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User manual Software version 2.00 release 9

1. 113 Tools menu The window that appears allows you to define the centre point of each of the four corner wells Each tab shows one of the four wells with a plot of the intensity of the light from the well This is colour coded with red showing the highest intensity Position the mouse cursor where you think the centre of the well is and click the left mouse button A black cross will appeared centred at the point of the cursor If you are not satisfied with the position of the cross move the mouse cursor and click the left button again Repeat this for each of the four wells When the centre of all four wells has been marked the Next button will be enabled Click this to continue Wallac 1420 Plate Dimension Wizard Numerical values are shown in the final window If you approve these click Finish to save the values and end the wizard If you feel the deviations are too large you can click Back to reposition the well centres You can alternatively click Cancel to end the wizard without saving the values Note for even better adjustment it is recommended that you repeat the scan with a smaller area selected see the lower figure on page 112 e g for a 1536 well plate this could be 1 mm per well 114 Tools menu Miscellaneous settings Miscellaneous Settings Well Names Instruments Generic 8x12 size pl Labsystems 8x12 DELFIA strip plate NUNC 8x12 DELF A strip plate Costar 35
2. Wallac 1420 Manager alr aj e 2 ve Tempra Wallac 1420 is operated by moving the mouse cursor to a point in this window a menu title a button or an icon and clicking the left mouse button If you click an icon or a button an operation will be started If you click a menu title the menu will appear You can then move the cursor to the item you want in the menu and click the left mouse button again to activate it 15 Operating Wallac 1420 Instrument control The main window has three tabs Instrument Control Live Display and Temperature The instrument control tab enables you to control the instrument by starting stopping or ending counting It shows you the protocol being used to control the measurement and it also shows status of the instrument If the system supports plate heating the status bar will display the current temperature of the plate and samples When run with a system not supporting plate heating this will not be displayed When the workstation is used without an instrument i e running in demo mode the status bar includes the text DEMO When Wallac 1420 workstation is connected to an instrument running in normal mode the demo mode indicator is blank You can switch between normal and demo modes using the Options selection in the Tools menu 16 Operating Wallac 1420 Live display The Live display shows you the live results in the form of a plate map with colour coding to indicate
3. sssseessssesssseseresrsrresesreerrereseees 80 Protocol name 482 24s 85 ee o EE E os a Es Inoi e islas lisina see 80 EW Lamp Enerdy AA E E E E E ET 81 AA NN 81 Polarizer aperture 22ER es A 81 Emission fillers 4 8 0 2 208 Bee lasse arten 81 EDUSSIOMAPCLUUES ui 81 COUNTING TIME ius as eA Rei A A AAS A ea 81 G factor nidad td 82 EANCE ctas 83 Protocol Mame cti tn 83 Fl sh Energy Ardo ideada 84 Flash Energy Level lina alpina 84 Excitation Alter 8 0 ee sa RR TEE EErEE 84 Light integration capacitors mino di 84 Note 3 3u 8082 20H As ER N GS Mis olavecet Heh pb steers Gants cee 85 Light Integration Reference Level oooooononicnnccniccnoonconoconocononnnc noc nono nccn nono nonnnconcrnonns 85 Emission filters sess ssesssccssdssees ates Se sassteseessavessssdave cove Sshosdenpee aves sgveasds dees sbea ie 85 A A fede enttoas sepsiech se a reamees E eaaeo eani R 85 Counting parameters oo so is6s sabes ecatepec sn sense eisen enges 86 Factory set values for the main counting window ooooococcconcnononononnconnonnnonnnnancnunonanonos 86 Label titan red 86 Delay lia ia in 86 Delay ass sched isn aiii bs Aes na 86 Window 1 tok reed Bele ea 86 Window 2s sci adieu iasdaane Gainnh isin aaais aonnh owe hws 86 Clio 86 Flash absorbancia ligadas 86 Beam SIZE Aisa ucts cei sae aioe ae Nae Mara Sate ee 86 Second measurement parameters eee cee cseeeeeeeeeeeeeeeseeseeeeeseeeesecnsecsaeesaessaeaee 87 LANCE abel Properties sessi
4. There is also a section where you can add additional information that you can connect with the filter properties Factory pre set CW lamp filter names begin with a letter indicating the technology for which they are to be use F is for fluorometry P is for photometry absorption After the letter comes the wavelength 90 Tools menu Filter slides E Emission filter slide A Emission filter slide B Cw lamp filter wheel Cw lamp filter wheel B There are two types of slide Emission filter and CW lamp excitation filter The former is a strip and the latter a wheel The instrument is loaded with slide A in each case but an empty slide B is also available for user filters as described below 91 Tools menu Emission filter slide Emission filter slide A Properties Available unused Filters m Filter Positions All filters can be dragged from the Available Filters list to an empty position in the slide or from one position to an empty one or back to the list 20000000 MM Factory Defined Eject Slide HB User Defined There are 8 positions in emission filter slides A and B The physical dimensions of the filter slots are diameter 25 5 mm depth 11 mm clear aperture 22 mm All standard filters with diameter of 25 4 mm 1 inch and thickness of 10 mm fit in the emission filter slides In emission filter slide A positions 1 to 4 and 8 are used for emission filters for
5. You can also add a new plate delete the currently selected plate or duplicate a plate An added plate has all the positions marked as Measured and will become the last plate in the sequence When you delete a plate it is the one currently visible that is deleted A duplicated plate is a copy of the current plate and will become the last plate in the sequence A map of all the wells appears An empty well 1 e one that will not be measured will be grey a well with a sample i e one that will be measured will be coloured If you move the cursor onto a well the sample type will appear If you click on the well a list of available well types will appear 39 Protocol editing To change the markings for the individual wells in a plate you can select a group of wells by using the mouse To start the selection click the left mouse button at the desired starting position and then drag the mouse cursor to the desired ending position on the plate without releasing the left mouse button Complete rows or columns can be selected by clicking on the character number on the plate frame corresponding to the row column More than one row column can be selected by clicking the mouse and dragging in the same way as when selecting free well areas The whole plate can be selected by clicking on the upper left corner of the plate frame To select a rectangular area of wells on the plate hold down the Shift key while dragging with the mouse The number of
6. pad to allow you to decrease or increase the number You can specify lt AssayIDpadX gt where X 1 7 or lt RunIDpadX gt where X 1 4 Examples If lt AssayID gt 1234 and you add pad 2 to get lt AssayIDpad2 gt the result will be 34 This would be useful with MultiCalc which could not accept an assay ID as large as 1234 If lt AssayID gt 234 and you add pad 4 to lt AssayIDpad4 gt the result would be 0234 This would be useful if you needed a four digits identifier The validity of possible keyword arguments is checked when you try to switch from this view to another If an invalid argument is found you are notified and the page switch is cancelled 51 Protocol editing General E Europium Protocol editor als 2 This tab gives you information about the protocol when it was created when it was edited most recently by whom it was created and edited and when an assay was last run with it The user names used come from the identifier given when Windows was booted Pre set protocols are created in the factory 52 Protocol editing File menu The protocol editor file menu contains the following items of which Save and Print have corresponding buttons Save When you select this item or click the button the current protocol information is saved Print When you select this item or click the button a dialogue opens up for you to select the printer type and other details of the printout Print pr
7. 30 Strip orientation 118 System requirements not met 131 T Temperature 9 16 19 47 60 Temperature tab 19 Time resolved fluorometry picture 69 Tools menu 65 In main window 11 Troubleshooting 129 130 Tubing 142 Tune Plate dimension 118 U Up button Measurement tab 43 Usage of MultiCalc 136 User level 65 66 V View results 57 58 Virtual memory dialogue 129 155 Index W Wallac 1420 MultiCalc assay protocols 135 Warning 131 Waste vial 140 Well columns 118 Well name Miscellaneous settings 118 Well names 115 Well operations 39 Well rows 118 Wizard Dual label normalization 98
8. CLOSED A OPEN THE CLIP B LIFT THE FILTER WHEEL z ion LIFT THE LAMP VERTICALLY OFF FROM THE CLIPS HOLDING IT REPLACE THE WHEEL AND CLOSE THE CLIP GE 2 put THE LAMP OFF FROM CAUTION THE LAMP SOCKET DO NOT TOUCH THE OPTICAL COMPONENTS AND THE LAMP BULB OR THE INSIDE OF THE WARNING LAMP REFLECTOR WITH BARE FINGERS LAMP AND OPTICS MAY BE HOT CODE 1420 401 Changing the CW Lamp If the CW Lamp needs replacing lift the cover to get access to the lamp Make sure the lamp is switched off and give the lamp chance to cool before removing it and replacing it Follow the instructions on the inside of the cover see the picture on the next page You will probably find it easier to use your left hand to remove the lamp from the clips holding it When you have replaced the lamp close the cover and switch power on 95 Tools menu Changing filters in the software Note if you change a filter in the software you must also change it physically in the filter slide or wheel When you select Properties a picture of the selected slide appears with the filters that are loaded in it marked in red or black as shown previously Those that are in red are factory pre set The excitation filter slide is in the form of a wheel and the emission filter slide is a strip To move a filter from the list to a place on the slide move the cursor to the filter name on the list drag it keeping
9. future applications Its size and alignment are about the same as the excitation area from the flash lamp 85 Tools menu The shutter is used at all times when no measurement is occurring to prevent stray light getting to the photomultiplier tube Counting parameters These determine the time factors involved in the measurement process all times are in microseconds Counting delay the time after the excitation pulse at which counting of the emission signal begins Counting window the time period within which counting occurs Counting cycle the time between excitation pulses within a measurement The second or additional counting window is fully independent of the first one and it useful when optimizing time resolved parameters or for calculations of the decay time of the label Factory set values for the main counting window Label Delay 1 Delay 2 Window 1 Window 2 Cycle High count 615 50 0 100 0 1000 High count 665 50 0 100 0 1000 Long decay 615 50 0 100 0 3000 Long decay 665 50 0 100 0 3000 545 50 0 100 0 3000 615 50 0 100 0 1000 665 50 0 100 0 1000 572 50 0 100 0 3000 FQA 615 50 200 100 100 1000 FQA 545 50 200 100 100 1000 Flash absorbance If you want there to be an absorption measurement as well as the usual time resolved fluorescence emission measurement select this parameter to be Yes Both measurements will then be made simultaneously Beam size This
10. if no are shown at the end 57 Result viewing Click on the result file and then click the Open icon 8 Wallac 1420 Explorer File Edit Tools Hele CA Protocols Wallac 3 15 03 00 14 03 44 15 03700 14 04 07 DEMO DATA GENERATE Ca Fluorometry EB Fluorescein 485nm 535nm 0 15 Fluorescein 485nm 535nm 1 0s Umbelliferone 355nm 460nm 0 1s Umbelliferone 355nm 460nm 1 0s GA Luminometry Ca Photometry Ca Time Resolved Fluorometry DELFIA ta Fluorescence polarization Ca Time Resolved Fluorometry LANCE Ea Users EA Projects 4 gt Protocol number N A La Note these results are in the form of raw numerical data The Result Viewer gives details of the actual measurement There are five tabs and these described below The Result Viewer also provides you the following functions by means of command line menus or in most cases buttons File Export Button Page setup Print Button and Exit View List Button Plate Button Protocol Button Errors Button Notes Button View all result data only when List is selected Toolbar Status bar Plate first plate previous plate next plate last plate all with corresponding buttons These buttons or menu items that allow you to step between plates only appear when the plate mode is selected Help information about Result Viewer 58 Re
