppr standard or epr GxP extension Emission Spectrum Scan Measures fluorescence or luminescence across a spectrum of wavelengths for emitted light for a fixed excitation wavelength The default value reported for each well is the wave length of maximum emission either fluorescence or luminescence End Time Reduction parameter used to omit the end of a Kinetic Spectrum or Flex run from data reduction Endpoint A single reading made at one or more wavelengths Excitation Spectrum Scan Measures fluorescence at a single emission wavelength across a spectrum of excitation wavelengths The default value reporte
139. lt is 0 End Time Specifies the time at which to stop showing data in the display Any values occurring after this limit are not reported in the display The default setting is the total assay time Absolute Values This setting displays OD RFU RLU values unshifted as opposed to the default that offsets the first point to 0 0 and shifts all other data accordingly Enabling Absolute Values can cause some data points that do not fall within the default limits to disap pear To see more data yet still display absolute values increase the limits for MinOD RLU RFU and MaxOD RLU RFU SPECTRUM READS Spectrum Reduction The Spectrum reduction formula is applied to the list of numbers in each well values at each wavelength after the wavelength combination formula is applied The default reduc tion for Spectrum readings is Lambda at Maximum SoftMax Pro Software User Guide 0112 0140 Rev A 69 70 5 Data Analysis 5 3 4 Maximum Reports the maximum absorbance optical density or percent transmittance T RFU or RLU within the reduction limits Minimum Reports the minimum absorbance optical density or percent transmittance YT RFU or RLU within the reduction limits Lambda at Maximum Reports the wavelength at which maximum absorbance optical density or percent transmittance T RFU or RLU within the reduction limits Lambda at Minimum Reports the wavelength of minimum absorbance optical density or pe
140. mands below Don t forget that the tag value from Register WindowMessage above is sent with the command string See Microsoft documentation regarding the SendMessage function for more information SendMessage hwnd WM COPYDATA tag LPARAM amp cd Either a completion message or return data is returned Remote commands ReturnStatus ReturnTiming and ReturnData return data In either case data is received through an asynchronous windows message inside Win32 COPYDATASTRUCT type data packet For example a typical OnCopyData window callback is shown below where the string data retrieved is finally stored into a Microsoft CString object Note the use of variable replyTag discussed above which is used to isolate the correct windows message returned BOOL CUserDlg OnCopyData CWnd pWnd COPYDATASTRUCT cd if cd gt dwData replyTag String pointing to status CString retStatus char cd gt lpData j SUMMARIZING REMOTE COMMANDING SoftMax Pro software has two groups of remote command instructions those that do not return data and those that do Remote commands that DO NOT return data Commands that do not receive data are further broken into gt The user wants to receive a completion message after the command has been sent SoftMax Pro Software User Guide 0112 0140 Rev A 9 4 Send Receive Remote Commands 9 4 4 After receiving a remote command that used the WM_COPYDATA message SoftM
141. matic is not available for Fluorescence Polarization reads if Automatic was previously selected Fluorescence Polarization defaults to Medium For the SpectraMax M5 and M5 the PMT Sensitivity is always set to Automatic for Luminescence and Time Resolved Fluorescence read modes For the SpectraMax L when the PMT sensitivity is selected to be Low the instrument operates in analog mode to detect very bright signals When the PMT sensitivity is selected to be High the instrument operates with a higher PMT voltage than in the Low setting and simultaneous measurements are made in both analog and photon counting modes SoftMax Pro Software User Guide 0112 0140 Rev A 33 34 4 Data Collection 4 2 11 4 2 12 Target Calibration Wavelength The SpectraMax L supplies the option to choose a target wavelength for your experiment based on the assay you are using TIMING For Kinetic reads you can configure the total Kinetic run time and the time interval between readings After entering a total run time and an interval between readings the total number of read ings is calculated and reported The minimum read interval is determined by the instru ment and depends on many factors including the number of wavelengths Automix status the number of strips and the distance the instrument filter wheel must move If you are writing a protocol to be used with a different model instrument 1 Select Edit Preferences 2
142. mber option If you decide later that you would like to view reduced data you can change the display options after the reading since this does not affect the actual data only how it appears on the screen and when printed Click OK to close the dialog box STEP 6 SAVE THE PROTOCOL 1 2 3 4 5 6 Choose the File gt Save command On the Mac select the popup menu command SoftMax Pro Protocol On Windows select Pro Protocol Files ppr from the Save as type list and give the file the name Flex Tutorial in the File name field Click Save to save a copy of the protocol file to the hard disk Close Untitled 1 You are asked whether or not to save changes say Dont Save Use File gt Open to open the ppr file you just created Congratulations You have just created a SoftMax Pro protocol file STEP 7 READ THE PLATE If you were reading an actual microplate at this time you would 1 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 Place the filled microplate in the appropriate drawer of the FlexStation A new set of tips would also be loaded in the tip drawer SoftMax Pro Software User Guide 0112 0140 Rev A 99 100 6 Tutorials 6 2 8 Since we are using a saved file it is not necessary to load anything in the actual instru ment 1 Choose File
143. ment one by one taking a reading through the air calibration hole on the plate holder drawer These val ues are stored in the non volatile RAM until the next calibration is done gt When you select Calibrate Cuvette the instrument steps through each wavelength one by one taking air calibration values for the cuvette It is therefore important that the cuvette port is empty AutoCalibrate for Fluorescence and Luminescence Reads In Fluorescence and Luminescence modes there are no air calibration values Instead ref erence reads are made on the reference chip on the carriage before each plate read These reference reads are used to set the RFU RLU scale at each wavelength to be used in the read by adjusting internal parameters including PMT gain When you turn off AutoCal ibrate the instrument uses the setting from the previous read or from the default settings if there has been no read since the instrument was turned on Turning off AutoCalibrate allows the instrument to complete readings more quickly and to begin the first reading of a Kinetic plate faster However you should allow the instru ment to perform AutoCalibration of at least one plate prior to disabling this function WELL SCAN EDITOR Well Scan reads allow readings to be taken at more than one location within a well The Well Scan Editor allows the user to specify how a Well Scan is performed You can specify the scanning pattern and the scanning density Only odd num
144. ments provide quick access to different types of information without the need to open or refer to multiple files SELECTING AN EXPERIMENT An Experiment can be selected by clicking the Experiment title bar the area that shows the Experiment number with the mouse cursor Selecting an Experiment changes its tool bar from light gray to dark gray and allows you to manipulate the whole Experiment such as deleting or duplicating it or adding new sections Multiple Experiments can be selected by holding down the Shift key while clicking the individual title bars of the Experiments SoftMax Pro Software User Guide 0112 0140 Rev A 3 6 Sections 3 5 2 3 6 3 6 1 3 6 2 MANIPULATING EXPERIMENTS New Experiment You can create a new Experiment by selecting the Experiment gt New Experiment com mand If an existing Experiment is active when this command is chosen all of the information from the previous Experiment except for the data is copied to the new Experiment Rename Experiment When an Experiment is created it is given a placeholder name like Experiment 1 You can rename an Experiment by double clicking on its title bar to open an edit dialog Duplicate Experiment You can make a copy of a selected Experiment excluding data in the same file by select ing the Edit gt Duplicate Experiment Name command You can duplicate multiple Experiments by Shif selecting them and then selecting the Edit Du
145. mp P Polarization mP and Anisotropy r Fluorophore A material that absorbs light energy of a characteristic wavelength undergoes an elec tronic state change and instantaneously emits light of a longer wavelength Four Parameter Logistic Curve Fit The equation used to generate this curve fit is y A D 1 x OB D G Factor G factor Grating factor is used in fluorescence polarization to correct polarization data for optical artifacts converting relative mP data to theoretical mP data Optical systems particularly with reflective components pass light of different polarization with different efficiency G factor corrects this instrumental bias Gain The amount of increase in signal power expressed as the ratio of output to input for a gnal p P P P photomultiplier tube Grayscale Data Display 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 shading 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 auto matically you may create others as required Incubator Choosing Incubator from the Control menu or clicking the Incubator button opens a dia log box allowing you to start or stop temperature regulation and to select an elevated
146. n saved as a tab delimited ASCII text file or in XML format can be imported to or exported from Plate or CuvetteSet sections A template file consists of seven columns of data separated by tabs Each line of the file provides information for one well in a plate or one cuvette When template information is exported the information for well cuvette A1 is written into the first line of the file information for well cuvette A2 is written to the second line of the file and so on until a line has been created in the export file for each well in a plate or all cuvettes If no template information exists for a well in a plate or for a cuvette that line is left blank The ASCII file terminates with the string End SoftMax Pro Software User Guide 0112 0140 Rev A 111 112 7 File Management amp Printing The order for importing template information must match the export order Similarly blank lines in the file are assigned to a well or cuvette providing a well cuvette with no template setting Table 7 1 ASCII Template File Columns and Descriptions Column Column Entry Description 1 Group Name Assigned in 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 2 Column format Assigned in the Group Settings dialog box Five text strings are supported in SoftMax PRO 5 x Empty Basic Standards Unknowns and Unknowns Dilution Bla
147. n the Formula Reference Guide 5 The formula from the Summary in the Data section of this Tutorial file uses a function InterpX that interpolates data from the curve fit in the Graph section The InterpX function returns the X values of the specified plot interpolated using the Y values and the fitted curve The complete formula is written InterpX Plot Graph Yvalues In this summary formula Plot 1 Graph 1 describes the plot from which the data is derived Yvalues in this example is 50 which describes the number to be interpolated So looking at the graph the Y value at 50 activation appears at an approximate con centration of 0 044 gt You can create a Summary formula either by clicking the Summary button in the tool bar or by choosing Group New Summary to open the Calculation dialog box 5 The same Calculation dialog box appears when you edit an existing formula For exam ple double click Summary 2 at the bottom of the Data section to see the Calculation dialog box SoftMax Pro Software User Guide 0112 0140 Rev A 6 2 Tutorial 2 EC 50 Protocol Tutorial Flex only 6 2 9 5 If you wanted to interpolate a value at a different percentage you could enter a differ ent value in place of 50 You could also reverse the formula to show the percent activa tion that is interpolated for a certain concentration 0 1 in this example by writing the formula this way InterpY Plot 1 Graph 1 0 1 gt The result is 62 867