11. parameter can be selected to be Normal or Narrow provided the Adjustable beam size option has been installed It enables measurements on plates with very small well sizes by focusing the excitation light beam to a smaller size 86 Tools menu Second measurement parameters In the case where you want to make measurements with two filters you can use this parameter to select the type of the second emission filter The options are the same as for the first measurement emission filter Two measurements are made first with the normal filter and then with the second which the system automatically moves into place LANCE Label Properties LANCE 615 100 flashes window 50 100 1000 615nm This label is for fast decay Eu chelates for example Eu W1024 in a TR FRET assays LANCE 665 100 flashes window 50 100 1000 665nm This label is for fast decay Eu chelates for example Eu W1024 in a TR FRET assays LANCE High Count 615 same as LANCE 615 but 1000 flashes Higher count rate LANCE High Count 665 same as LANCE 665 but 1000 flashes Higher count rate LANCE Long Decay 615 same as LANCE 615 but 3000 cycle time This label is for long decay Eu chelates for example Eu W8044 in a TR FRET assays LANCE Long Decay 665 same as LANCE 665 but 3000 cycle time This label is for long decay Eu chelates for example Eu W8044 in a TR FRET assays LANCE 545 same as LANCE Long Decay 615 but 545nm emission LANCE 572 same as LANC
12. rectangle and it reserved mainly for future applications Its size and alignment is about the same as the excitation area from the flash lamp The shutter is used at all times when no measurement is occurring to prevent stray light getting to the photomultiplier tube Counter position The counter position can be selected to be Top or Bottom Counting time This parameter determines how long the excitation continues Second measurement parameters In the case where you want to make measurements with two filters and or two different energies you can set these parameters in this field Click the check box Second measurement to activate them There are three of them CW Lamp energy CW Lamp Filter and Emission Filter See the corresponding sections for the first measurement for more details When you select one of more of these parameters two measurements are made the first with the normal settings and the second with these new settings The change is automatic 75 Tools menu Photometry A reference measurement is made before the plate is moved to the measurement position This enables the absorbance to be calculated Photometry Label Properties P405 Slot A1 v P405 Slot A1 The parameters that can be set for photometry are the following Protocol name This is the name by which the protocol is identified A password can be associated with it to prevent parameters being changed by an unauthorized pers
13. stackers The button light will be blinking Light is blinking 2 Click the button briefly One by one all the plates will be loaded and measured If a plate has a barcode then it will be measured using the protocol specified by the barcode If it has no barcode it will be measured using the protocol specified by the previously read barcode 3 Only when the input stacker is empty will operation stop The button light will be blinking Remove the stackers and load new ones if required 4 Click the button briefly to start operation again 29 Operating Wallac 1420 Ending operation Stop button Clicking the Stop button in the workstation interrupts the measuring process immediately The plate being measured is E brought out to the loading position You cannot resume measurement after clicking Stop but must begin the assay from the beginning if required Clicking the End button in the workstation ends the assay after the current plate has been completely measured including any possible repeat measurements The plate is brought out as described above 30 Operating Wallac 1420 Button on Wallac 1420 This button has different functions depending on how long you press it for and what is happening when you press it If a plate has been measured you can remove it load a new plate and briefly press this button to continue the assay with the same protocol as the plate just measured However if you press this butto
14. tab 38 50 Excitation aperture 74 77 Excitation filter LANCE 84 Excitation filter TRF 70 Exit 125 Exit Protocol editor 53 Explorer 35 65 Explorer folders 36 Explorer icons 36 Index Export file per plate 120 Export results Result Viewer 62 F File export 62 File menu 125 File menu Protocol editor 53 File types for export 62 Fill 143 144 Filter CW Lamp 74 76 81 90 Filter emission 71 74 76 78 81 85 89 Filter excitation LANCE 84 Filter excitation TRE 70 Filter properties 97 Filter slide settings changing 96 Filter slides 91 Filter wheel CW Lamp changing 95 Filters 65 88 Filters window buttons 88 Fine tuning plate dimensions 110 Flash absorbance LANCE 86 Flash absorbance TRF 72 Flash absorption measurement 72 86 Flash Energy Area TRF 70 84 Flash Energy Level Time resolved fluorometry 70 84 Fluorescence count time F polarization 81 Fluorescence counting time Fluorometry 75 Fluorescence polarization 80 Fluorometry 73 Fluorometry tab 73 Flush 143 144 147 Folders 36 G General Protocol editor 52 General options 119 General tab 38 G factor 82 152 H Heating 47 Height 46 47 Height of the plate Plate type 118 How to change filter slide settings 96 ID Protocol editor 41 ID tab 38 41 Injector position during measurement 45 Input Output 122 Installation of software 10
15. the measurement is shown by the progress bar The Next button becomes available when the measurement has been completed When the measurement is completed click Next to move the wizard directly to the Normalization confirmation page otherwise the wizard moves to the Measurement warning page Measurement warning page This is used to inform you that something out of the ordinary has happened during the measurement of the normalization assay This page gives you enough information to decide whether the results can be accepted regardless of the problem that caused the warnings Clicking Next on this page advances the wizard to the normalization confirmation page Clicking Cancel closes the wizard and the normalization factors are not recorded in the database 100 Tools menu Normalization confirmation page Wallac 1420 EuSm Dual label normalization Wizard The normalization measurement is now completed Check the results and press Finish to accept the new normalization factors or Cancel to ignore them Normalization factors Ratio 0 3887 Blank 5089 Supplementary values Sample _ Label Mean CV Blank Samarium 2255 6 5 Blank Samarium in Europium window 5159 56 Europium Samarium 2130 je Europium Samarium in Europium window 4836 7 9 Cancel Help This appears after normalization has been successfully completed It shows the measured normalization values and requires you to confirm that you accept these val
16. the mouse button pressed down until it is over the empty slide position that you want it to occupy then release the mouse button The name of the filter will appear in black in the slide position To move a filter from one position to another click on it and drag it to the empty place you want it to occupy To remove a filter from the slide click on it and drag it back to anywhere in the area of the Available filters list then release the mouse button 96 Filter properties Tools menu The filters used in the instrument have the properties shown in the table below The following abbreviations are used CWL Centre wavelength BW Bandwidth Full Width Half Maximum Tpk Maximum transmittance at the peak wavelength Filter type Tr fluorescence excitation Tr fluorescence excitation Europium chelate emission LANCE option installed Europium chelate emission LANCE option installed Samarium chelate emission Terbium chelate emission Dysprosium chelate emission Colorimetric label emission Colorimetric label emission Colorimetric label emission Fluorescein excitation Fluorescein emission Umbelliferone excitation Umbelliferone emission Name D340 D320 D615 D665 D642 D545 D572 P405 P450 P490 F485 F535 F355 F460 Centre Wavelength CWL 340 nm 320 nm 615 nm 665 nm 642 nm 545 nm 572 nm 405 nm 450 nm 490 nm 485 nm 535 nm 355 nm 460 nm Bandwidth BW Full
17. time resolved fluorescent chelates if this option is included position 5 and 6 for emission filters for fluorometry labels umbelliferone and fluorescein respectively Position 7 is kept open for luminometry measurement If you want to add you own filters you must either use slide B which is fully customizable or change some of the existing filters Factory pre set filter names begin with a letter indicating the technology D is for Time resolved fluorometry including LANCE F is for fluorometry P is for photometry not used for emission filters L is for luminometry After the letter comes the wavelength 92 Tools menu User defined filters can be named freely but there is only space for five letters to be shown in the figure of the filter position The full name will be shown if you move the mouse cursor onto the slot where the filter is Physically changing the emission filter slide Click the Eject slide button on the Emission filter slide dialogue A message will appear telling you to unscrew and remove the plate on the side of the counter When you have removed the plate click OK to cause the slide to be ejected Pull the slide out carefully Avoid touching the filters with your fingers When you have got the slide out you can then change the filters or load filters into emission filter slide B When the slide is ready carefully return the slide to its position Push it in as far as it will go Replace the filter slide co
18. versions Tab delimited text or MultiCalc If the Include in output list box is checked then it specifies which optional parts of the assay run information should be included in the file saved when the measurement is completed These are the same as for printer output List Plate Protocol description Error description Notes You must also type in the file name This can include keywords connected with the file as described in Events The button to the right of the filename box gives you a list of all valid placeholder arguments to choose from Other filenames possibly used in other protocols are also available through this button Note you will not be allowed to change to another tab if an invalid placeholder argument is found However if a previous file with the same file name exists it will be overwritten by the new file without warning 49 Protocol editing Events f Fluorescein 485nm 535nm Protocol editor This tab allows you to give a command that will be executed when a protocol has been run The keyword should be in triangular brackets lt gt exactly as the examples given in the tab show These commands are then automatically replaced by the appropriate name or number etc after the assay run ends The first box specifies the executable to run while the second box specifies the command line arguments to use The button to the right of the first box lets you browse to find the executable file you
19. wells selected is shown in the plate indicator text after the plate order number When you have selected the wells and released the left mouse button a drop down menu of sample types will appear These will be the types defined in the Wallac 1420 Manager Tools menu item Miscellaneous settings By selecting one of these types you specify what the type of the selected area is to be 40 Protocol editing ID f Fluorescein 485nm 535nm Protocol editor Nove E This tab allows you to select the protocol number and name The number can be selected from a drop down list of available numbers The protocol name has to be typed in In the case of a factory pre set protocol you cannot change the number Notes or additional comments can be connected with the protocol These appear at the bottom of the Explorer window 41 Protocol editing Measurement The measurement tab specifies the measurement sequence to be used in the protocol f New User 1 protocol Protocol editor ale 2 al 2 s Note If the protocol being edited is pre set the measurement sequence cannot be changed Measurement mode This field allows you to specify on what unit the complete measurement sequence should be performed before moving on to the next similar unit The possible unit is a single well a strip or a plate Plate and strips modes are disabled when using a fast kinetic or scan measurement i
20. 12 3x4 wells Costar 3524 4x6 wells Falcon 3047 4x6 wells NUNC 384 NUNC 136528 Terasaki 72 EA Wallac 1450 514 Isoplate NUNC 24005 petridish 60x15 mm Fr 4 gt Add Copy Remove Properties In the dialogue that appears when you click this item in the Tools menu there are three tabs for Plate types the available plate types are listed including a number of default types Well names the available names are listed There are up to five default types two Empty and to be Measured that are generally valid and three LanceBlank LanceCrosstalk and LanceHigh for on plate LANCE normalization measurements these require that LANCE is installed Note well names cannot be copied Instruments when you select this tab you can see names of instruments Selecting a name and properties gives more information The following buttons appear in Miscellaneous 115 Tools menu Add add a new item to the list available e g a new plate type Give the name and edit the properties It will appear with a new icon showing it is different from a default item Copy select an item to activate this Make a copy of an item It will appear with a new icon showing it is different from a default item and the name will be preceded by the words Copy of You can give it a different name and edit other parameters by selecting Properties Remove select a user created item to activate this Remove the selected item from the list
21. 1420 922 05 August 2000 User manual mua 1420 Software version 2 00 release 9 Wallac 1420 Manager ars a 2 PerkinElmer life sciences Wallac Oy P O Box 10 FIN 20101 Turku Finland Tel 358 2 2678111 Fax 358 2 2678357 Website http www wallac com Contents Contents Introduction esssesssossssnsnssnssnsnnsnssnsnnsnssnsnnsnnsntnnsnssnsnnsnsnsnnnnnsnnsnssnsnnsnssnsnnsnssnsnnsnsensnnsnnsnsnnenn 7 SYSTEM Witt dis circa 9 Software installation docto enebro escote reinados cones 10 Operation oveivieW nnss est ine insb EEE STRES ER S 11 Operating Wallac 1420 crssonssossonssnssnnesnsesnnsnonssonsnnnsnnssnnssnnssnnsnnnennnssnnssnssonssonssonsnnssnnsenne 13 Startins p Wall c 1420 2 2 22 2 2 5222 asa 15 Instrument control 2 u 2 12 2 54285888 Rei hans Innen Back 16 Lave display ss an hiess E eee 17 Tempera E vec e ae ra eset pits Rs BRHHienHBpnprrimmnenee 19 Barcode OPUS unse het iniciada 20 Plate Orienta cirio EE 20 Starting operation with the Start button u uuesesseessesnnesnnesnnesnnennnesnnennnenennnennnn nenn 21 Starting operation with the Start Wizard useeserseesseesnesnnesnnesnnesnnesnnennennnesnnennnn nennen 24 Starting operation and using a protocol number barcode uusuussseennesnneennesne nenn 26 StACKEr AAA ans spe E e eeoa esnobe oseas ia eiis 27 Starting operation no protocol number barcode oooconocnnoccocnconononnnnncn
22. 25 to 48 appear on the next page Only rows of one plate will be printed on one page If you press OK in the print dialogue the selected parts of the run information will be printed on the printer that has been selected in the print dialogue Export results If you select Export in Result Viewer it allows you to export the results of the active plate A window appears in which you can give the path the file name type it in the file type select it from the list a Save button and a Cancel button The file types you can save files in are Excel 5 0 or later Tab delimited text or MultiCalc To help you select where you want the file to be saved you can browse to find the folder you want Buttons allow you to go up one level create a new folder and display the folder information as a list just showing folder names or with additional details The white area in the centre of the window is where the folders appear when you start to browse to find the path 62 Tools menu 63 64 Tools menu Tools menu Note These operations can affect the whole system and should only be undertaken by someone qualified to do them There are up to twelve items in this menu depending on the options installed Explorer for protocol editing and result viewing see pages 35 and 57 Start Wizard for starting when you first need to define plates and wells see page 24 Results of latest assay run the result viewer is started and the latest re
23. 6 Operating Wallac 1420 Stacker operation A stacker is able to hold up to 20 or 40 in the larger version microplates No lids loose frames or very loose strips are acceptable See Appendix 2 for a list of plates that fit in the stackers Starting operation no protocol number barcode Caution Do not put your fingers into the sample loading area In the stacker A model the loading area is uncovered when the stackers are not in place so you must avoid the danger of getting your fingers trapped by the plate lift mechanism If you need to do something in that area switch power off first 1 In stacker operation plates are loaded into the input stacker with the first plate to be measured at the bottom See the picture on page 20 for correct plate orientation 2 The empty output stacker the one with the tabs for the quick release mechanism should be fitted first into the multilabel counter it occupies the left hand position 3 Fit the input stacker with plates in the right hand position 1 Load the output 2 Load stacker the input stacker Release tab for the stacker Note Make sure the stackers have clicked into position properly 27 Operating Wallac 1420 Stackers are loaded ready for operation Quick release mechanism for plates 4 Select the protocol and click the Start button or use the Start Wizard 5 The plates will be moved one by one from the input stacker to the measurement positio
24. 7 Appendix 3 Dispenser operation uuesseessesnnesnnesnnesnnesnnennonnnensnensnensnnnnennnnnnennnnnnennnenn 139 A RN 139 Emptying the waste al rs rd 140 Preparing the tubing iia ad ee 142 Dispenser maintenance uunucesseessessnesnnesnnesnnesnnennensnensnensnennonnnnnnnensennnonnnennnnnnnennnennnenn 143 MENO NOS 146 ME O O 147 Needle maintenance om esse es 147 leita E E S E E 149 Trademarks DELFIA is a registered trademark and LANCE is a trademark of Wallac Oy Windows and Excel are registered trademarks of Microsoft Corp in the U S and other countries Introduction Introduction System overview Note This manual describes all the features of VICTOR2 Some versions of the instrument do not have one or more of these features You can ignore the parts of this manual not relevant to the version of VICTOR2 you have Wallac 1420 multilabel counter is a complete platform for quantitative detection of light emitting or light absorbing markers Depending on the model of the instrument you have it is suitable for flash or glow luminometry fluorometry fluorescence polarization high sensitivity time resolved fluorometry homogeneous time resolved fluorometry LANCE option flash absorbance and photometry It is a compact bench top unit with features such as dispensers shaking temperature control top or bottom reading and scanning The software is a 32 bit application running under Windows 95 Windows 98 or Windows NT U
25. E Long Decay 665 but 572nm emission LANCE FQA 615 same as LANCE 615 but including 2 window 200 100 This is a label for Eu TRF Quenching assays LANCE FQA 545 same as LANCE 615 but with 545 emission filter and 2 window 200 100 This is a label for Tb TRF Quenching assays 87 Tools menu Filters In this window there are three tabs Emission Filters CW Lamp Filters 1 e continuous wave lamp excitation filters and Filter Slides including filter wheels Filter Slides allows you to specify the slides for emission and CW lamp filters The names of the filters or slides in the currently selected tab are displayed Names of user defined filters can be freely defined by the user but names of factory preset filters cannot be changed There are four buttons at the bottom of the window Add add a new item to the list available e g a new filter not available for slides Give the name and edit the properties It will appear with a new icon showing it is different from a default item Copy select an item to activate this not available for slides Make a copy of an item It will appear with a new icon showing it is different from a default item and the name will be preceded by the words Copy of You can give it a different name and edit other parameters by selecting Properties Remove select a user created item to activate this not available for slides Remove the selected item from the list You cannot re
26. Installation of Wallac 1420 in MultiCalc 136 Instrument Control 16 Instrument name 118 Instrument options 66 121 Instrument properties 118 Interrupt request 122 IRQ 122 K Keywords 50 51 Kinetics button Measurement tab 45 L Label button Measurement tab 44 Labels 65 67 Labels selecting 42 Labels window buttons 67 LANCE 83 LANCE normalization 102 108 LANCE normalization wizard 65 Latest assay run 57 65 Latest assay run icon 17 Legend 18 Level user 66 Light integration capacitor LANCE 84 Light integration capacitor TRF 70 Light Integration Ref Level LANCE 85 Light Integration Reference Level TRF 71 Light on the button 31 Linear scale 18 List Result Viewer 59 Live display 17 22 Live Display 16 Logarithmic scale 18 Luminescence counting time 79 Luminometry 78 M Measurement 42 Measurement Protocol editor 42 Measurement height Plate parameters 46 Measurement mode Plate 42 Single well 42 45 Strip 42 45 Measurement mode Measurement tab 42 Measurement operations list Meas tab 43 Measurement status page Eu Sm norm 100 Meas status page LANCE norm 105 Measurement tab 38 42 Meas warning page Eu Sm norm 100 Meas warning page LANCE norm 106 Memory out of 129 Miscellaneous 65 Miscellaneous settings 115 Miscellaneous settings window buttons 116 MultiCalc 135 136 MultiCalc assay protocols 135 136 N
27. Measurement technologies esccecssecescecsseceeeeecssceeseeecacceneecaeceeeeecaeceeneecaeeeeneees 122 Comm nicatlon 223 3 Rl As ee ee A ee LS 122 XIE cs ssdesecasnasescoanesaceossscsocaseasesss deaseunesuassassancssvedevassanscasseasessseve sass scaseasbevossonesweacesesedosasons 123 Fille Menor ie ti sit ita 125 Troublesho0tiE 2 5 cssicc sseassesssssscecssvccsossasedvssoseesvassadecsesivossssescodedusasessasasevessosesossbooseasoaaoaes 127 O t of MEMOLY 225 5 dese sok eles een en ooh leven pension 129 Display problems mico ete as Ga eee ee ee ann 130 Performance degradation seat an 130 Processing Croacia 131 System requirements not Meteo cece eee eeeeeeeeeeeseeeeeeesecesecsecaecsaecsaecsaecseesaeseseeeeeeas 131 Appendices acsenesiseneuiesneninnnnssahissstaneniiunsiesibins ii nischilinsschssn 133 Appendix 1 Connecting MultiCalc to Wallac 1420 uuessessnesnnesnersnensnensnennnennennnnnn 135 A O O AO 135 Wallac 1420 MultiCalc assay protocolsS ooooconocnnonocononononanconananonanonnn conc cononancnnncnncconoo 135 Installation of Wallac 1420 output option in MultiCale ooooonccnocinonccononanonninnnnananinono 136 MultiCale assay protocols iniciis resiti ee ss tape 136 Usage Of Multi Cale cami da 136 Appendix 2 Plate types for stackers oooonocononiconoconocononononnnconcnanonnnron oran oran ono nono canon necnnoos 137 Wallace do ae A ee Ba a ige 137 NUNC cepo 137 A NN 137 GIEINER ui aia asada 13
28. N m F485 Fluorescein Slot 45 F535 Fluorescein Slot 45 Protocol name This is the name by which the protocol is identified A password can be associated with it to prevent parameters being changed by an unauthorized person 73 Tools menu CW Lamp energy Using this parameter you are able to change the excitation energy supplied by the CW Lamp CW Lamp Control Mode There are two ways of ensuring that the light incident on the plate from the CW Lamp remains constant stabilized energy mode or constant voltage mode In the stabilized energy mode a photodiode close to the light path has part of the incident light directed onto it This sends feedback to the lamp controller which alters the lamp voltage to adjust the intensity of the light produced so that it remains constant In the constant voltage mode the lamp voltage is not changed but the software corrects for changes in the lamp output You can select the control mode you want from the option buttons The default mode is stabilized energy The use of the constant voltage mode allows the CW Lamp lamp to be run continuously at maximum power and it is faster than the stabilized energy mode This enables optimum performance in cases where the light intensity is critical as in fluorescence polarization or where speed is critical as in fast kinetics CW Lamp filters You can select the CW lamp excitation filter from the drop down list of available filters See
29. NKOW ce esseceeeceeseceeeeeceeceeneecsaeeeeneers 72 Plash absorbance sas 72 A SN 72 Second measurement parameters ee eee eeceeeceseceecesecssecseeaecsaecsaecaeeeseseaeeeeeee 72 Fluorometty 2n r 282 an SEHE sp Rlpee 73 Protocol Name a BEE ee E 73 CW Lamp energy ise si ccsscescetestessceases snes seta thts is cossdsovessstee sot sasvesdbteiesvensssesdeopetaesdeness 74 CW Lamp Control Mode citan ice 74 CW Lamp filters is casi ia 74 Excitation aperture Wallac 1420 040 041 only eee eee cee cseeceeeeeeeeeeeeeeeeeeenaes 74 Emission Filters oiiire 8000 Bassisten a ee 74 Eimissioh aperture n ea is 75 Counter POSITION isis 75 Counting Meri reist A Reis es HS Bee 75 Second measurement parameters ee eeceecesecesecesecseeceecaeesaecseecseesaeeeeeeeeeeeenes 75 Phot melr yet eiehusenstienfnibeinerbesispstlessent 76 Protocol NAME 2 css hive deeds sea ad 76 EW Eamp filters coi 76 Excitation aperture Wallac 1420 040 041 only oooccnoccnoccconacononanononoconocanocnocnnonanono 77 Reading tme tt iin eisen hie ii duvterwberees 77 Second measurement uueusesssesnnesnnesnensnensnensnnnnnnnnnnnnnnnnsnnnsnnesnnesnnesnnnsnensonseensennnnnn 77 Luminometty A 78 Protocol name ciclista BE ERSa Rasen 78 Emission luis picas 78 Emission pert res esen sense en einen 78 Counting TIME Ni doi 79 Contents Second Measurement ier eeno teas ecenin keo ses iiiter ss 79 Fluorescence polarization Wallac 1420 040 041 only