148. nd has a low salt concentration less than 0 5 M the Water Constant is adequate The Water Constant is determined during manufacture and is stored in the instrument If the sample contains an organic solvent such as ethanol or methanol we recommend using the cuvette reference It is important that the solvent does not absorb in the 900 nm 1000 nm range to determine whether or not a given solvent would interfere see the discussion of interfering substances below When a noninterfering solvent is added to the aqueous sample the water absorbance decreases proportionally to the percentage of organic solvent present For example 596 ethanol decreases the water absorbance by 596 and results in a 596 underestimation of the pathlength You can avoid the error by putting the same water solvent mixture in a cuvette and using the Cuvette Reference To use the Cuvette Reference place into the cuvette port a standard 1 cm cuvette contain ing the aqueous solvent mixture that is used for the samples in the microplate The cuvette must be in place when you read the microplate When you click the Read button the instrument first makes the 900 nm and 1000 nm measurements in the cuvette and SoftMax Pro Software User Guide 0112 0140 Rev A 4 2 Instrument Settings then makes the designated measurements in the microplate The cuvette values are stored temporarily and used in the PathCheck calculations for the microplate samples Use of Cuvette
149. needed to do this would be Mean Mean Group2 Many possibilities exist for creating and editing column formulas see the Formula Refer ence Guide for a complete discussion Copy and Paste Column You can copy a Group column and paste it within another Group in the same Experiment or into a Group of another Experiment 1 Highlight the column to be pasted 2 Select Edit gt Copy 8 C Ctrl C 3 Activate the destination section If you highlight a column in the destination section the column you are pasting is placed to the right of the highlighted column otherwise it is appended to the right of all existing columns 4 Select Edit gt Paste V Ctrl V Columns and Summaries in Group sections can also be copied and pasted to other pro grams SoftMax Pro Software User Guide 0112 0140 Rev A 75 76 5 Data Analysis Showing Existing Column Formulas To show Group formulas in a Group table select the Group gt Show Formulas command To see formulas briefly hold down the Shift Ctrl keys Modifying Column Formulas When a column is selected you can use Group Edit Column or double dick the col umn to edit the formula associated with the column and the column name Adding or Editing a Summary in a Group Section Click the Add Summary button or use Group New Summary to create a Summary or double click an existing Summary and enter a formula for this Summary for the Group Information in the
150. ng or not applying PathCheck to the absorbance values as you choose If you did not have PathCheck turned on during the plate read you cannot apply PathCheck after the read Background Constant Subtraction and Blanking Considerations Raw OD measurements of microplate samples include both pathlength dependent com ponents sample and solvent and a pathlength independent component OD of micro plate material The latter must be eliminated from the PathCheck calculation in order to get valid PathCheck normalized results There are 3 ways to accomplish this described below beginning with the easiest Use a Plate Blank This method can only be used if all samples in the microplate are the same volume and you are not depending on PathCheck to correct for variability in volumes To use this method 1 Designate at least one well preferably several as Plate Blank see instructions under Template SoftMax Pro Software User Guide 0112 0140 Rev A 38 4 Data Collection 2 Pipette buffer e your sample matrix into those wells and read along with your samples SoftMax Pro automatically subtracts the average of the blank wells from each of the samples The OD of the microplate material is subtracted as part of the blank 3 Make sure that Apply Plate Blank is checked under Reduction Use Plate Background Constant If your sample volumes are not identical or if you choose not to use a Plate Blank then you can use the Plate Ba
151. ng button for the Experiment to change to undecided a Toggling the Experiments print setting button overrides any changes made to individual sections The File gt Print dialog box contains a Print All option that overrides the print settings made to sections or Experiments CHANGING SECTION ORDER You can change the order that sections appear in an Experiment by drag and drop Select the title bar of the section and drag it to its new position in the Experiment marked by a thick black line CHANGING TEXT FORMAT Use the Edit gt Text Style dialog box to format text in Notes and Group sections IMPORT EXPORT EXPORT The File gt Import Export gt Export command creates a data file from the data in one or more Plate sections CuvetteSet sections or Group table sections within a SoftMax Pro file The exported data and format depends on the options in the Setting Preferences dialog box Please contact Molecular Devices Technical Support for a copy of the SoftMax Pro XML schema SoftMax Pro Software User Guide 0112 0140 Rev A 107 108 7 File Management amp Printing Data is exported as it is displayed if raw data is displayed raw data is exported if reduced data is displayed reduced data is exported The File gt Import Export gt Export dialog box contains additional export options that allow you to export selected plates and cuvettes or all plates and cuvettes and selected groups or all groups Export F
152. ngth or if you choose Ref The Pathlength reduction reports the pathlength in each well With all SpectraMax Gemini and FlexSta tion instruments additional wavelength combinations become available depending on the number of wavelengths chosen For any instrument you can choose Custom and create different reduction formulas see the SoftMax Pro Formula Reference Guide for more on custom formulas The Pathlength option is only applicable to Plate sections when using the SpectraMax 190 340PC 340PC384 Plus Plus384 M2 M2 M5 or M5 However the wavelength reduction display is not instrument dependent in SoftMax Pro so this choice is always shown regardless of the instrument PathCheck Pathlength correction allows microplate data in Endpoint reads for those instruments that support PathCheck to be normalized to a 1 cm pathlength When PathCheck is turned on in Plate gt Settings for the plate Apply Path Check is displayed as an option in the Reduction dialog box KINETIC READS Wavelength Combination The default wavelength combination for Kinetic readings is Lm1 and is the only choice besides Custom for all instruments except the SpectraMax For the SpectraMax the choices depend on the number of wavelengths chosen For exam ple if you read at two wavelengths choices default to Lm1 and Lm2 along with Custom SoftMax Pro Software User Guide 0112 0140 Rev A 5 3 Data Reduction Kinetic Reduction Kin
153. nk groups have an Empty column format If this field is left blank no sample name will be assigned to the well although the sample name will be included in the group 3 Sample 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 sample name will be included in the group 4 Number of description 0 for Empty and Unknown column formats 5 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 6 Sample descriptor Assigned in the Group Settings dialog box Any text string can be entered in this field or it may be left blank 7 Descriptor value Assigned in the Series dialog box Can be set to any number or left blank The tab delimited ASCII files can be opened with spreadsheet programs such as Microsoft Excel but if edited in Microsoft Excel they cannot be imported back into Soft Max Pro SoftMax Pro does not import files in the Microsoft Excel XLS format SoftMax Pro Software User Guide 0112 0140 Rev A 7 4 File Protection 7 3 4 7 3 5 7 4 EXPORTING GRAPHS The Graph gt Export Graph command exports a graph to file On the Mac the graph is saved in PICT format on Windows it is saved in EMF Enhanced Metafile format Well Graphs can
154. nknowns and Unknowns Dilution creates a different set of columns in a new or existing group The table below shows the default columns created with each selection type SoftMax Pro Software User Guide 0112 0140 Rev A 49 50 4 Data Collection Table 4 1 Columns Created and Their Formulas Unknown Name Formula Basic Standard Unknown TO Dilution Sample ISampleNames Y Y Y Y Wells IWelllDs Y Y Y Y Sample Index Y Concentration IConcentration Y Mean Value Average Values Y Y Values IWellValues Y Y Y Y R Outside Standard If Values gt MinStd Y Y Range Standards and Values lt MaxStd Standards R Result InterpX STDO Y v StandardCurve Values MeanResult Average Result Y Y Std Dev Stdev Result Y Y Y CV Cv Result v Y Y Dilution IFactor Y Adj Result MeanResult Factor Y Edit The Edit button opens the Group Settings dialog box for a selected group in the Group list The name sample descriptors and column format can be changed for the group that you are editing Changing the column format causes the columns in the group to revert to their defaults If you have added columns or changed column information in the section for the group you are editing these changes are removed SoftMax Pro Software User Guide 0112 0140 Rev A 4 3 Template Edftor 4 3 3 4 3 4 4 3 5 4 3 6 SAMPLE
155. ns the groups Standards and Unknown and the destination Experiment already contains the group Standards the group Unknown is created but the group Standards in the destination Exper iment is unchanged If you copy and paste the template within the same Experiment no new groups are created Group names can be edited in the pasted template but these changes are made in the original template also New Group Names The second way to copy and paste a template allows you to keep the same sample lay out and sample names and concentrations but also allows you to change the Group names independent of the source template 1 Open the Template Editor and select all wells 2 Select 8 C or Ctrl C to copy the layout 3 Open the Template Editor for a new Plate section and select all wells 4 Select 8 V or Ctrl V to paste the layout Export Template Use Plate Export Template to export a template to a tab delimited text or XML file The file format must be specified first in the global Preferences dialog Import Template Use Plate gt Import Template to import a template from a tab delimited text or XML file GROUP SECTION A Group section is a table that shows all of the information for a particular sample group that has been defined in a Plate template Unlike other section types Group sections are created automatically when you create or select groups for an Experiment in the Template Editor Group sections can also be
156. o appends ppr to any file name passed in gt If the file is not found SoftMax Pro does nothing Example OpenAssay myFile OpenAssay mySubDirectory myFile OpenAssay c temp testprotocol OpenCDrawer FlexStation only Tell SoftMax Pro to open the compound drawer SoftMax Pro Software User Guide 0112 0140 Rev A 9 6 SoftMax Pro Commands OpenDocument Tell SoftMax Pro to open a data file The file should be in SoftMax Pro s application folder unless the full file path is specified If the file is not found SoftMax Pro does nothing Example OpenDocument myDataFile OpenDocument c temp testdataOpenDrawer Tell SoftMax Pro to open the instrument drawer OpenTDrawer FlexStation only Tell SoftMax Pro to open the tips drawer Quit Tell SoftMax Pro to exit the application Read Tell SoftMax Pro to read the current Plate or CuvetteSet section If the current section is neither a Plate nor CuvetteSet section next Plate or CuvetteSet section will read ReturnCalc formulaName sectionName experimentName This command will return the result from specified formula This command requires the name of the formula name of either Notes or Group section and the name of the Exper iment If the name of the Experiment is not specified it will search from the first Experi ment of the document Example ReturnCalc MyFormula MyGroupSection MyExperimentSection ReturnData This command asks SoftMax Pro to send data back from t
157. o save data or protocol files AUTOSAVE SoftMax Pro software can be configured to save one or more copies of your data automat ically immediately after each plate read has completed See AutoSave in Section 2 5 Setting Preferences for more information SoftMax Pro Software User Guide 0112 0140 Rev A 7 2 Printing 7 2 7 2 1 7 2 2 7 2 3 7 2 4 7 3 7 3 1 PRINTING As explained in Chapter 3 SoftMax Pro is designed for WYSIWYG report printing the software interface is identical to the report PRINTING REPORTS You can create customized reports by including or excluding Experiments and individual sections changing the order in which sections print and adding text describing each Experiment in Notes sections You can also create Summaries consisting of reduction for mulas 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 Bl disabled a and vice versa Changing the print setting option for the Experiment changes the state of all sections within that Experiment Changing the print setting option for one or more sections within an Experiment causes the print setti