30. Name of Instrument 118 Name of plate 117 Needle maintenance 147 Nickname of instrument 118 Normalization 98 102 Norm confirmation page Eu Sm norm 101 Norm confirmation page LANCE 107 Normalization wizard dual label 98 153 Index Notes Result Viewer 62 Number of well columns Plate type 118 Number of well rows Plate type 118 O Options 65 119 Options Protocol Explorer Tools 53 Options installed 121 Orientation of strips 118 Out of memory 129 Output 9 Outputs Protocol editor 48 Outputs tab 38 48 51 File output 49 Printer output 48 Overview 9 P Padding keywords 51 Password 69 73 76 78 80 83 Perform on first plate repeat only 44 Performance degradation 130 Photometry 76 Plate Add 39 Delete 39 Duplicate 39 Plate dimension wizard 65 Plate dimensions fine tuning 110 Plate dimensions Plate type 118 Plate format 48 Plate heating 16 19 Plate heating Plate parameters 47 Plate height 118 Plate map options 120 Plate map results picture 60 Plate name 117 Plate name Plate type 117 Plate orientation picture 138 Plate repeat parameters Plate parameters 46 Plate tab 38 46 Index Plate type 38 47 Plate type Miscellaneous settings 117 Plate type selection Eu Sm norm 98 Plate types for stackers 137 Polarizer aperture 81 Preparing the plate Eu Sm norm 99 Preparing the plate LANCE norm 104 Preview print 53 Print Prot
31. Sm 50 100 1000 Tb 500 1400 2000 Dy 30 30 1000 Factory set values for the second counting window are always 0 0 The counts in this window are displayed only if the values deviate from zero Flash absorbance If you want there to be an absorption measurement as well as the usual time resolved fluorescence emission measurement select this parameter to be Yes Both measurements will then be made simultaneously Beam size This parameter can be selected to be Normal or Narrow provided the Adjustable beam size option has been installed It enables measurements on plates with very small well sizes by focusing the excitation light beam to a smaller size Second measurement parameters In the case where you want to make measurements with two filters you can use this parameter to select the type of the second emission filter The options are the same as for the first measurement emission filter Two measurements are made first with the normal filter and then with the second which the system automatically moves into place 72 Tools menu Fluorometry Fluorometry light of a particular wavelength is selected with a filter from the spectrum of a continuous wave lamp and used to excite the fluorochrome in the sample The parameters that can be set for fluorometry are the following Fluorometry Label Properties Fluorescence Sos al F485 Fluorescein StA5 E 2 ol s F535 Fluorescein Slot A5 ha 9 ol
32. Width Half Maximum ca 50 nm ca 80 nm ca 8 5 nm ca 7 5 nm ca 6 5 nm ca 7 5 nm ca 7 5 nm ca 10 nm ca 6 nm ca 6 nm ca 15 nm ca 25 nm ca 40 nm ca 25 nm Maximum transmittance at peak wavelength Tpk ca 60 ca 70 ca 80 ca 80 ca 80 ca 70 ca 70 ca 45 ca 30 ca 30 ca 60 ca 50 ca 35 ca 50 Note the time resolved filters are available only if this option is included with the instrument 97 Tools menu EuSm Dual label normalization Note TR FIA must be installed for this to be enabled Before dual label europium samarium time resolved fluorescence measurements can be made the instrument must be normalized for the plate type s to be used To begin this procedure select EuSm Dual label normalization from the Wallac 1420 Manager Tools menu This will activate a wizard that will guide you through the normalization procedure First comes a welcome to the wizard page Wallac 1420 EuSm Dual label normalization Wizard Welcome to Wallac 1420 EuSm Dual label normalization Wizard Normalization Wizard is a tool you can use to quickly normalize a plate to be used in EuSm Dual label measurements You can select that this does not appear in the future After this the main steps in the normalization are Plate type selection You must select from the list the plate type you are going to use This is necessary because normalization is plate specific i e you must have the same siz
33. You cannot remove a preset item from the list nor an item that is used in a protocol This command requires confirmation because you cannot undo Remove Properties select an item to activate this View the properties of the selected item If the item is user defined then you can edit the properties but if it is a factory pre set you cannot edit them 116 Tools menu Plate type When you click the Plate types tab the available plate types are listed This list includes a number of default types To define a new plate click Add You can also click on an existing plate type and then click Copy to save the plate type with a new name If you wish you can edit the parameters to make a new plate type Click Properties to see the plate properties Plate Properties Name user plate 1536 Parameters Number of well rows 2 1 32 Number of well columns 48 1 48 Height of the plate mm fos Strip orientation Horizontal Vertical Dimensions from the comer of the plate to the center of the first well and from the center of a well to the center of the next well mm In the case of Properties the dialogue that appears gives information about the plate Each of the parameters in this dialogue must be defined when you add a plate The software does not allow you to give unreasonable values e g a well separation that would mean that all wells do not fit in the plate The maxi
34. actor is used in the calculation of the corrected mP for samples as CorrectedS G x CorrectedP CorrectedS G x CorrectedP mP 1000x where CorrectedS S sample S buffer and CorrectedP P sample P buffer 82 Tools menu LANCE Note this requires the LANCE option to be installed In LANCE homogenous time resolved fluorescence a flash lamp is used to illuminate the sample In the most common form of LANCE this excites the donor molecule which after a delay transfers the energy to the accepter molecule which then emits light Two labels must be defined one for the donor and the other for the accepter The picture shows the parameters that can be set for a LANCE label Lance Label Properties D615 Europium LANCE_615nm Slot 7 Protocol name This is the name by which the protocol is identified A password can be associated with it to prevent parameters being changed by an unauthorized person 83 Tools menu Flash Energy Area There are two options for this parameter Low and High The energy of the flash lamp used is dependent on the discharge capacitor value The Low Flash Energy Area selects a basic capacitor of 10 nF The High Flash Energy Area selects a second 10 nF capacitor in the lamp s discharge circuit thus doubling the energy If you select High you will reduce the counting time number of flashes but the counts will remain about the same Fla
35. are or rounded You also need to select the label by clicking the Label button and then selecting from the label dialogue The scanning button is enabled only when using well mode and there are no other labels in the measurement sequence However copying and pasting of the same scanning label is possible Injector position during measurement At the bottom of the Measurement tab are two check boxes for controlling the injector head These will only be enabled if you have selected the by wells mode You can select if you want the measuring head to be kept above the well during measurement This means that measurement can be started immediately after injection without waiting for the injector head to move aside If you check this box then a second check box is enabled where you can select if you want injecting and measurement to happen simultaneously This is for use in very fast kinetic measurements 45 Protocol editing Plate f Fluorescein 485nm2535nm Protocol editor alel SU Generic 8x12 size plate Plate repeat parameters The number of times each plate is to be measured 1 99 If this is more than 1 then you must give the time between the end of one measurement and the beginning of the next 0 3600 secs This allows you to do slow kinetic measurements Measurement height Standard This selection is only enabled when using a plate type that has its dimension data stored in the instrument Th
36. ash excitation system available for newer instruments is especially designed to cope with this Excitation filter There are two non changeable excitation filters in the flash excitation path Both are colour glass filters The main filter is D 340 which has a maximum transmittance at 340 and half bandwidth of about 35 nm This filter is used for factory set protocols The second filter D320 has a maximum transmittance at 320 but its bandwidth is much broader it includes the same excitation band as D340 but its lower edge goes down to 280nm allowing excitation of some special Tb chelates Light integration capacitor A reference circuit monitors the total excitation energy When the selected total amount of energy has been reached the flashes stop The reference circuit makes use of three light integration capacitors numbered 1 2 and 3 any one of which can be selected The capacitor selected determines the total excitation energy in the same proportions as the capacitor numbers within component tolerances This means that selecting capacitor 2 instead of capacitor 1 approximately doubles the excitation energy for one measurement i e the number of flashes for one measurement doubles hence the time used for one measurement doubles and the number of counts doubles Selecting capacitor 3 instead of capacitor 1 70 Tools menu triples the excitation energy the measurement time and the number of counts The number of flashes can be seen f
37. atabase The values obtained are the blank measurement in the acceptor window crosstalk i e the blank corrected contribution of the donor to the acceptor window signal as a proportion of the donor signal the maximum acceptor signal in the acceptor window and the maximum donor signal in the donor window These values are then used in a measuring sample to obtain the actual acceptor value i e the blank and crosstalk corrected acceptor signal normalized to the maximum donor signal thus eliminating quenching effects See the Calculation methods in the instrument manual for more details Note to avoid accidental clicking of the Finish button it only becomes visible after five seconds have elapsed since the page was opened During the time the Finish button is disabled the text of the button is Wait t where t is the remaining waiting time and it is updated every second When you click Finish the new values replace the old values in the database 107 Tools menu Using LANCE normalization samples in the assay Instead of running the LANCE normalization wizard and saving values that can be used for any subsequent LANCE assay with that particular protocol you can include LANCE normalization samples on the plate as part of the assay samples and thus obtain corrected values for that particular plate When you define the protocol make sure that both LANCE labels are defined in the Measurement tab as shown below If only one label is select
38. ate Pernia dida 47 Temperature level Checking ooocnccnnnnccncccnonccconcconnncnnnccnnnnnnnnccnnnnnonncconnnnnnn corn nnrnnnccnnes 47 OUtDULS pi nia 48 Events A 50 KEY Word usina 51 General O ON 52 Pl cad 53 Ai ai t n ii 53 PMC tddi 53 E O 53 Print SEtUP 3 22 HE a tae eee dee cee ke a Mad tes Meee Ae EAR 53 Stalt soni ON 53 EX MR o o 53 Tools MOM tinta di 53 AAA AAA TN 55 Latest results 2 IA AA TER ese TS 37 Results im the Explorer estir 57 E to 59 Plate MP 60 Protocol cristina scr 61 Noti A 62 ETT A ias 62 Pr RE 62 Export results comicios retrete d 62 Tools menu ssessesesnenneneonennenesnennenesnennenesnennenennennenennennenennennesennennenesnennesesnennesesnennssesnenassennen 63 TOS AMEN rt it 65 MO A A AT 66 Contents Labelsciurisi ina Rah DE lassen 67 Time resolved fluorometry 22u222022nensnensensennnennnnnnnennnesnnesnnesnnesnnnsnnnnnonsnensnnnsnnnnnnnn 69 Protocol name 2 sitiado ERsai Rain Rsene 69 Flash Enere y Arcana sanieren 70 Fl sh Energy Level cui ae e E EEO EEEE EEE E E ESE 70 Excitation filter 2 nn A A ed 70 Light integration capacitor isesi esse ee 70 N te E E T 71 Light Integration Reference Level ooooonnnccnnnccioncccnoccnonononnccononononocono nooo nocono nooo nccnnnnoos 71 Emission Mrs 71 Emission APETITO c ss cis esses ssce shi tics soaccavusteo E RREA E E OSO REER AEAEE T 71 COUNTING PaLaMete Spurs 72 Factory set values for the main counting WI
39. ccnnos 102 Protocol Selection ias 103 Preparing the plate erei ries e e EE En E Dilo least es 104 Me s rement status pagesia nrn a A A ustemaiventeeees 105 Measurement warning page uursnersnenseensennnnnnnnsnnesnnennnesnnesnnesnensnonsensennnnnnnnnnennnernnenn 106 Normalization confirmation page uesseessessnesnnesnnesnnesnensnensnensnnnnonnnennnnnnnennnennnennnn 107 Using LANCE normalization samples in the asSay ooconncnnnnnonnconnnonnnnnnnnonancnnncnnncnnos 108 Plate dimension Wizard nenne Aae rE aE REER E ES 110 Plate selectionner pienenee e bus ca dies E EEEE diia E 111 Technology selection E E EE EE ES EEE 111 Preparing the plate s ei ria e E tee ae 112 Define the centre point of the corner Wells oooooncnnncninnnccnnonnonnconoconoconcconocnoroconnnonnos 113 Miscellaneous settings 2nen sts pp 115 Plate pe iii eds 117 Plate name ioconniadoninaoio ado nie 117 Number of welliow8 u a ass 118 Number of well columns 220022402040202 ner cece cseecaeeeeeeeaeeseeeseeeseensecsaeenaessaeeaes 118 Height of the platero Tree Ess 118 A yes os obeusededsbeben BREI 118 Plate dimensions 2 2 stream AA 118 RUM ia 118 Well MES it 118 Instrument propert eS sirere aoeeoe ees ches HR Ea TEESEEISE k DEEST E Paa TES ETES ETSE 118 OPUS eri 119 Generales sce iii sit aes 119 Instrument hard Wale ooooccnonnconoconcnononnnonnnonnnonnnnnnnnnonnno nn crono neon IIE Er e r oaa ri srr sE 121 Contents
40. d Measurement In the case where you want to make measurements with two filters you can click the Second measurement check box and then set this emission filter It has the same options as the normal emission filter When you select this two measurements are made the first with the normal settings and the second with this new setting The change is automatic 79 Tools menu Fluorescence polarization Wallac 1420 040 041 042 043 only Fluorescence polarization light of a particular wavelength is selected with a filter from the spectrum of a continuous wave lamp This light is then polarized and used to excite the fluorochrome in the sample The emission light is then viewed through two polarizers one parallel to the incident polarization S plane and one perpendicular to it P plane These are each in turn moved automatically into the emission light path The ratio 1000 Parallel G Perpendicular Parallel G Perpendicular is calculated to get the polarization in units of mP G is a correction factor see the end of this section The parameters that can be set for fluorescence polarization are the following Fluorescence polarization Label Properties r Name FP Fluorescein Password Last Edited GT 15 03 00 15 21 42 r Parameters Cw Lamp Energy 65535 AA IA AUS Cw Lamp Filter Fass Fluorescein Slot 45 y Polarizer Aperture C Small Normal Emission Filter F535 Fluorescein Slot 45 y Emissi
41. d using the last defined plate map Additional plates possible all wells will be measured further plates will be measured with no plate map taken into consideration i e every well will be measured There is also a check box Upon assay completion a new assay measurement is automatically started using the same protocol This check box is only enabled if the Barcode stored to results option has been selected If you are using stackers then measurement of the next plate with the previously used protocol will begin automatically This feature is useful for automatically producing a separate export file for each plate Define your protocol to have only one plate and select this feature Each plate will be run as a separate assay producing its own export file The external software can then process these files as from the same assay because each will have been produced by the same protocol If you are not using stackers the instrument waits until the button on the instrument is pressed and then begins measuring the current plate with the previously used protocol 120 Tools menu Instrument hardware Pees Pleo ise ile cil Romp cl UTN GUSTA leal This tab can only be accessed if Advanced level has been selected in User Level It shows which options are installed These include Stacker option Barcode reader options Temperature control Adjustable beam size Counting from below High power flash driver Disp
42. e Latest assay run icon Instrument Tools CY a 1 rument contral Live The Result Viewer is started and the latest results shown after the measurement has been completed This command performs following actions 1 If there are no assay runs in the database yet a warning is given and the Result Viewer is not invoked 2 If this button is clicked while an assay run is already in progress that assay is displayed in the Viewer 3 If this button is clicked and there is no current assay running the latest assay run is displayed on the Viewer Results in the Explorer Alternatively once results have been received and stored in the computer you can view them by means of the Wallac 1420 Explorer See the picture on the next page The protocol connected to the latest results will be selected automatically but you can select any protocol that was used to make the measurements that you want to view The results files associated with the protocol will then appear on the right side of the window Each entry for a results file comprises the following items Assay ID the date and time when the assay began and when it ended Notes and Errors Note to change a column width in the Results Viewer move the mouse cursor to the line between two columns The cursor form will change to a vertical bar with two arrows Hold down the left mouse button and move the column division in the direction you choose The information in a column is complete
43. e a name for a well for identification purposes e g standard unknown etc You can define a short form single letter for the name and select a colour for it from a scroll bar of colours The short form is checked to see that it is not used for any other well name The default names are Empty and Measured Instrument properties In the Instrument tab of Miscellaneous settings when you select properties of an instrument the nickname you selected appears and can be changed Also the official name and product number of the instrument as well as its serial number will be displayed You cannot change these 118 Tools menu Options This can only be accessed if you have Advanced level selected There can be four tabs but only three show on Advanced level the fourth requires Service level General Assay ends after last plate in the plate map ha E Upan Han easuremnentis automat You can define the number of results to be held in the database for one protocol When this number is exceeded the oldest results for that protocol will be automatically deleted from the database to make room for new ones The maximum number of results is 100 If you want the start to be always by means of the Start Wizard check this box This means that when you click Start the Start Wizard will be activated This is useful if e g you need to define the number of plates before each measurement If you just want to run the softwar
44. e and type of plate as you plan to use for the actual dual label measurement The list shows Plate type name Date when the normalization for this plate type was last done Name of the user who ran the last normalization Correction and background values from the last normalization run 98 Tools menu Wallac 1420 EuSm Dual label normalization Wizard Generic 812 size plate 0 49 170 25 Labsystems 8x12 DELFIA strip plate NUNC 8x12 DELFIA strip plate Costar 3512 3x4 wells Costar 3524 4x6 wells Falcon 3047 4x6 wells NUNC 136528 Terasaki 72 Wallac 1450 514 Isoplate NUNC 163118 Terasaki 60 normal The wizard explains what samples you should put in the plate and where you should put them Plates must have 3 or more strips maximum of 8 with 1 12 wells per strip The strips must be horizontal e g Al to A12 etc 99 Tools menu Strip A should be full of blank samples Strip B should be empty and Strip C should be full of europium samples Measurement status page Wallac 1420 EuSm Dual label normalization Wizard The measurement has now been started Measurement status Assay is running Measurement progress Measurement in progress please wait lt Back Hers Cancel Help This is shown during the actual normalization measurement and it gives you feedback about the measurement The measurement status pane shows the current status of the measurement The progress of
45. e as a demo without having the counter connected check the Run Workstation in demo mode check box 119 Tools menu Note if you change to Demo mode your PC has to be rebooted before the change takes effect Make sure you have no other programs running when you make this selection because they will be closed during the shutdown procedure If do not have the barcode reader option then the line Barcode reader switched off will be selected If you have the reader and want to use the barcode as a plate number to be connected with your results select Barcode is stored to results In both these cases you must select the protocol manually see Start or Start Wizard for more information If you have the reader and you select Barcode is used as a protocol number then the barcode will determine the protocol used to measure the plate If a plate does not have a barcode then the protocol used to measure the previous plate will be used See the Plate orientation picture on page 20 for where to fix the barcode You can specify what happens when the end of the plate map definitions is reached for a protocol There are three possibilities Assay ends after last plate in the plate map no more plates will be measured until a new start instruction is given or a new barcode read If there are more plates an error message will appear Additional plates possible latest plate map is used further plates will be measure
46. e default height set is about 8 mm above the bottom of the plate 46 Protocol editing User defined If you do not want to use the standard measurement you can give a new value here You must click the user defined button The minimum value is 3 0 mm 8 0 mm if the dispenser is used and the maximum 18 0 mm depending on the plate type and if the dispenser is used or not The height is measured from the bottom of the plate This is useful if you are measuring samples on filter paper and you need to optimise for the sample being at the top or at the bottom of the wells Plate type Select the one you want from the list of available plate types under the scroll bar If the plate size and or format is different from the current one all the plates in the plate map will be deleted and one plate of the selected type will appear Make sure you select the plate type before defining the plate map to avoid losing your settings Plate types are read from the Wallac 1420 database when the Protocol Editor is started so it will not recognize plate types added while the Protocol Editor is open Note the generic 8 x 12 plate selection does not check the height of the plate before a measurement Note If the type you selected has a different number of wells than the current one all the previous plates will be deleted and one plate of the selected type will appear Temperature level checking This setting allows you to define what the target tempera
47. e plate whose dimensions you want to fine tune Remember that this should be the plate type with the name you chose specifically for the technology to be used Note that you must click in the Plate name field to select the plate When you have done this Next will be enabled Click it to go to the next dialogue Technology selection You must select the technology to be used to count the plate Note the options that appear will depend on what is installed in the instrument Open the folder and select the actual protocol for which the plate dimensions are to be fine tuned The Next button will then be enabled and you can click it to go to the next dialogue of the wizard 111 Tools menu Wallac 1420 Plate Dimension Wizard Fluorometry EB Fluorescein 1 0s ER Umbellferone 1 0s Fluorescein 0 1s Umbellif 0 15 Preparing the plate Wallac 1420 Plate Dimension Wizard Prepare the plate by dispensing appropriately labelled sample to each of the four corner wells of the plate All the other wells should be empty Specify the area of the well to be scanned The example shows the default value for a 1536 well plate 112 Tools menu When the plate has been prepared load it and click Next Wallac 1420 Plate Dimension Wizard After scanning is completed the Next button will be activated Click it to continue Define the centre point of the corner wells Wallac 1420 Plate Dimension Wizard
48. ed properly When this is done the needles are rinsed at the same time However it is good to regularly clean the outside of the needles 147 Appendix 3 Dispenser operation Avoid touching the needles with your fingers To clean the needles use e g a stick with a tip of soft material dipped in ethanol or use a spray bottle filled with ethanol Be very gentle when touching the needles so that they are not bent out of shape Rinse with water The position of the needles is very critical for high quality performance If a needle is bent call the service engineer to get the needle replaced Do not try to re adjust the damaged needle because the quality of dispensing could be affected Both the needle and the tubing will be replaced as a set It is good to check the flow of liquid dispensed during flushing If the flow is not normal it might indicate that the needle is partly blocked If this has happened due to dried reagents use water and e g ethanol to dissolve the plug If that does not help the needle needs to be changed contact the service 148 Index 149 150 A Active protocols 22 Advanced level 66 119 Aperture slide 71 75 78 81 85 Barcode 20 25 26 27 29 31 51 120 Barcode options 20 120 Barcode reader option 20 Beam size LANCE 86 Beam size Time resolved fluorometry 72 Button light 31 Button on Wallac 1420 31 Buttons Measurement tab 43 Buttons in the filters window 88 Button