158. ocument is con strained by the width set for the printed page size You can change the dimensions and orientation of the printed page and consequently the display size in the File gt Print Setup Windows or File gt Page Setup Mac dialog boxes STATUS BAR Below the menus at the top of the SoftMax Pro window is a Status Bar that displays instrument controls The Status Bar can be shown and hidden using View gt Show Status and View gt Hide Status respectively Depending on the type of instrument that is connected to the computer or selected in the Edit gt Preferences dialog some or all of the following items are in the Status Bar INSTRUMENT ICON Indicates that the instrument is communicating properly with the computer If an appears on top of the icon the computer is not making proper contact with the instru ment Clicking the instrument icon opens the Edit gt Preferences dialog SoftMax Pro Software User Guide 0112 0140 Rev A 13 14 3 SoftMax Pro Interface 3 3 2 3 3 3 3 3 4 3 3 5 TEMPERATURE DISPLAY Reports the current temperature within the microplate chamber in degrees Celsius With the SpectraMax M2 M2 M5 M5 Plus and Plus384 the SoftMax Pro Status Bar shows the temperature within the microplate chamber This may be different from the front panel of the instrument which displays the temperature within the cuvette chamber The readings should be very similar to on
159. of cells similar to a microplate format gt Instrument settings shown to the right of the data display gt Reduction settings shown at the bottom of the data display When colors appear in the name labels above individual cuvettes when displayed as one or three cuvettes per row or when colored areas appear when the CuvetteSet section is displayed as a grid this means a template has been defined for the CuvetteSet section Each group defined in the template has a different color the icon of the corresponding group table has the same color SoftMax Pro Software User Guide 0112 0140 Rev A 23 24 3 SoftMax Pro Interface 3 6 7 If your experiment requires multiple cuvettes you can add up to 96 cuvettes in a single CuvetteSet section or create more than one CuvetteSet section The commands described in the Plate section above also apply to CuvetteSet sections GRAPH SECTION Graph sections are used to plot information from groups as scatter plots or bar graphs Once a graph has been created new plots can be added and deleted the axes can be cus tomized and the size of the graph can be changed Grid lines can be enabled or disabled default is enabled Graph sections are divided into gt Tool bar gt The body of the Graph section 5 Plot information below the graph You can create more than one Graph section within an Experiment data file and plots in the Graph section can be created from any Experim
160. of reaction When a lag time is specified in SoftMax Pro data collected prior to the lag time is not included in data reduc tion Large Display A display choice available with 384 well plates This setting is useful when looking at large numbers if you are using a monitor that is 17 inches or larger Linear Curve Fit The linear function fits the best straight line to the data The equation for this fit has the form o y A Bx where A is the y intercept of the line and B is the slope A linear fit should be used when ever the values appear to lie on or scattered around a straight line 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 B D SoftMax Pro Software User Guide 0112 0140 Rev A 141 142 A Appendix 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 radi ation Mask Lets you hide selected data so that they are not used for calculations and are not reported The Mask function is commonly used to suppress outliers Maximum Reduction for a Spectrum scan that reports the maximum signal within the reduction parameters MaxOD MaxRFU or MaxRLU The limit for the maximum value you want to use for displaying and analyzing Kinetic or Spectrum data Any values above th
161. omized for any instrument whether or not SoftMax Pro is connected to an actual instrument see Section 2 5 Setting Preferences SoftMax Pro Software User Guide 0112 0140 Rev A 11 12 2 Installation amp Setup This can be useful in the following circumstances 5 If you want to create protocol files on a computer that is running SoftMax Pro but is not connected to an instrument 5 If you want to create protocols for an instrument other than the one connected to the computer SoftMax Pro Software User Guide 0112 0140 Rev A 3 1 3 2 3 3 3 3 1 3 SoftMax Pro Interface This chapter describes some of the main elements of the SoftMax Pro interface and their logical relationships to each other For detailed instructions on setting up an experiment and acquiring and analyzing data see Chapters 4 through 6 THE SOFTMAX PRO APPLICATION WINDOW The SoftMax Pro application window consists of two elements gt A Menu Bar and Status Bar at the top of the application window 5 An untitled document showing an Experiment data file divided into a number of sec tions The displayed sections depend upon the configuration of the default protocol and the type of instrument chosen in Edit gt Preferences or connected to the computer WYSIWYG DISPLAY SoftMax Pro has been designed to provide you with a WYSTWYG What You See Is What You Get preview of printed reports Consequently the width of a d
162. only This enables the remote logon of a user with remote logon permission to SoftMax Pro GxP The logon ID and password are separate by single space SoftMax Pro Software User Guide 0112 0140 Rev A 127 128 9 Robotics amp LIMS Integration Example Logon mylogonID myPassword NameCurSection This renames the current selected section If no section is selected nothing happens gt This command is used typically to identify different plates in an automated environ ment gt The data copied follows the display settings for the current plate normally Raw Data Example NameCurSection Plate 002345 New Have SoftMax Pro send a File gt New command This creates a new document and reads in the values contained in the Default Protocol ppr protocol file NewExperiment Create a new Experiment section NewNotes Tell SoftMax Pro to send an Experiment gt New Notes command This creates a new Notes section within the current Experiment NewPlate Tell SoftMax Pro to send an Experiment gt New Plate command This creates a new Plate section within the current Experiment OpenAssay filepath Tell SoftMax Pro to open a protocol file The file should be in SoftMax Pro s Protocols folder if the full file path is not specified The Protocol folder can be set using the Protocols menu Protocols can also be stored in subdirectories of the Protocols folder gt Only protocol files can be opened gt SoftMax Pr
163. ords all authorized users and their associated information including gt User names user login IDs and passwords gt Assigned user permissions to SoftMax Pro GxP features 5 Global settings pertaining to all listed users password aging non network use guest access etc SoftMax Pro Software User Guide 0112 0140 Rev A 115 116 8 SoftMax Pro GxP 8 2 8 2 1 gt Audit histories of all administrator actions within GxP Admin user creation modifica tion etc gt Electronic signatures If set by the administrator SoftMax Pro GxP users do not require continual access to a User Accounts edb file in order to use SoftMax Pro GxP for data collection and analysis when no network connection is available corporate network failure use of SoftMax GxP while traveling etc HOW THE PROGRAMS WORK TOGETHER SoftMax Pro GxP GxP Admin and the MDC File Server applications work together as follows gt The administrator installs GxP Admin and MDC File Server software on a Windows PC 5 The administrator creates a User Accounts EDB file with GxP Admin after entering a special Creation Key number 5 Using GxP Admin software the administrator creates SoftMax Pro GxP users and assigns the appropriate user permissions gt SoftMax Pro GxP software is installed on individual computers that are used for data collection or data analysis Each Windows log on account must be linked to a User Accounts file
164. ormat The first line of an exported text 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 or CuvetteSet 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 following table describes each of these fields Field Field Entry Description 1 Section kind Either Plate or Cuvette 2 Section name Defined in Section dialog 3 Export version 1 3 in SoftMax Pro 5 x 4 Export format PlateFormat or TimeFormat set in Preferences 5 Read type Endpoint Kinetic Spectrum Well Scan or Flex 6 Data mode For absorbance plates Absorbance or Transmittance For others Fluorescence Luminescence or Time Resolved Fluorescence 7 Data type Raw or Reduced 8 Pre read included TRUE or FALSE always FALSE for Cuvettes 9 Kinetic points Reduced plates and Endpoint plates 1Kinetic Spectrum plates number of reads 10 Kinetic or Flex read time Kinetic Flex read time in seconds or Well Scan Well Scan read pattern read pattern horizontal vertical X or fill otherwise blank field 11 Kinetic or Flex interval Kinetic or Flex read interval in seconds or Well Well Scan density Scan density otherwise blank field 12 Start wavelength Spectrum start wavelengt
165. ose values are stored in the non volatile RAM until the next calibration is done When you select Calibrate Cuvette the instrument steps through each wavelength one by one taking air calibration values for the cuvette It is therefore important that the cuvette port be empty These air calibration values are used for Absorbance reads only in the following circum stances gt When the Instrument Settings gt AutoCalibrate option is disabled gt When a Spectrum reading is performed using the Speed Read option Molecular Devices recommends that you periodically update the air reference information that is stored in the instrument This is particularly important if you are using Speed Read reading cuvettes without a reference or running Kinetics in microplates with read intervals that are too short to allow AutoCalibration DATA DISPLAY DURING A READING The values read by the instrument appear in real time in the data display as they are received from the instrument Depending upon the read mode and type the number of wavelengths the plate type and the display options selected the data display shows one or more numbers or plots During a Kinetic or Spectrum reading you can enlarge the display of one or more wells To enlarge a single well to a Well Graph double click the well To display the data from multiple wells in one enlarged Well Graph 1 Hold the Shift key while selecting more than one well 2 Choose the Plate gt Graph
166. osize or double click the column dividers to set the width of selected columns to accommodate the longest text in the column Hide Group Hide hides selected columns from display You can show hidden columns with Group gt Show All Hide Replicates Group gt Hide Replicates hides data for all replicates You can show hidden data with Group gt Show All Show All Group gt Show All shows all hidden columns or replicates GRAPH SECTION The Plot Dialog Box The Plot dialog box opens when you create a new Graph section with Experiment gt New Graph or when you add a plot to an existing graph from the Graph gt Graph Options dia log box In this dialog box you can name the plot assign specific information to be plot ted on the X and Y axes and choose which icon and color will be used All groups that have been created or assigned within the file are listed on the left side in the Source field To create a new plot 1 Select a group in the Source field 2 Select group columns to assign to the plot in the X Axis and Y Axis lists Summaries and user defined formulas can also be graphed gt Click the Formula button for either the X axis or Y axis to open a Formula dialog box gt To plot a Summary enter the Summary name in this dialog box gt Formulas can be plotted for comparison 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 t