49. ed then the measurement will be treated as normal time resolved fluorometry B LANCE protocol Protocol editor BE Eile Help ale 2 Samples ID Measurement Plate Outputs Events General By wells By strips Measurement mode Operations Operation properties o LANCE_615nm e LANCE_665nm When you set up the plate you can select the LANCE well types from the menu that appears when you click the left mouse button Use this feature to include at least one of each of the three LANCE well types on the plate You can choose the number of replicates of each well type and you can choose where they are located on the plate as the following example shows 108 Tools menu LANCE protocol Protocol editor When you have defined the plate and saved it you can run the assay in the normal way When the LANCE samples are detected on the plate they will automatically be used to make the necessary corrections for this particular LANCE measurement They will overrule any values that may have be stored in the instrument memory from a normalization with the LANCE wizard However these on the plate normalization values will not be stored and used for any other assays than the current one Only values resulting from use of the LANCE wizard are stored 109 Tools menu Plate dimension wizard A description is given here of how the Plate dimension wizard can be used to fine tune the p
50. ence 44 Protocol editing When using well or strips mode the label button will be disabled when there is already a label in the measurement sequence However copying and pasting of the same label is possible For detailed information about labels see Labels on page 67 Kinetics button Clicking this causes a dialogue to appear that lets you specify the parameters for a kinetic measurement You can give the number of repeat measurements up to 100 and the delay between each measurement 0 to 600 s You need to also select the label by clicking the label button and then selecting from the label dialogue If the repeat is less than 26 and the delay between repeats is 0 results will come in one group The kinetics button is enabled only when using well mode and there are no other labels in the measurement sequence However copying and pasting of the same kinetic label is possible Scanning button Clicking this causes a dialogue to appear that lets you specify the parameters for a scan measurement This means that several measurements are made for a single well and the measurements are made from different points of the well These points are in the form of an array for which you can give the number of horizontal steps the number of vertical steps and the point displacement i e the distance between each point on the well where the measurement is made The total number of points can be a maximum of 100 The distribution of points can be squ
51. enser and pumps 121 Tools menu Measurement technologies The final section of this tab tells what measurement technologies are installed and available Fluorometry FP Fluorescence polarization Luminometry Photometry TR Fluorometry dissociation enhanced time resolved fluorometry LANCE homogeneous time resolved fluorometry Communication This tab can only be accessed if Service level has been selected in User Level The Instrument communication interface card settings Interrupt request and Input Output port base address can be selected from the scroll bar options provided You must reboot before these new selections become effective 122 Exiting 123 124 Exiting File menu The File menu in the main window contains one item Exit This allows you to exit from the workstation software You need to confirm the operation If you try to exit while an assay run is taking place you will get an error message telling that this is not possible 125 Exiting 126 Troubleshooting 127 128 Troubleshooting Out of memory If free disk space for the database drops below a specified limit a warning message appears in the top right corner of the Manager window A warning sound is also made once a minute These warnings are continued until enough free disk space is available again An assay run cannot be started from the user interface while the disk space is below the limit however the Start butto
52. epeats Delay between repeats Measurement height Protocol notes Ben Default Name of the label Fluorescein Label technology Prompt fluorometry CW lamp filter name F485 Fluorescein CVV lamp filter slot AS Emission filter name F535 Fluorescein Emission filter slot AS Plate map of plate 1 MM o z ZRZRZZZZZ zzzzzrzrzz zzzzzzzz zzZzzZzzzzz zzzZzzzzzz ZERRZZZZZ ZzZZz2 2 22 zzzzzzzrz ZERZRZZZZZ MM MM MM MM MM MM MM 4 gt list Plates A Plate A Protocol A Notes Selecting Protocol causes a list of protocol parameters to appear There is also a plate map showing which sample types were on the plate Any notes written in the protocol editor Notes box will appear here 61 Result viewing Notes This tab allows you to see any notes that are connected with the results being viewed Error Error gives you information about the general error situation and specific information about any errors that occurred for each measurement type Print Clicking this icon leads to an extended print dialogue which in addition to the normal print options gives you a choice of printing selected sheets or the entire workbook The former is the default You can also click the Preview button to get a preview of the pages to be printed In the case of high density plates the results from the first columns 1 to 18 or 1 to 24 appear on one page and the rest 19 36 or
53. eview This item allows you to see what will be printed out You can select whether you want a side by side two page view or a single page view You can also zoom in magnify the page to see more details alternatively you can zoom out reduce the magnification if the page is already magnified You can step to the next page or back to the previous one When you have finished viewing the pages that are going to be printed out you can click the Print button to get the printout or Close to return to the protocol editor Print setup You can define the printer type that will be selected You can also set the paper size and orientation Start Starts measurement with the currently selected protocol Note if the protocol has been changed you will be prompted to save the protocol before measurement is started Exit This closes the protocol editor If the protocol has been changed it will prompt you to save it first Tools menu The Tools command line menu has one item Options When you click this a dialogue with a check box appears If you do not check it then whenever you click the Start icon measurement of the selected protocol will begin directly If you do check it then whenever you click the Start icon the Start Wizard will be used to start the measurement of the selected protocol 53 Protocol editing 54 Result viewing 56 Result viewing Latest results The results from the latest assay can be viewed by clicking th
54. f some special Tb chelates Light integration capacitors A reference circuit monitors the total excitation energy When the selected total amount of energy has been reached the flashes stop The reference circuit makes use of three light integration capacitors numbered 1 2 and 3 any one of which can be selected The capacitor selected determines the total excitation energy in the same proportions as the capacitor numbers within component tolerances This means that selecting capacitor 2 instead of capacitor 1 approximately doubles the excitation energy for one measurement i e the number of flashes for one measurement doubles hence the time used for one measurement doubles and the number of counts doubles Selecting capacitor 3 instead of capacitor 1 84 Tools menu triples the excitation energy the measurement time and the number of counts The number of flashes can be seen from the result viewer by selecting View all result data Note Each flash contributes to the total excitation energy Flashes keep on occurring until the required total excitation energy is achieved However because the energy comes in discrete flashes the last flash will cause the total excitation energy to exceed the required amount by a maximum of not more than the energy in one flash This will not be significant if there are many flashes but could be significant if there are only a few flashes E g in standard measurements the amount of flashes is about 1000
55. g a Move the mouse cursor onto it and then click the left mouse button to select it then click the File menu and click Open b Move the cursor on to it click the right mouse button and select Open from the menu that appears c Double click the mouse when the cursor is on the icon At the bottom of the window on the status line the protocol ID will appear if defined otherwise it is not available N A and also any notes that have been included in the protocol When you create a protocol you can save it e g as a user protocol in the Users folder or you can save it according to the technology The choice is yours but the default structure gives you a starting point and helps you to find the pre set protocols You can move a user made protocol to a different folder by selecting it holding down the mouse button dragging it to the target folder and releasing the mouse button Note The dual label protocol only available if TR FIA is installed should not be copied and edited e g to make a single label europium or samarium protocol You should begin with an existing single label protocol For dual label operation you must select the europium label first and then the samarium label After you have selected the second label a third samarium in the europium window will be created automatically When running with these three labels you must have a valid Eu Sm normalization see the Tools menu for how to make such a normalization Fo
56. g Explorer folders In the Wallac folder there are as a default folders named according to the technologies installed In each of these folders there are protocols using the appropriate technology On the same level as the Wallac folder there are two other folders Users and Projects To open a folder click on it and then select Open from the File menu Alternatively you can open a folder by double clicking the left mouse button when the cursor is on the folder icon You can also click once on the mark next to the folder to see the protocols contained in it To close a folder either double click on it or click the sign next to it If you are working on the Advanced or Service level you can create rename or delete folders A sub folder of Users or Projects can be renamed by selecting it and clicking the right mouse button and selecting Rename Explorer icons Factory pre set protocols and groups have a lock symbol to show they cannot be changed User made protocols and groups do not include the lock because the user can change them See the examples below The icon for a factory pre set folder The icon for a factory pre set protocol The icon for a user made folder DE pA The icon for a user made protocol 36 Protocol editing Protocol selection You can Open copy paste start or create a protocol delete a user made protocol plus its associated results To open an existing protocol you can do one of the followin
57. h it to prevent parameters being changed by an unauthorized person 69 Tools menu Flash Energy Area There are two options for this parameter Low and High The energy of the flash lamp used is dependent on the discharge capacitor value The Low Flash Energy Area selects a basic capacitor of 10 nF The High Flash Energy Area selects a second 10 nF capacitor in the lamp s discharge circuit thus doubling the energy If you select High you will reduce the counting time number of flashes but the counts will remain about the same Flash Energy Level The Flash Energy Level sets the voltage of the discharge capacitor the range is from 1 to 255 corresponding to 500 to 800 volts Note that although the lamp input energy depends on the squared power of the discharge voltage the output optical power is not linearly dependent on the input energy By changing this parameter you will fine tune the Flash Energy If you increase the number you will reduce counting time but counts from your sample will remain about the same CAUTION If software version 2 0 release 8 or higher is installed on an instrument having a serial number less than 4201396 then you should avoid the combination of settings Flash Energy Area High and Flash Energy Level high values e g 255 along with 384 well or higher plates The reason is that this high power continuous usage combination may damage the older type of flash lamp The cooled fl