167. ou can enlarge the well plots in Kinetic Spectrum Well Scan or Flex reads to get a close up view of the data 5 Double click a well or cuvette in the data display or gt Select the wells or cuvettes and select Plate gt Graph Kinetic and Spectrum Well Graphs Well Graphs plot the data for the individual wells in a microplate or for individual cuvettes and show the well ID and reduction information including the goodness of fit R2 value if appropriate The default display for the X axis shows time for Kinetic plots and wavelength for Spectrum plots OD 96T RFU RLU or mP is displayed on the Y axis for both plots Well Scan Well Graphs Well Graphs for Well Scan data default to displaying raw data in a grayscale plot SoftMax Pro Software User Guide 0112 0140 Rev A 63 64 5 Data Analysis Well Graph Options Print Opens the Print dialog box Reduction Opens the Reduction dialog box By default the ranges on the 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 can change these settings but changes made within well graphs are applied to all wells or cuvettes in the Plate or CuvetteSet section When viewing Raw data you can scale the graph to see data outside the reduction limits lines are included on the graph indicating the limits and the area outside of them will be shaded sli
168. ou have a Spectrum scan with 20 measurements and X is 10 it reports the OD RFU RLU of the 10th wavelength measured For a full discussion of custom formulas see the Formula Reference Guide When you finish your entry in the Ca culation dialog box and click OK the formula is displayed and becomes the default selection for the Custom option RECALCULATION OPTIONS By default SoftMax Pro performs continuous recalculation of the data when you read plates create or change formulas and so on At certain times it is useful to disable recal culations so that you can edit the Experiment without waiting for recalculations to be completed Edit Suspend Recalculation disables automatic recalculation While suspended no recal culation occurs regardless of what you may change add or delete from the Experiment This is useful when creating or changing column formulas within Group sections Select Edit Suspend Recalculation again to re enable automatic recalculation When recalculation has been suspended you may want to see the results of the changes you have made but may still not wish to enable continuous recalculation Choose Edit gt Recalculate Now to recalculate all data once without enabling continuous recalculation GROUP AND GRAPH SECTIONS GROUP SECTIONS Unlike other section types Group sections are created automatically when you create and assign a Group to a Plate section within the Template dialog after clicking
169. ould interfere with Path Check AUTOMIX Automix is a patented feature that allows you to set automated shaking of the microplate The options available for Automix depend on the read type chosen and the type of instru ment Before First Read This setting can be used with any read type Whether you read at a single wavelength or at multiple wavelengths checking Before First Read shakes the plate for the set amount of time before the first wavelength reading only SoftMax Pro Software User Guide 0112 0140 Rev A 39 40 4 Data Collection 4 2 14 4 2 15 Between Reads This option is available only when Kinetic read is selected For readings at a single wavelength Between Reads shakes the plate for the specified amount of time prior to each reading at that wavelength For readings at multiple wavelengths enabling Between Reads shakes the plate for the specified amount of time before each reading at the first wavelength only BLANKING PRE READ PLATE The blanking specified in the Plate gt Settings dialog box is known as pre read plate blank ing but pre reading the plate is only one of a number of types of blanking available in SoftMax Pro Pre read plate blanking adds considerable time to a read and is generally not required for modern plates that have high uniformity If you choose pre read plate blanking SoftMax Pro prompts you to read the blank plate after you click the Read button The pre read value
170. ows you to enter a different integration time Values are reported as relative luminescence units RLU the default reduced value is Lm1 Kinetic In a Kinetic read the data are collected over time with multiple readings taken at reg ular intervals To achieve the shortest possible interval for Kinetic readings choose wavelengths in ascending order The default reduced values calculated for Kinetic data are VMax units sec or units min Time to VMax seconds Onset Time seconds Time at Minimum Time at Maximum Time at 1 2 Maximum Slope and Area Under Curve Spectrum Depending on the read mode selected a Spectrum read measures optical density 96 Iransmittance if this has been selected in the Reduction dialog box relative fluo SoftMax Pro Software User Guide 0112 0140 Rev A 4 2 Instrument Settings rescence units RFU or relative luminescence units RLU across a spectrum of wavelengths All Spectrum readings are made using the scanning monochromator of the instru ment Well Scan A Well Scan read takes one or more readings of a single well of a microplate at single or multiple wavelengths Every option available for Endpoint reads is available for Well Scans Values reported are optical density Transmittance relative fluorescence units RFU or relative luminescence units RLU Flex Flex is used to set up reads on FlexStation instruments where one has to configure compound additions
171. p 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 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 7 Click OK to save the series and close the dialog box To increment the names of a series of samples without applying a concentration or dilu tion 1 Create a series in the Series dialog box 2 Uncheck the Sample Descriptor field To increment the names of a series of samples with a constant concentration or dilution 1 Create a series in the Series dialog box 2 Check the Sample Descriptor field 3 Enter the concentration or dilution and select the multiply operator with a factor of 1 If sample names are set to increment automatically using the Series function be aware that SoftMax Pro 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 numeric it is not truncated If sample names are very long and they m
172. periments 17 ReturnData 129 ReturnStatus 129 ReturnTiming 130 Robotics 121 S Sample 51 Save Data After Read 9 SaveAs 130 Section Name 18 Sections Changing Section Order 107 Select Section Num Robotic Command 131 SelectAll Robotic Command 130 SelectSect Robotic Command 130 SelectSectNum Robotic Command 131 Semi Log 144 Sensitivity 33 Series 144 Define 51 Set Password 114 SetFolderAs Robotic Command 131 SetPort Robotic Command 131 SetTemp Robotic Command 131 Settling Time 47 SetUserPassword Robotic Command 129 131 132 Shake 32 Show All 77 Slope 68 144 Smoothing 71 SoftMax Pro GxP 115 SoftMax Pro Window 13 Spectrum 28 145 Spectrum Well Graphs 63 Speed Read 47 Standard Curve 83 145 Start Wavelength 70 Statements Menu 119 Stokes Shift 145 Stop 131 Strips Wells to Read 43 T Template 20 Template Editor 48 Group 49 Selecting Wells or Cuvettes in 48 Templates Copying and Pasting 55 Exporting and Importing 56 Text Format Changing 107 Threshold Data Display 145 Time at 1 2 Maximum 68 145 Time at Maximum 68 Time at Minimum 68 146 Time Resolved Fluorescence 30 145 SoftMax Pro Software User Guide 0112 0140 Rev A 151 Time to VMax 67 146 Timing 34 Tip Columns 45 Transmittance 137 Triturate 44 146 Tutorials Tutorial 1 Quantitative Endpoint Assay Tutorial 88 Tutorial 2 EC 50 Assay Tutorial 96 U User Accounts file 115 User Name 146 V Variable We
173. perly connected or is turned off the instrument icon in the upper left hand corner of the window will appear with a question mark Check that the connection is secure and that the instrument is powered on LMAX II The LMax II and LMax 11384 instruments require manual selection of the instrument in the Preference dialog in order to establish communication This is due to the different baud rate requirement for LMax II instruments INSTRUMENT PREFERENCES If you have problems communicating with a connected instrument open the Edit gt Pref erences dialog box and select the appropriate communications port ports that are not available are disabled gt Windows use COMI through COMO gt Mac use a USB to serial adapter with an available USB port 5 If you do not have an instrument connected to the computer but want to use SoftMax Pro select None gt Click OK to close the Preferences dialog and the instrument icon should appear without a question mark If the serial port setting is correct and the instrument is on yet communication is still fail ing quit SoftMax Pro turn off power to both the computer and the instrument and check that the cable connections between the instrument and computer are secure Turn both machines on again and restart SoftMax Pro For more on instrument preferences see Section 2 5 Setting Preferences SoftMax Pro Software User Guide 0112 0140 Rev A 2 5 Setting Preferences
174. pes except Well Scan are available for all read modes Not all read modes are supported by all instruments Fluorescence RFUs Fluorescence is light emitted by a substance resulting from the absorption of incident radiation The governing equation for fluorescence is Fluorescence extinction coefficient x concentration x quantum yield x excitation intensity x pathlength x emission collection efficiency Absorbance ODs Absorbance is the amount of light absorbed by a solution In the absence of turbidity Absorbance optical density reflection Luminescence RLUs Luminescence is 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 With the SpectraMax L M5 and M5 selecting the Luminescence read mode displays the integration time box next to the Read Type selection Time Resolved Fluorescence RFUs Most fluorescent substances are not suitable for this type of reading However the fluores cence emitted by lanthanide dyes is delayed long enough to measure fluorescence after the lamp is turned off Time resolved fluorescence is used to reduce the amount of back ground noise that interferes with fluorescence The excitation lamp flashes and after it is off the delayed emission is collected for a set period of time before the lamp is flashed again With the SpectraMax M5 and M5 Time Resolve
175. plate Regions Contiguous regions of templates 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 The real value in this method is the ability to copy a complicated template and then to be able to analyze the data separately from the original Plate To copy and paste a region of a template 1 Open the Template Editor of the source Plate and select the wells to copy 2 Select C or Ctrl C to copy the layout 3 Open the Template Editor for a new Plate section and select all wells 4 Select 8 V or Ctrl V to paste the layout SoftMax Pro Software User Guide 0112 0140 Rev A 55 56 4 Data Collection 4 3 9 4 4 4 4 1 Only the highlighted wells 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 is 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 EXPORTING AND IMPORTING TEMPLATES Templates can be exported or imported by opening a Plate sec
176. plicate Selection command Delete Experiment You can delete a selected Experiment by selecting the Edit gt Delete Experiment Name command You can delete multiple Experiments by Shift selecting them and then selecting the Edit gt Delete Selection command Reorder Experiments You can change the order that Experiments appear in a file by drag and drop Select the title bar of the Experiment and drag it to its new position in the file marked by a thick black line SECTIONS A section is a part of the SoftMax Pro Experiment window intended to perform a specific set of functions There are five types of sections Notes Plate CuvetteSet Group and Graph and each type of section has a specific icon that identifies it ACTIVE SECTION MENUS When a section is active a section specific menu is added to the SoftMax Pro main appli cation menus between the View menu and the Protocols menu Notes Plate Cuvette Group or Graph MANIPULATING SECTIONS Like Experiments sections are either active or inactive Make a section active by clicking it with the mouse or by selecting it from the View Experiment sub menu SoftMax Pro Software User Guide 0112 0140 Rev A 17 18 3 SoftMax Pro Interface When active the section title bar is colored dark gray Multiple sections can be made active at the same time by Shift selecting them New Section You can create a new section except Group sections by selecting the a
177. plicate to ensure that anomalies can be excluded prior to generating a stan dard curve Replicates can be created using the Assign button or with the Series dialog box 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 In the Template Editor highlight a rectangular selection of wells to be part of the series SoftMax Pro Software User Guide 0112 0140 Rev A 51 4 Data Collection 2 Click the Series button 3 Specify the name of the first sample in the series in the box Subsequent replicates use this name as a base and either increment the number within the name or append a number to it 4 The Sample Descriptor information is derived from the information entered in the Group Settings dialog box Specify the starting value for the first sample in the series 5 Choose the operator for the series or from the Step by list and enter a value for the increment For example with a starting value of 0 5 the division operator and the increment 2 the series will start with 0 5 and divide each subsequent sample by 2 to produce the series 0 5 0 25 0 125 erc 6 Choose a fill direction to describe how the series fills the wells and a number of replicates The maximum number of replicates depends on the selection of wells if you are filling down from the to
178. pply injections 1 Enable P injection or M injection on the Injection and Delay section of the Settings dialog box 2 Select the Injection type on the Injection Wells section of the Settings dialog box by clicking the P injector or M injector icon Only the wells specified in Wells to Read are displayed 3 Click the wells to which you want the injections to apply SENSITIVITY GEMINI SPECTRAMAX L M2 M2 M5 M5 FLEXSTATION FLUORESCENCE LUMINESCENCE ONLY Readings This setting determines the number of readings made in each well of the microplate or each spot in a well for Well Scan reads Readings are averaged and the average reading is displayed The default number of readings per well varies with the read mode for Fluorescence the default is 6 for Luminescence the default is 30 for Time Resolved Fluorescence the default is 20 The number of readings can vary from 1 to 30 PMT Sensitivity The PMT photomultiplier tube is 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 PMT sensitivity can be set to low for samples having a higher concentration medium or high samples having lower concentration In Endpoint reads an Automatic setting is available allowing the instrument to adjust the PMT voltage automatically for varying concentrations of sample in the plate Auto
179. pproach is that it may be universally applied to Visual C and Visual Basic modules Excel macros etc An example of retrieving clipboard data is shown below where the string data retrieved is finally stored into a Microsoft CString object if OpenClipboard NULL return FALSE Error opening clipboard HANDLE handle GetClipboardData CF_TEXT if handle NULL f CloseClipboard return FALSE Error no data in clipboard get clipboard data store in MFC CString object CString returnStr CString char handle close the clipboard SoftMax Pro Software User Guide 0112 0140 Rev A 9 4 Send Receive Remote Commands 9 4 2 CloseClipboard THE WM_COPYDATA APPROACH Call the Win32 RegisterWindowMessage function to register a unique message tag Soft Max Pro uses this tag information when the command is received to distinguish between a Windows generated message and an external command The value returned should be a non zero value UINT tag RegisterWindowMessage SOFTMaxPROMsg Call the Win32 RegisterWindowMessage function to register another unique message tag Your software can use this tag information when the asynchronous windows message is returned and can distinguish between a Windows generated message and an external command The value returned should be a non zero value UINT replyTag RegisterWindowMessage SOFTMaxPROReplyMsg All messages must be sent to the Soft
180. ppropriate New command from the Experiment menu New Notes New Plate New CuvetteSet New Graph If an existing section is active when this command is chosen all of the information in the section except data is copied to the new section Group sections are created automatically when you define new groups in the Template Editor and can also be created with the Duplicate command Section Name When a section is created it is given a name based on its section type like Notes 1 You can rename a section by double clicking on its title bar to open the Section Name dia log or select Section Name from the Section Notes Plate Cuvette Graph menu Duplicate Section You can make a copy of a selected section excluding data in the same file by selecting the Edit Duplicate Section Name command You can duplicate multiple sections by Shift selecting them and then selecting the Edit gt Duplicate Selection command Duplicating a CuvetteSet section creates a copy of the previous CuvetteSet section including the last reference that was read Delete Section You can delete a selected section with the Edit gt Delete Section Name command You can delete multiple sections by Shifi selecting them and then selecting the Edit gt Delete Selection command Deleting a Group section automatically deletes references to that group from the Tem plate View Section Each Experiment in a file has its name added to the View menu a
181. rcent trans mittance T RFU or RLU within the reduction limits Area Under Curve Estimates the area under the curve as defined by the data plots 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 coordinates of the data points The areas defined by each of the trapezoids are then computed and summed Spectrum Limits Limits define the data that are viewed and included in data reduction If you alter a limit to show less data you can always display the excluded data again by changing the limit Start Wavelength nm Specifies the limit for the minimum wavelength setting to report Any values from the reading that are under this limit are not shown and are excluded from data reduc tion End Wavelength nm Specifies the limit for the maximum wavelength setting to report Any values from the reading that are above this limit are not shown and are excluded from data reduc tion WELL SCAN Wavelength Combination Lm1 is the only choice besides Custom Well Scan Reduction Choices are Maximum Minimum Average default and Custom Average provides the average value for all points in the Well Scan SoftMax Pro Software User Guide 0112 0140 Rev A 5 3 Data Reduction 5 3 5 FLEX READS Smoothing Smoothing is used to reduce noise in Flex readings This setting takes the moving average of the raw data and displays the Flex plot
182. rd curve may be set to a linear semi log log log quadratic 4 parameter logistic log logit 2 parameter logistic exponential cubic spline or point to point curve fits Stokes Shift The difference between the wavelengths of the excitation and emission peaks Template Editor A representation of the microplate or a set of cuvettes 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 various wells of the microplate or cuvette Threshold Data Display Data is displayed as plus for values above asterisk for values within and minus for values below user defined limits Time Resolved Fluorescence Most fluorescent substances are not suitable for this type of reading However the fluores cence emitted by lanthanide dyes is delayed long enough to measure fluorescence after the lamp is turned off Time resolved fluorescence is used to reduce the amount of back ground noise interfering with fluorescence The excitation lamp flashes and after it is off the delayed emission is collected before the lamp flashes again Time at 1 2 Maximum Reduction for a Kinetic or Flex run that reports the time at half the maximum signal within the reduction limits as follows SoftMax Pro first determines the point within the reduction limits that has t
183. re obe aeu tbt debe pL JJI JJJ JJJJJINNnN Sede ador Carica Pa 53 Copying and Pasting Templates a KK KK KK KK KK KK KK KK KK Es 55 Exporting and Importing Templates 342 53 098 KK KK KK KK KK KK KK KK KK KK 56 Reading a Microplate or Cuvette eit KK KK KK KK KK KK KK KK KK KK KK KK KK k 56 Data Collection from a Microplate pis cased des uae KK KK KK KK KK KK KK KK 56 Data Collection from a Cuvette ssc ick KK KK KK KK KK KK KK KK KK KK KK KK KK as 57 Calibration Qe sky ef esr ate Walk eA al bawann E a keyen lida de aba tata 59 Data Display During a Reading cnt o kk kK KK KK KK KK KK KK KK KK KK KK k 59 Data Analysis T tfodBe lO H N ND NR HH HRH HHR EE gg gl N Ead 61 Dara Displayer katie Tr CM 61 The Display Dialog Box aaa KK KK KK KK KK KK KK Aa 61 Graphing Wells cada tas st l nan vies ER in EAD KWA bal Ld 63 Masking Wells or Cuvettes i o xoa D KK KK KK KK KK KK KK KK KK KK KK KK KK ke 65 SoftMax Pro Software User Guide 0112 0140 Rev A DataXRed cttonos 14e Ma ous d k heater O Ke a Ma ees Soke Ade td ad 65 Endpoint Reads siii ai 66 A 66 SPCC eds ia a ic e 69 Well Seam enden Cet RES pe Ret eec HN gt n R de adque vata d 70 Flex Reids os eo Cite b a ude E rrr 71 Data Mode Transmittance Absorbance kk kK KK KK KK KI KRI KK 72 Custom Reduction Formulas 2 254 ico AS kk ER KK KK KK KK KK KK KK KK KK K 72 Recalculation Options 234 2 22993 VIO ROO De KK KK KK kK KK kk kK kk aa 74 Group and
184. roup section hold down Ctrl Shift while the group is active or select Group gt Show Formulas A row of formulas is displayed beneath the column titles gt If formulas are long they may not fit in the columns To see the complete formula for a particular column click once on a column and the formula is reported in the Group section s tool bar 5 To modify or change a formula for a selected column select the Group gt Edit Column command or dick the Formula button on the section tool bar Resizing and Autosizing Columns gt You can resize columns by double clicking or dragging column dividers 5 To Autosize columns select the columns and use the Group gt Autosize command Adding a Column 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 high lighted column or if no columns are selected after the last column in the group To add a new column to the Standards group 1 Select the groups tool bar to make it active 2 Deselect any highlighted columns by clicking outside the column area 3 Choose Group gt New Column or click the New Column button 4 We are creating a new column that displays the maximum value obtained for each sample Enter MaxValue in the Name field 5 Enter Max Values in the Formula field and click OK A new column is displayed at the right of the previous columns showin
185. ru ment and the current status of the instrument is displayed in the Status line in the Stak Max software StakMax is compatible with SpectraMax L M2 M2 M5 M5 Gemini EM and XPS and the new models of the following instruments compatible models are identifiable by their serial numbers SpectraMax Plus384 Serial MNR xxxxx 340PC384 Serial LNR xxxxx 190 Serial NNR xxxxx VersaMax Serial BNR xxxxx Click the Help button in the StakMax software user interface to open the StakMax User Guide which provides complete documentation on the use of StakMax SoftMax Pro Software User Guide 0112 0140 Rev A 3 4 Instrument Settings and Control 3 3 6 3 3 7 3 3 8 3 4 3 4 1 3 4 2 INCUBATOR BUTTON Displays the Control gt Incubator dialog box allowing you to set and regulate the tempera ture of the microplate cuvette chamber The temperature setting can be left at the default or can be specified by entering a different value The incubator setting is independent of the protocol being run Running an assay does not automatically set the temperature set point After a reading however the temperature set point range and average temperature are recorded in the saved file AUTOMIX BUTTON Shakes the microplate This is a manual shaking of the plate as opposed to an automatic shaking that can be chosen in the instrument settings and is not available for the EMax DRAWER BUTTON Opens or closes t
186. ry data pertaining to the Exper iment You can enter text in this section by typing directly or by creating Summaries con taining formulas for displaying reduced data A Summary is part of a Notes section that is generated from formulas applied to data in the Experiment Text in Notes sections can be formatted font face font size and font style using the Edit Text Style dialog box You can create multiple Notes sections within the same Experiment New Summary Notes New Summary opens a dialog box allowing you to gt Name a new Summary gt Enter a formula to be used for the Summary Edit Summary Use Notes gt Edit Summary to edit the name and formula of an existing Summary See the SoftMax Pro Formula Reference Guide for a complete guide to formulas SoftMax Pro Software User Guide 0112 0140 Rev A 19 20 3 SoftMax Pro Interface 3 6 4 PLATE SECTION Plate sections are used to collect data from the instrument and to define data display and data reduction If you read the same physical plate twice with different instrument set tings you would have two Plate sections The Template Editor in the Plate section is used to create a map of the contents of the microplate Plate sections are divided into gt Tool bar 5 Data display shown in a microplate grid format 5 Instrument settings shown to the right of or below the data display depending on the plate type and printer settings 5