58. ially useful for models using stackers In this case the first plate to be loaded from the stacker must have a barcode Measurement can be started simply by briefly pressing the button on the counter See page 118 for more details on barcode usage Plate orientation The figure shows a typical 96 well plate and how it is to be orientated when loading it either directly into Wallac 1420 or into an input stacker The Al position must be in the left corner furthest from you i e the side that enters the counter first If a barcode is used it should also be on the side that enters the counter first The barcode can then be used to identify the plate or to select the protocol Optional barcode Loading direction Position Al j Plates used in a stacker must have this ledge at least on the short sides 20 Operating Wallac 1420 Starting operation with the Start button 1 Open the loading position lid 2 Load your first plate and close the lid See the picture on page 20 for correct plate orientation Load first plate 21 Operating Wallac 1420 3 Select the protocol you want to use by scrolling through the list of active protocols amp Wallac 1420 Manager 5 Sopa rel le 4 Use the mouse to click the Start button on the user interface You can follow the measurement process by selecting the Live display 22 Operating Wallac 1420 5 When the plate has been mea
59. ialogue select the ID tab Select the protocol number to be the same as the MultiCalc protocol ID number Then under the Outputs tab include all items in output and select the File type as MultiCalc The File Name should be as follows c wiacalc Ocom 1420_2 lt ProtocolNumber gt lt RunIDpad2 gt Note Max 3 labels can be selected Plate repeat must be 1 Kinetics or scanning measurements are not possible Plates having more than 144 wells are treated sequentially in MultiCalc 135 Appendix 1 Connecting MultiCalc to Wallac 1420 Installation of Wallac 1420 output option in MultiCalc Copy the files 1420_2 C02 and 1420_2 h2 from the distribution CD ROM and paste them into c wiacalc Ocom Set the properties for 1420_2 in MultiCalc to AOSPMTV by selecting on level 5 ETC SYSTEM ETC COMM PROT PROPERTY select 1420_2 type the letters AOSPMTV and press Enter Escape to the main menu and then restart MultiCalc Note Property A stands for Automatic evaluation whenever an assay file is available Property O is for the connection type Offline MultiCalc assay protocols All assays with Wallac 1420 have to be of the FLUORO type for all measurement technologies used in Wallac 1420 The response from labels 1 and 2 will appear as COUNTS and COUNTSB and from label 3 as COUNT Usage of MultiCalc Leave MultiCalc in the counter mode evaluation will occur automatically 1f property A has been set as described above Evaluat
60. ing extra information Time resolved fluorometry number of flashes Fluorescence measurement time Luminometry counting time Photometry A D converter value and the number of readings 59 Result viewing Plate map This plate map shows you the results arranged from top to bottom in the order they appeared in the list from left to right See List format for the alternative way of viewing results Assay 3 2 11 1999 12 39 59 Wallac 1420 Result Viewer BEES File View Plate Help aja BP e WP roo A 2 X 3 4 5 6 7 8 9 10 MA Plate Repeat Endtime Starttemp Endtemp BarCode 1 1 12 40 22 N A N A N A Fluorescein Counts 112 sersas 201371 er06s3 sse159 _485074 _356788 es7oo2 824611 749138 182367 880354 713396 518399 310954 24834 100488 370807 155839 174239 _988639 451235 127892 14622 18822 384101 s36346 575472 605746 611094 174571 666414 _456281 358601 66468 611607 785485 804580 524683 _308930 8677213 729409 956341 926460 543360 150914 467459 242974 863617 217505 _781859 _845217 996827 sa9eg7 615384 _398513 273550 304307 841742 33505 382107 101697 _680433 _ 65652 18701 919602 283128 280167 592023 694271 839234 729228 490089 213305 746298 473774 463381 g49664 746993 117198 603057 391382 737657 612876 576681 367725 1600391 232853 430901 804852 521934 s90090 754033 352105 177291 660734 496978 z
61. ion can also be started manually by selecting COUNTER 1420_2 in MultiCalc and pressing Enter Re evaluation is also possible there after the Recover function has been used 136 Appendix 2 Plate types for stackers Appendix 2 Plate types for stackers All stackers are tested with following plates until further notice given Note Lids or loose frames can not be used in the stacker Wallac 1244 550 DELFIA microtitration strip plates 554 160 Rigid low fluorescence plate C120 105 Anti mouse coated plates C122 105 Streptavidin coated plates 1450 510 Rigid sample plate 1450 410 Rigid sample plate 1450 514 515 Isoplate Nunc 475515 FluoroNunc Black strip plate 437591 FluoroNunc White strip plate 45161 FluoroNunc Clear non strip 477178 8 x 12 strip plate clear 242757 93 384 well white 242757 384 well clear Costar White clear bottom Black clear bottom Black White Greiner 655101 Clear non strip 655074 White non strip 655077 Black non strip 655180 Clear sterile Only without lids Falcon 3915 Clear non strip White non strip 137 Appendix 2 Plate types for stackers Labsystems 95029550 Combiplate white 9502817 Cliniplate black Acceptable range of plate dimensions for plates in a 1420 216 Stacker Length 127 2 128 2 mm Width 84 5 86 0 mm Height 14 0 25 0 mm Ledge height 1 5 6 5 mm Note the length and width include the size of the ledge 138 Appendix 3 Dispenser opera
62. ion of the preceding operation and the start of the one succeeding the delay operation in the operation list You use a slide bar to do this Clicking the OK button inserts the delay operation into the measurement operations list A single delay can be from 0 1s to 3600 s At the bottom of the dialogue is a check box Perform on first plate repeat only If you only want delay for the first of the plate repeats check this box If you want delay for every repeat then leave it unchecked Shake button Clicking this causes a dialogue to appear that lets you specify the parameters for a shake operation You can specify the duration of the operation the speed slow normal or fast the extent the shaker moves and the path that it follows straight line elliptical or figure of eight linear orbital or double orbital respectively Clicking the OK button inserts the shake operation into the measurement operations list At the bottom of the dialogue is a check box Perform on first plate repeat only If you only want shaking for the first of the plate repeats check this box If you want shaking for every repeat then leave it unchecked Label button Opens the Label Selector to let you specify a label to be inserted in the measurement operations list There can be up to 10 labels in the list Note for LANCE measurements two labels must be selected If only one is selected then the measurement will be treated as normal time resolved fluoresc
63. late dimensions This is needed in the case of user defined high density plates 384 or higher to ensure exact positioning of the plate The factory default plates cannot be changed If the same plate type is used for more than one technology it is recommended that you repeat this procedure for each technology In this case when defining plates give each plate type a different name for each technology First a welcome page appears Wallac 1420 Plate Dimension Wizard Welcome to the Wallac 1420 Plate Dimension Wizard This wizard helps you to adjust the dimensions of a plate type by scanning some predefined wells and showing the results graphically This allows you to fine tune the well positions in a plate When the plate dimensions are set correctly you get more accurate results from the instrument NOTE You can only modify the dimensions of a user defined plate type If you want to change a factory preset plate type make a copy of it and run the wizard again You can select that this page does not appear any other time by removing the check mark from the check box Then click the Next button 110 Tools menu Plate selection Note This step requires that there is at least one user defined plate Wallac 1420 Plate Dimension Wizard Select the plate type which dimensions are to be adjusted Wells Horiz Ver Hor Ver ARES 32 48 11 025 7 925 2250 2 250 lt Back Cancel Help Select th
64. m the plate crosstalk of the donor signal into the acceptor window and quenching The final result is normalized to the maximum donor intensity To begin this procedure select LANCE normalization from the Wallac 1420 Manager Tools menu This will activate a wizard that will guide you through the normalization procedure First comes a welcome to the wizard page Wallac 1420 TRF LANCE Normalization Wizard Welcome to the Wallac 1420 TRF LANCE Normalization Wizard LANCE Normalization Wizard is a tool you can use to create a persistent normalization for a L NCE protocol You can select that this does not appear in the future Click Next to proceed After this the main steps in the normalization are 102 Tools menu Protocol selection You must select from the list of LANCE protocols the one you want to do the normalization for Highlight the one you want and then click the Next button Wallac 1420 TRF LANCE Normalization Wizard LANCE 615 665 LANCE 615 665 with normalization LANCE High Count 615 665 LANCE Long Decay 615 665 LANCE 545 572 Copy of LANCE 615 665 103 Tools menu Preparing the plate The wizard explains what samples you should put in the plate and where you should put them The strip to be used must be horizontal e g Al to A12 etc The plate used is the one defined in the protocol you previously selected Blanks should be in Al and A2 these are used to correct for the plate backgrou
65. mments appear in order to confirm that it is the one you want Note the Al position must be in the left corner furthest from the user i e the side that enters the counter first If a barcode is used it should also be on the side that enters the counter first Click Finish to start measurement See the Live display during measurement When the plate has been measured remove it load the next plate and briefly press the button on the counter 25 Operating Wallac 1420 Starting operation and using a protocol number barcode 1 When you load a plate the button will be blinking See the picture on page 20 for correct plate orientation Load the first plate Light is blinking 2 Press the button briefly Operation will start The button light will be lit without blinking The barcode on the plate will be read and the protocol specified by the barcode will be selected and used for measuring the plate When the measurement has finished and the light starts blinking again remove the plate and load the next one and briefly click the button If the plate belongs to the same assay as the previous one no barcode is needed If it is barcoded for a new assay then the appropriate protocol will be used for measuring the plate 1 Remove the gt measured plate 2 Load the next plate Continue measuring plates this way Note that the button light will remain blinking as long as the protocol number barcode option is selected 2
66. move a pre set item from the list nor can you remove a filter that has been assigned a position in a slide This command requires confirmation because you cannot undo Remove Properties select an item to activate this View the properties of the selected item If the item is user defined then you can edit the properties but if it is a factory pre set you cannot edit them 88 Tools menu Emission filters Emission Filters CW Lamp Filters Fiter Slides D545 D572 D615 D642 D665 F460 F535 No filter Add apy Remove If you select properties of an emission filter or you want to define the properties of a new filter a further dialogue appears where the name of the filter and the technology for its uses can be defined There is also a section where you can add additional information that you can connect with the filter properties Factory pre set emission filter names begin with a letter indicating the technology for which they are to be used D is for time resolved fluorometry LANCE filters are marked as such F is for fluorometry L is for luminescence After the letter comes the wavelength 89 Tools menu CW Lamp filters Filter Slides Add Copy Remove Bere If you select properties of a CW lamp filter or you want to define the properties of a new excitation filter a further dialogue appears where the name of the filter and the technology for its use can be defined