187. s etc PROGRAMMER NOTES gt COM ports 1 9 can be used for robotics purposes Molecular Devices recommends using COM1 or COM2 These are typically the most stable gt An Autosaved file from the robotics run can be saved as either a text txt or SoftMax Pro experiment data file pda This selection is made in the SoftMax Pro Edit gt Pref erences dialog box gt More random access memory RAM is required to run SoftMax Pro using remote commands Depending on the robotics used and the type of file created with SoftMax Pro software a computer configured with the minimum amount of RAM may restrict the size of the file that can be generated gt It is important to put a short pause between remote commands An Experiment with more than one plate of data could adversely affect SoftMax Pro s effi ciency If increasing the computers memory is not an option Molecular Devices recom mendis using only one plate per file and also ensuring that the computer is not being used for other purposes while SoftMax Pro is running SoftMax Pro Software User Guide 0112 0140 Rev A 125 126 9 Robotics amp LIMS Integration 9 5 9 6 REMOTE CONTROL IN SOFTMAX PRO GXP If the target SoftMax Pro is the GxP version R is required in the program command line i e SoftMaxProGxP exe R when launching SoftMax Pro GxP in order to avoid the Log On dialog which requires manual entry of user ID and password Instead the Logon remote
188. s a command and all its sections are available in cascading sub menu commands Selecting a section from one of these sub menus opens the section in the current window Expand and Collapse Sections Newly created Experiments and sections are initially shown expanded To simplify naviga tion within an Experiment data file you can collapse sections to show just their tool bars SoftMax Pro Software User Guide 0112 0140 Rev A 3 6 Sections 3 6 3 To expand or collapse a single section click its triangular indicator located in the top left corner of the tool bar To expand or collapse all sections in an Experiment or multiple Experiments in a file use the View Expand and View Minimize commands or their associated hot keys Ctrl and Ctrl Reorder Sections You can change the order that sections appear in an Experiment by drag and drop Select the title bar of the section and drag it to its new position in the Experiment marked by a thick black line Open Section in New Window To open an active section and edit it in a separate window select New Window from the active section menu Plate Cuvette Graph Notes or Group Closing the new section window or expanding the section in the original window saves changes made to the section Print Section The File Print Section Name commands enables you to print just the active sections NOTES SECTION Notes sections are used to record text or to report Summa
189. s and a CuvetteSet section and read all three with AutoSave enabled gt Filel contains data for PlateSectionl gt File2 contains data for PlateSection AND PlateSection2 gt File3 contains data for both Plate sections AND the CuvetteSet section This functionality applies to both SoftMax Pro and text file formats SoftMax Pro Software User Guide 0112 0140 Rev A 2 6 The Protocols Menu 2 5 4 2 6 SoftMax Pro File When selected your data will be AutoSaved as a proprietary SoftMax Pro file format pda standard or eda GxP file format If your protocol contains more than one Experiment section only the Experiment containing the plate cuvette being read is AutoSaved XML File XML is supported for data export and AutoSave XML is the optimal file format if you plan on importing all read data into other data collection and storage applica tions specifically LIMS Laboratory Information Management System or SDMS Scientific Data Management System packages Tab Delimited File When selected your data will be AutoSaved as a txt file format which can be opened by any word processor spreadsheet or database program If you are saving data to a text file use Append to File to save all data for all Plate and CuvetteSet sections to a single txt file or use Create New File to save the data from each plate to a new file PRINT DOCUMENT AFTER READ Checking this box enables automatic printing of a report after
190. s are stored in the data file and they are subtracted from the individual well values obtained during sample reading Pre read blanking can be disabled in the Reduction dialog box so the data can be reviewed with and without pre read blanking sub tracted The purpose of pre read plate blanks is to correct for well to well variability over a whole plate Pre read blanking is available for all read types but is most useful for Endpoint read ings When used in Spectrum scan reads SpectraMax instruments only the pre read scan is subtracted on a point by point basis from the normal scan If pre read is selected using Kinetic read not generally recommended a pre read with the same Kinetic read interval and duration as the normal reading is done and the OD RFU RLU at each time point of the pre read is subtracted from the OD RFU RLU of the cor responding time point of the normal reading Background constant subtraction is available for Endpoint reads as part of PathCheck cor rection If you pre read a plate make sure that background constant subtraction is dis abled AUTOCALIBRATE This checkbox allows you to disable or enable automatic pre read calibration for all instruments AutoCalibrate for Absorbance Reads For Absorbance reads an air calibration value is used to provide the Ip value for the Log 1g I calculation Air calibration values for all wavelengths offered by the instrument are stored in nonvola tile RAM and are loaded
191. se reports using Criterion Host 2 0 or ScreenPlay 2 5 for all modes ABS FI FB Epi ABS Lum and the dual method saved as text txt When importing 1536 well data from the Analyst instrument only the first quadrant of 1536 well plate data the top left section of 24 by 16 wells can be imported This data is displayed in the standard 384 well plate format Instrument parameters for each plate in the text file are imported into a Notes section as text Raw data is imported into a Plate section The plate ID from the text file is used to name both the Plate and the Notes sections and the file name is used to name the Experi ment More than one text file can be imported into the same SoftMax Pro file by creating or copying another Experiment into an existing file All analysis features of SoftMax Pro can be used to analyze Analyst files Import FLIPR File gt Import Export gt Import FLIPR allows you to import FPD files that have been gen erated using FLIPR instrument control software version 2 0 and above Files are exported from the FLIPR software using the Plug Ins gt Import Export gt FLIPR command In order to import FLIPR files they must be exported by the FLIPR software into the specific FPD format Standard FLIPR files of the FID and FWD formats cannot be imported into SoftMax Pro All analysis features of SoftMax Pro can be used to analyze FLIPR files IMPORTING AND EXPORTING TEMPLATES Template informatio
192. selection should receive fluid gt The fluids are dispensed from left to right gt The fluid transfer targets are cumulative from transfer to transfer that is the second transfers targets start with the next available clean tip and untargeted compound col umn rather than reusing tips and compound columns targeted by the preceding fluid transfer SoftMax Pro Software User Guide 0112 0140 Rev A 4 2 Instrument Settings 4 2 24 4 2 25 4 2 26 5 Each fluid transfer uses a new tip When the number of columns in the Wells to Read area is the same or greater than the number of columns of both Tips and Columns the algorithm fills the Tips and Columns targets as before that is each assay plate column is targeted with a new tip column and new compound column until all tips and columns have been targeted SoftMax Pro does not fill beyond the limitations of the available Tips and Compound col umns Once all available tips and compound columns have been assigned SoftMax Pro stops assigning targets Settling Time SpectraMax M5 and M5 Only In Fluorescence Polarization mode Settling Time specifies a well to well read delay After the read head moves to the next well the read is delayed by the settling time to allow the fluid in the microplate wells to settle thereby minimizing sloshing For all other read modes the selected delay is added between the reading of each column there is no delay between each well in a
193. self With the 28 x a small 1 is embossed in the plastic of the adapter 1 Connect the MDC serial cable to PORT 1 on your USB to serial adapter 2 Connect the USB to serial adapter to a open USB port on your computer 3 Turn your MDC microplate instrument on 4 Launch SoftMax Pro software 5 Select Edit gt Preferences menu item and make sure the correct serial port is selected Note f you are using a multiple port adapter with more than one MDC microplate reader be sure to use the serial cables that shipped with your MDC microplate instrument Non MDC serial cables will NOT work with MDC microplate instruments Note These USB to serial adapters do NOT work without installing the correct software driver If you are not sure you have or are using the latest driver available please check with the manufacturer directly Drivers can normally be downloaded from the manufacturer s SoftMax Pro Software User Guide 0112 0140 Rev A 2 Installation amp Setup 2 4 2 4 1 2 4 2 website Be sure you have installed the latest software drivers before using a USB to Serial adapter with your MDC microplate instrument TROUBLESHOOTING INSTRUMENT CONNECTIONS If the instrument is properly connected and turned on you will see the instruments icon in the upper left corner of the SoftMax Pro window below the menus and the correct instrument is selected in the Edit gt Preferences dialog box If the instrument is not pro
194. sion to produce enough electrons to generate a useful current Pipette Tips FlexStation only The FlexStation instrument requires the use of pipette tips Molecular Devices suggests that you load a complete set of pipette tips each time you make a run to ensure that suffi cient tips are always available Please see the instrument manual for the FlexStation regarding the types of tips that can be used and how they should be loaded Plate Section The Plate section is used to display data to specify how microplates are 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 that determines the type of microplate being read The specification of the plate type depends on the well format and manufacturer of the microplate Point to Point This curve fitting option fits a linear equation to each pair of data points Point Count Number of Reads per Well The number of integrations performed on a single well in a Fast Kinetic read Protocol File Contains the template s instrument settings and reduction parameters Protocol files are useful if you repeat a particular type of protocol frequently Quadratic Curve Fit A mathematical description of a parabola y A Bx Cx Ranged Data Display Raw or reduced data is assigned proportionally to integer valu
195. ss precision SoftMax Pro Software User Guide 0112 0140 Rev A 2 2 Data Display 5 2 2 gt 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 5 Rotated Same as the Normal view except that it is rotated 90 degrees clockwise well A1 is in the upper right corner gt 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 File gt Print Setup 5 Interleaved 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 in the upper left corner but it is followed horizontally by well A3 A5 A23 A2 A4 etc and vertically by well C1 El OL B1 DL erc This display is most useful when the 384 well plate is com posed of 4 daughter plates of 96 wells each Polarization Display Polarization Display options are available in the Display dialog box when the read mode is Fluorescence Polarization You can choose to display Polarization mP Anisotropy r or Raw S amp P values The G factor in the Polarization and Anisotropy calculations default is 1 can be manually adjusted within the range of 0 01 to 99 GRAPHING WELLS Y
196. t all automated shaking of the micro 8 plate is performed during a reading AutoRead This feature enables automatic reading of subsequent Plate sections in the order in which they appear within an Experiment You can set intervals delay times between the plate readings if desired Baud A data transmission rate measured in bits second for communication devices Bottom Read Available in the Instrument Settings this selection causes instruments with bottom read capability to read up through the bottom of the microplate rather than down from the top Compound Source Compounds are transferred from the compound source plate to the assay plate Cubic Spline Curve Fit This curve fitting method 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 Cutoff A filter used to condition the light entering or exciting the monochromators In automatic mode the instrument sets the cutoffs automatically based upon the wavelengths chosen for reading With some read modes and types you can choose a different filter wavelength manual setting for the emission monochromator CuvetteSet Section CuvetteSet sections determine how the cuvettes will be read display data as it is acquired from the instrument and define how the data will be displayed and used in reduction CuvetteSet sections can display samp
197. t parameters for 4 and 5 parameter curves Parameters can be fixed to a constant value z e a number or they can reference by formula other information in the SoftMax Pro file 4 e a curve fit parameter from another Graph section This option can be used independently of weighting and parallel line analysis selec tion Parallel Line Analysis This feature accessible through the Parameter Settings dialog box is only available for the 4 and 5 parameter curve fits Determining parallelism between a standard and test compound and subsequent estimation of relative potency are fundamentals of bioassay use in biopharmaceutical development and testing For linear data parallelism is evaluated by examining the similarity between the slopes of straight lines SoftMax Pro Software User Guide 0112 0140 Rev A 83 84 5 Data Analysis Bioassay data and immunoassays in particular has been demonstrated to fit well with a sigmoidal shape defined by a 4 or 5 parameter curve fit These S shaped curves are not parallel within the classic definition of never intersecting However the basic question of whether a curve with a specified shape can fit both standard and test data sets when shifted along the X axis can still be addressed following a method first detailed by Gottschalk and Dunn Journal of Biopharmaceutical Statistics 15 437 463 2005 SoftMax Pro begins by fitting the standard and test samples to non linear curves m
198. tMax Pro protocol file and data file SoftMax Pro Software User Guide 0112 0140 Rev A 6 1 Tutorial 1 Quantitative Endpoint Protocol Tutorial STEP 7 READ THE PLATE If you were reading an actual microplate at this time you would 1 Prepare the samples in 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 2 Place the filled microplate in the drawer of the instrument Since we are using a simulator you don t need to load the instrument 1 To enable the simulator select the Plate 1 group hold down Ctrl Shift and choose Control Enable Simulator 2 To read a plate selected Control gt Read or click the Read button in the Status Bar at the top of the window 3 Values appear in the data display in the Plate section as if they were being read by the instrument although faster than in real time 4 The display for each well defined by a group boundary shows a raw value The reduction formula is shown at the bottom of the display Wavelength Combination Lm1 5 Click the Display button in the Plate section tool bar Select Reduced Choose Grayscale from the Options Display list The dialog box updates to show the low and high limits for grayscale display values which are automatically set to the minimum and maximum data values 6 Click OK The data display in the Plate section up
199. ted by SoftMax Pro can be exported for use with other software or for archiving Time Plate XML For Plate sections you can choose one of the following three formats gt Time exports data in single column text for each well 5 Plate exports data in a text matrix corresponding to a microplate grid gt XML exports data in an XML file format CuvetteSet sections are automatically exported in the time format Include Labels Includes microplate column labels for all exported data This is most useful for Group section data since the column headings in the Group table are shown including any other calculated data in Summaries etc Labels included with Plate and CuvetteSet sections show 1 12 headers for 96 well Plate section data exported in Plate format 384 well plate data have 1 24 headers CuvetteSet section labels show A1 A2 etc representing the cuvettes shown in the CuvetteSet sec tion Include Filename and Date Includes the file name and date in the file with the exported data Include Account Info Includes the name of the user who generated the file This option is available only if you are using SoftMax Pro GxP Interpolate Wells FlexStation Only Interpolation is used to normalize the Kinetic read times of wells against the read time of the first well in each column Because injection occurs at the same time in all wells of a SoftMax Pro Software User Guide 0112 0140 Rev A 2 5
200. ter a defined period of inactivity gt Guest Access The administrator can specify whether or not guest users are allowed to launch SoftMax Pro GxP If enabled guests can only open view and print existing SoftMax Pro data files gt Forced Password Change When set globally all users are required to change their pass words when they first log on to SoftMax Pro GxP gt Lock Software after three log on attempts inactivate specific user accounts within a User Accounts file after three failed login attempts The administrator can reactivate users remotely by using GxP Admin software gt Offline Use When specified by the administrator SoftMax Pro GxP automatically copies an encrypted read only copy of the User Accounts file for each user after each user has successfully logged on at least once This local copy of the User Accounts file under administrative control then allows users to work without a persistent network connection thereby allowing users to continue using SoftMax Pro GxP when no net work connection is available For information on the specific use and configuration of GxP Admin features please con sult the GxP Admin User Manual USING SOFTMAX PRO GXP USER LOG ON Authorized Users Once SoftMax Pro GxP has been installed all authorized users can log on to SoftMax Pro GxP and begin working Simply launch SoftMax Pro GxP and all users will be prompted to enter their UserID and password Guests New users who
201. th the requirement that the first and second deriva tives 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 Exponential The exponential function used to generate this curve fit is y A B 1 exp x C Minimum Number of Standards The number of standards used to generate a standard curve depends on the particular curve fit selected and on the judgment of the operator The table below gives the mini mum number of standards required mathematically by each of the curve fitting algo rithms Table 5 3 Minimum Number of Standards Required for Curve Fits Curve Minimum Number of Standards Linear 2 Quadratic 3 Semi Log 2 Log Log 2 Log Logit 4 4 Parameter 4 5 Parameter 5 Point to Point 2 Exponential 3 Cubic Spline 4 For optimal results you should always exceed the minimum number of standards required for any given fit There are other requirements on the standards for the log logit and 4 parameter and 5 parameter logistic curves The fits for the logistic curves are based on the assumption that the curve has a high and a low asymptote and a certain steepness for the linear por tion 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
202. the SoftMax Pro icon to start the program Choose the File gt New command to create an untitled document This will select your default Protocol format if you had previously selected one If you are not connected to an instrument and want to emulate a FlexStation instrument choose Edit Preferences and choose a FlexStation instrument from the Reader list The Experiment has a Notes section Notes 1 and a Plate section Plate 1 STEP 2 DEFINE INSTRUMENT SETTINGS 1 2 Select the tool bar of Plate 1 to make it active Click the Settings button in the tool bar or select Plate gt Settings Endpoint is selected by default This tutorial uses a Flex read so select Flex Clicking the setting name bold heading on the left side of the Instrument Settings dialog box shows the choices available for that setting in the right pane allowing you to make changes Following are the details about the EC50 protocol settings we will use to create a protocol file using SoftMax Pro Make sure all settings match those in the table Table 6 2 Example Settings Read Type Flex Wavelengths One Excitation 485 Emission 525 Auto Cutoff Sensitivity 3 readings PMT set to High Timing Time 200 seconds Interval 2 seconds Total number of readings 101 Minimum interval 1 28 seconds Assay Plate Type 96 Well Standard clrbtm Wells to Read A1 H10 Compound Source Beckman 96 2 3 mL Compound Transfer None
203. the cen ter of the curve and is commonly known as the slope factor A large value of B describes a sharper transition Typically B has a magnitude of about 1 for inhibitory experiments and 1 for excitatory experiments SoftMax Pro Software User Guide 0112 0140 Rev A 5 4 Group and Graph Sections Both the log logit and 4 parameter equations are displayed as ys A D 1 x C B D 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 highest values of x are used The corresponding y values are assigned to 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 The 5 parameter logistic equation has an extra exponential term G that is sometimes described as an asymmetry factor y A D 1 x C B G D As with the 4 parameter logistic equation D is the high asymptote A is the low asymptote and B is the slope factor Unlike the 4 parameter logistic equation how ever C is not the IC50 value The IC50 value needs to be calculated separately This is described in Chapter 3 of the Formula Reference Guide Use the 5 parameter logistic equation inste
204. ting and analyzing data is as follows Launch SoftMax Pro software Create Plate and CuvetteSet sections as needed Specify instrument settings using Plate Settings Define templates reduction parameters and display parameters Prepare the microplates or cuvettes to be read and place them in the instrument Start the reading Save the data file If required modify templates reduction parameters or display parameters o o JO OC hb Y DN Analyze the data using Group section tables and Graph sections 10 Save the final data file Instrument settings are associated with a Plate or CuvetteSet section in a SoftMax Pro Experiment You can create more than one Plate or CuvetteSet section in a single Experi ment and the instrument settings associated with each Plate section can be different You must specify instrument settings wavelengths read mode etc prior to reading a microplate or cuvette When you have defined instrument settings a summary is displayed to the right of the data display in the Plate or CuvetteSet section INSTRUMENT OPERATION Instrument settings are made in the Plate gt Settings dialog box You must select a Plate sec tion to make this menu available The operation of individual instruments is only described here to illustrate the functional ity of the software For complete information about the function of the instrument or how to set it up for a reading consult the instrument manual
205. tion and Plate gt Import Template or Plate gt Export Template Template files can be prepared outside SoftMax Pro and then imported as needed when ready to run samples The easiest way to determine the correct format for importing a template is to export a template from SoftMax Pro READING A MICROPLATE OR CUVETTE DATA COLLECTION FROM A MICROPLATE 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 instrument are raw absorbance fluorescence luminescence or fluorescence polariza tion values and are not affected by settings in the Template Editor To read a microplate 1 Open the drawer of the instrument by clicking the Drawer button in the Status Bar or selecting the Control gt Open Drawer command 2 Insert the prepared microplate 3 Click the Read button in the Status Bar or choose the Control gt Read command 4 The active Plate section is read If no Plate section is active a dialog box is displayed in which you can choose a Plate section to read 5 If you have chosen to pre read the plate for blanking you are offered the choice of a Pre read or a Normal read e no pre read After reading the blank plate you must start the read process again for the normal plate 6 When a reading begins the Read button changes to Stop allowing you to terminate a reading if desired SoftMax
206. tte d D EE Installing an Instrument a vesicae KK KK KK a Computer System Requirements o ooooooooommo mom Installing SoftMax Proc ooi ub a KK KK KK KK KK KK Uninstalling SoftMax Pro cue e de reg KK KK KK KK KK KK Running the Software for the First Time oooooooooo Communicating with an Instrument 000 Launching SoftMax Pro Software o u uc ea bar KK KK Entering Registration Informati0N ooooo oooomoooo Connecting the Instrument via USB WW RR eee Single Port Adapters 2254 5192 025 KK KK KK KK ce KK KK KK KK Multiple Port Adapters sia pex KK KK KK KK KK KK KK KK KK Troubleshooting Instrument Connections ooooommmo o Setting Preferences peas eras Reader Settings sor 20 p O d A EU a ars Manual Export Format 42 226 eL kK es KK KK KK KK KK KK es Save Data After Read AutoSave ooooooooomm o o Print Document After Read 0 KK RR RR RR KK The Protocols Menu kk kk kk kK KK KK KK KK KK KK KK KK KK KK SoftMax Pro Interface The SoftMax Pro Application WindoW 0ooooococooooo WYSIWYG Display 2 lt ee rta sla ett alah V n il ee La aces Statis D DD AE NS Instrument TG ttc 553 av SK Qedir OK ada e a rue tede arr Rs SoftMax Pro Software User Guide 0112 0140 Rev A Contents Close Drawer Open Drawer A kK KK KK KK KK KK KK KK KK KK gud Incubatat jo 43 2 aec UP READ PRENEE REESE ME EEp se PIRE Experiments osse bore RUE a U
207. uded in the printed report You can exclude sections from printing as described in Chapter 7 File Management amp Printing 2 Choose File gt Print to print the whole Experiment 3 Select the appropriate settings for your printer and then click Print or OK to print the Experiment report Congratulations You have completed this tutorial TUTORIAL 2 EC 50 PROTOCOL TUTORIAL FLEX ONLY Tutorial 2 is designed for a FlexStation instrument using a Flex read It assumes that you have read the previous chapters in the manual and have a basic understanding of the logic and operation of SoftMax Pro The following steps are required to create and run an EC50 protocol using a graph of con centration vs RFU Create a new data file Define instrument settings Define template Set reduction parameters Set display parameters Save the protocol Read the plate you won t actually do this instead open a saved file Data analysis Group sections o o JO C FP Y NM Data analysis Graph 10 Data analysis Summary formulas 11 Print a report The following steps guide you through creating a protocol file for an EC50 protocol using a Flex read Reading an actual plate is not required as SoftMax Pro can generate simulated data SoftMax Pro Software User Guide 0112 0140 Rev A 6 2 Tutorial 2 EC 50 Protocol Tutorial Flex only 6 2 1 6 2 2 STEP 1 CREATE A NEW DATA FILE 1 2 Double click
208. ues By default they put the data in the clipboard but optionally they use the WM COPYDATA mechanism to return data directly to the caller In order to have the SoftMax Pro response returned in this fashion the commands must be sent using WM COPYDATA so that SoftMax Pro knows what to send the response to SoftMax Pro then replies with another WM COPYDATA message that contains the response MFC EXAMPLE CODE The sample MFC code is taken from the Wisual C 1RemoteCmdTest v2 project in the Automation Examples folder which is installed in the SoftMax Pro application directory by default To send a command to SoftMax Pro RSE OOO ARO IA ASAIO IA IIIA SendCmdToSMP Send a single command to SoftMax Pro 1 Retrieve the handle to the SoftMax Pro window 2 Register the window message 3 Construct the COPYDATASTRUCT 4 Send the command to SoftMax Pro O void CMySampleClass SendCmdToSMP CString cmdStr SoftMax Pro Software User Guide 0112 0140 Rev A 9 8 MFC C Interface to SoftMax Pro Remote Commands that Return Values HWND hwnd FindWindow SOFTMaxPROMainWnd SoftMax Pro UINT smpMsg Register WindowMessage SOFTMaxPROMsg use WM_COPYDATA char temp 255 strcpy temp LPCSTR cmdStr COPYDATASTRUCT cds cds dwData ULONG_PTR this gt m_hWnd cds cbData DWORD strlen temp 1 cds lpData amp temp SendMessagel hwnd WM_COPYDATA WPARAM smpMsg LPARAM amp cds
209. ues above the high limit an asterisk for values within the limits and a minus for values below the low limit Ranged When Ranged is chosen raw data that falls between the high and low limits is scaled on an integer scale from 0 to 9 Values above the high limit are displayed as a plus 4 and values below the low limit are displayed as a minus Grayscale Grayscale presents the raw data in eight shades of gray changing from light for val ues less than or equal to the low limit to dark for values greater than or equal to the high limit Plots All Read Types Except Endpoint The Plots option presents Kinetic or Spectrum data as plots of OD RFU RLU versus time or wavelength Show With Reduced Number When you select this option you can see both the raw and reduced numbers displayed Raw Data in 1000 s Use this option to display Luminescence readings for example as multiples of 1000 to make the numbers in the wells easier to read e g 20 000 would be shown as 20 384 Well Display Data in the Plate section is displayed in a grid array microplate format If you are using a 384 well plate you have the following additional options for displaying the data gt Normal Shows the data in standard left to right format with well Al 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 le
210. um data the blank value is averaged and subtracted from each point in the read Template blanks are subtracted after the cuvette reference reading is subtracted Group Associated Blanks Plates and Cuvettes Blank wells can be assigned within any group other than the Blank group in the Template Editor 1 Open the Template Editor for the plate 2 Select the wells or cuvettes to be used for the group associated blank These should be wells or cuvettes that are already assigned to a Group not the Blank group 3 Select BL from the Sample list The average reduced value of the blank wells or cuvettes within that group is subtracted from individual reduced values within that group only Use Group Associated Blanks when samples on the plate have been prepared differently and therefore require individual blank correction 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 and you do not have the option of reviewing the uncorrected data To see the reduced number without the group blank subtracted you must mask the group blank wells or cuvettes see Section 5 2 3 Masking Wells or Cuvettes Group blanks apply to all wells or cuvettes in a group and thus can be subtracted from wells or cuvettes in more than one Plate or CuvetteSet section SoftMax Pro Software User
211. ur Experiment To delete a group you must delete the Group section Clearing a group from the Template Editor removes only the assignment of wells to that group name it does not delete the group Name Use either the default name Group X where X is an integer that is initially set 1 2 3 etc as other groups are created or enter a custom name up to 32 characters in length to 1 and is incremented f e Sample Descriptor Check Sample Descriptor to apply a descriptor and its units to the group Select a set of units from the list or type them directly in the Units field Sample Descriptor is automatically selected when either the Standards or Unknowns Dilution column format see below is selected The Sample Descriptor field can also be used for time or fraction number information No sample descriptor is assigned automatically for the Basic or Unknowns column format You can assign a sample descriptor to these column formats manually but the information does not appear automatically in the Group section To see it create a new column containing the formula Sampledescriptor factor or concentration For more information about creating columns see Adding Columns on page 75 Column Format The Column Format setting defines the default columns for the data calculated and reported in the associated Group section of the current sample group Each of the four column formats Basic Standard U
212. using the averaged values The default is no smoothing setting of 1 averaged point A setting of 3 provides the least amount of smoothing the value of the center point is added to the values of the points on either side the sum of these points is then divided by three and the resulting value is substituted for the center point A setting of 9 averages the center point with the four points on each side When multiple wavelengths are read smoothing is performed before performing wave length combination calculations such as ratios or multiplication For example if you read at two wavelengths and choose to take a ratio of those wavelengths smoothing is per formed on each individual wavelength and the wavelength ratio is calculated using smoothed data Baseline Options Baseline options affect the way that the data is graphed Absolute Shows the raw data without any change in the way it is graphed Raw data is used in subsequent reduction calculations Zero Baseline Offsets the graph slightly If you enter 5 for the number of points for example the average of the first 5 points is used as the baseline value and all raw data are subtracted from this value Baseline subtraction is applied only to the reduced data raw data plots do not change To view the plot with baseline subtraction choose Reduced Plot in the Display dialog box or select Show Reduced in a zoomed well Graph window If two or more wavelengths
213. ust be kept long for reference you will have to set up your replicates manually Hold down Ctrl Shift keys to display the values for all wells SoftMax Pro Software User Guide 0112 0140 Rev A 4 3 Template Edftor 4 3 7 BLANKING In addition to the pre read plate blanking configured in Plate gt Settings and a reference reading of a cuvette in the Template Editor you can configure Plate blanks and group associated blanks The different blank types have these features gt Pre read plate blank not in the Template Editor The pre read value of each well of a microplate is subtracted from any reading made in the well It is used to correct for well to well variability across a whole plate generally not required for current plates that are highly uniform 5 Plate blanks used to correct readings when all samples on a plate have been prepared in the same buffer or matrix and therefore can use the same correction value Plate blanks are subtracted from the raw data or after pathlength correction if PathCheck is selected gt Group associated blanks used to correct readings when samples on a plate have been prepared in a different buffer or matrix and therefore have to be individually corrected Group blanks are subtracted after wavelength and Kinetic reduction Blanks can be used in combination and have cumulative effects Plate Blanks Plate Sections Wells can be assigned to a Blank group in the Template
214. ve the data for this column MeanValue Summary 1 100 This formula divides the numbers from the MeanValue column by the value of Sum mary 1 and then multiplies the result by 100 to construct a percentage 5 Close this dialog box by clicking OK Resizing and Autosizing Columns gt You can resize columns by double clicking or dragging column dividers gt To Autosize columns select the columns and use the Group gt Autosize command Adding a Column 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 high lighted column or if no columns are selected after the last column in the group To add a new column to the Data group 1 Select the groups tool bar to make it active Deselect any highlighted columns by clicking outside the column area Choose Group gt New Column or click the New Column button Rh 0 N We are creating a new column that displays the maximum value obtained for each sample Enter MaxValue in the Name field 5 Enter Max WellValues in the Formula field and click OK A new column is displayed at the right of the previous columns showing the maximum value obtained for each sample SoftMax Pro Software User Guide 0112 0140 Rev A 101 102 6 Tutorials Step 9 Data Analysis Graph 1 Scroll down the SoftMax Pro window until you see the section tool bar for Graph
215. xed wave The value for the fixed em or ex wavelength 25 Reads per well Number of times a well is read for a single reading 26 PMT setting Automatic High Medium or Low 27 Start integration time Time to start integration for Time Resolved Fluorescence otherwise blank field SoftMax Pro Software User Guide 0112 0140 Rev A 109 110 7 File Management amp Printing 7 3 2 Field Field Entry Description 28 End integration time Time to end integration for Time Resolved Fluorescence otherwise blank field 29 First row read First row read 1 to max always 1 for cuvettes 30 Number of rows Last row read 1 to max always 8 for cuvettes 31 Time tags Indicate whether the exported data has read time tags The information for the first Plate or CuvetteSet section is provided in the lines following the descriptors The number of data lines depends on the export format and the type of read that was run A minimum of one data line is exported when the Time format is used with a single wavelength Endpoint read If your instrument has a temperature associated with the data all Readers except EMax VMax and UVMax the temperature at the time of the reading appears before the first data point in the reading The second line of a file for a Group table section includes fields that correspond to the number and types of columns found in that section they are labeled as the table is

0112-0140_Rev _SoftMax Pro Software user guide.book

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