67. mum number of rows and columns are shown Plate name This is the name that will appear on the list of plate types You can change this name by editing the current name or by marking it and deleting it and typing a new name 117 Tools menu Number of well rows This is the number of wells in a row The product of this with the number of well columns gives you the number of wells in the plate Number of well columns This is the number of wells in a column The product of this with the number of well rows gives you the number of wells in the plate Height of the plate This defines the height of the plate from its base to its top The units are millimetres Strip orientation You select whether the strips are aligned horizontally i e in rows so that the first strip enters the instrument first or vertically i e in columns so that first well of each of the strips enters the instrument first The normal setting is Horizontal The order of the measurement is according to the strip definition Plate dimensions The picture shows what dimensions are needed to define a plate The units are millimetres and you can specify the dimension to the nearest hundredth of a millimetre Note the distances should be measured from the outermost part of the plate Tune When you click the Tune button the Plate dimension wizard is started See the description starting on page 108 for how this works Well names In the dialogue that appears you can giv
68. n After each plate has been measured it is moved to the output stacker When the assay has been completed operation will stop If there are still plates in the input stacker it means that you must start a new assay by selecting the protocol and clicking Start as described above 6 When all plates have been moved to the output stacker it can be removed To do this press the release tab at the bottom of the stacker then lift it up After you have removed the stacker pull up the handles of the quick release mechanism to allow the plates to be emptied easily from the stacker Note make sure you do not operate this mechanism when the stacker is loaded in Wallac 1420 because you may jam the conveyor If the button on Wallac 1420 is blinking it means that the assay has not finished and you must remove the empty input stacker load an empty output stacker and then an input stacker which has the remaining plates of the assay plus plates for other assays if you want 28 Operating Wallac 1420 Starting operation and using a protocol number barcode Caution Do not put your fingers into the sample loading area In the stacker A model the loading area is uncovered when the stackers are not in place so you must avoid the danger of getting your fingers trapped by the plate lift mechanism If you need to do something in that area switch power off first This configuration makes best use of the stackers operation is automatic 1 Load the
69. n aperture slide which has three different apertures and a shutter position The normal aperture is circular with a diameter of about 4 mm and it is used for factory set labels The damped aperture is also circular but only 1 mm in diameter This allows you to shift the dynamic range of the emission signal for a particular user selected label which may otherwise exceed the linear range of the instrument This damped aperture is used for the top fluorometry factory set labels The band aperture is an approximately 1 mm x 3 mm rectangle and it reserved mainly for future applications Its size and alignment are about the same as the excitation area from the flash lamp 71 Tools menu The shutter is used at all times when no measurement is occurring to prevent stray light getting to the photomultiplier tube Counting parameters These determine the time factors involved in the measurement process all times are in microseconds Counting delay the time after the excitation pulse at which counting of the emission signal begins Counting window the time period within which counting occurs Counting cycle the time between excitation pulses within a measurement The second or additional counting window is fully independent of the first one and it useful when optimizing time resolved parameters or for calculations of the decay time of the label Factory set values for the main counting window Label Delay Window Cycle Eu 400 400 1000
70. n for more than 2 s the current assay will be stopped and a new assay will be started with the current protocol This will not work if the protocol barcode option is selected If a plate is being measured and you press this button nothing happens If no assay is running and you press the button for more than 2 s then a new assay will be started with the current protocol This will not work if the protocol barcode option is selected Note make sure in all cases that you have loaded the correct plate before pressing the button because the plate in the loading position or the empty plate holder will be taken into the counter Light on the button The following table shows you what the light on the button will be like depending on what is happening with Wallac 1420 and whether or not the protocol barcode option is used Condition Barcode none or plate Barcode for protocol number number Not measuring no assay Off Blinking Not measuring waiting next Blinking Blinking plate Measuring loading or On On unloading 31 Operating Wallac 1420 32 Protocol editing 34 Explorer Through the Explorer you can access the Protocol Editor and the Result Viewer Click the Explorer icon to open the Explorer 1 Wallac 1420 Ma Protocol editing B ER nm 0 18 Fluore nm 1 0s Umbelliferone 355nm 460nm 0 15 Ba 15 03 00 14 03 44 15 03 00 14 04 07 DEMO DATA GENERATE 35 Protocol editin
71. n on the instrument still starts the run Please try one or more of the following to correct the situation Check that Windows is set to handle your virtual memory settings Do the following to check this Open the Control Panel in Settings in Windows Double click on the System icon Switch to the Performance tab Click the Virtual Memory button Check that the option Let Windows manage my virtual memory settings recommended is set See Out of memory picture Other things to check Check that there is at least 20 MB available on your C drive Try running with 256 colours See System Requirements This picture shows the windows you need to open to get to the Virtual memory dialogue 129 Troubleshooting ardware rograms Aa a 0 Find Fast Fonts Keyboard Mail E Virtual Memory Adobe Passwords PostScript Printers g a Sounds System lei Manman Jo Disable virtual memo not recommended Display problems If you get display problems when running a DOS application in graphics mode while Wallac 1420 software is running you might have a problem with your display driver Please contact your display driver manufacturer for an upgrade Performance degradation If you find your Wallac 1420 software is not running as fast as it used to please run the Wallac 1420 Database Maintenance program You should anyway do this on a regular basis This program will compact your database and will in some si
72. n the operation sequence Well mode will be disabled when using more than one label in the operation sequence However copying and pasting of the same label is possible Plate mode 42 Protocol editing can be used for slow kinetics by making repeated counting of the plate with delays in between Measurement operations This field lists the operations that have been specified to be used in the assay The operations are listed in the order they will be performed If you select an item in the list and then click the right mouse button a menu will appear to allow you to Copy Delete or see the Properties of the selected item A row of buttons beside the Operations list enables you to add items to the list Click the button referring to the operation you want Set the parameters and click OK to add the item to the list Down button This moves the selected operation in the operation list one step closer to the end This button is enabled when the operation currently selected in the measurement operations list is not the last operation in the list Up button This moves the selected operation in the operation list one step closer to the beginning This button is enabled when the operation currently selected in the measurement operations list is not the first operation in the list Delete button This deletes the operation selected in the operation list This button is enabled when there is an operation selected in the measurement operations li
73. nconoconocnnocncnanannos 27 Starting operation and using a protocol number barcode ooooconccniccnonnconoconoconocnonnnonnos 29 Ending Operation te 30 Stop DUTON as eee o iaa 30 End buttona a oi 30 Button on Wallac 1420 ea HR IE Rote steeds 31 Light on the Dutton 24 22 22 ms 31 Pr tocol editing cssssassscssesssusssussnsesnetssssnsersssannenneinnensncssnnsninnenshheskunsuhsehesehtenseinseisherssteste 33 Explorer Eat na eA eee ees 35 Explorertolder Sumito 36 Explorer Icons ns cota isdsvesdubeasess jessedssccosteseesessdavesesteae 36 Protocol selection sun stehlen tia ile 37 Protocol editor u east Bene oE ESEE E EE E EEOAE 38 Samples a a R ri 39 TDD EEEE EE 41 Measurement ni 42 Measurement mode eclosionar REES nen 42 Measurement Operations coooococnnonnnoncconccononononnnonnnonn nono nnnnnnn non nn cone cn neon aran nc nono nn nc nnnrnncrnnnnns 43 Down Buttons sc cooler ri 43 OaM oi LACON o PENNANEN EEEE E EAE E EE iis 43 Contents Delete Dutton siniese eea dd 43 Dispense button Re ee A Re OE es 43 Delay Dutton iia orita aati 44 Shake b utton ii 44 Label Dll aaa 44 Kinetics buttons AAA AA AA e i 45 Scanning buttons eeii ea les Binnen iss iii 45 Injector position during measurement uueneessessnesnnesnnennensnensnensnensonnnnnnnnnnnnnennnnn 45 Pl A ons 46 Plate repeat parameters viii dia 46 MENS dsce6tssest ca bsesanet setusteodasnceescesnes se tsesaoeteavassssdaunsopheveesbosdaeeseet odes 46 Pl
74. nd in the acceptor window These are made by dispensing buffer only Spectral crosstalk samples 1 e donor only samples in A3 and A4 these are used to calculate the amount of the blank corrected donor signal in the acceptor window as a proportion of the donor signal Only donor and buffer should be dispensed to these wells High intensity samples in A5 and A6 these are to normalize the final result to the maximum donor value The ratio of the high intensity donor value to the sample donor value corrects for possible quenching in the sample These samples should include all the reagents to give the maximum acceptor signal All other wells should be left empty Then load the plate and click Next Wallac 1420 TRF LANCE Normalization Wizard Prepare the Generic 8x12 size plate plate so that il blank series is placed in wells 41 and A2 rj O O 2 cross talk series is placed on wells 43 and 44 EB high series is placed in wells A5 and 46 e OOS and all others wells are left empty OJOJO Load the plate into the instrument ge d Click Next to start the measurement of the plate 104 Tools menu Measurement status page Wallac 1420 TRF LANCE Normalization Wizard Assay 17 This is shown during the actual normalization measurement and it gives you feedback about the measurement The measurement status pane shows the current status of the measurement The progress of the measurement is shown by the progress ba
75. ocol editor 53 Print Result Viewer 62 Print preview Protocol editor 53 Print setup Protocol editor 53 Printer export 62 Processing error 131 Product number 118 Protocol Result Viewer 61 Protocol editor 38 Protocol ID 37 Protocol name 69 73 76 78 80 83 Protocols Number of Max 40 38 Pumps 139 R Reading time Photometry 77 Reagent bottle 145 Repeat for plate 46 Result viewer Error 62 Export 62 Notes 62 Plate map 60 Preview 62 Print 62 Protocol 61 Result Viewer 57 58 59 62 Results export 62 Results of latest assay run 57 Results picture 60 Results viewer File menu 58 154 List 59 Plate menu 58 View menu 58 Results viewing 57 Routine level 66 Rows of wells 118 S Samples protocol editor 39 Samples tab 38 39 Save Protocol editor 53 Scanning button Measurement tab 45 Second Emission filter Luminescence 79 Second meas parameters Fluorometry 75 Second meas parameters LANCE 87 Second meas parameters TRF 72 Select where output goes Protocol editor 48 Serial number 118 Service level 66 119 Shake button Measurement tab 44 Shaker Path 44 Slide emission filter changing 93 Slide aperture 71 75 78 81 85 Slowdown in program 130 Software installation 10 Stabilized energy mode 74 Stacker 26 27 29 138 Start Protocol editor 53 Start button 21 22 28 129 Start Wizard 24 28 53 65 119 120 Stop button
76. on CW Lamp filters You can select the CW lamp filter from the drop down list of available filters See the chapter on Filters for more information about how to define filters Factory pre set CW lamp filter names begin with a letter indicating the technology for which they are to be used P is for photometry After the letter comes the wavelength 76 Tools menu Excitation aperture Wallac 1420 040 041 only There are three sizes of excitation aperture controlling the size of the incident light beam You can select with the option buttons Small Normal or Large Normal is the default Reading time This parameter determines how long the sample is read to determine the absorbance Second measurement In the case where you want to make measurements with two filters you can click this check box and then set the parameter CW Lamp filter It has the same options as the parameter with the same name above When you select this two measurements are made the first with the normal settings and the second with this new setting The change is automatic 77 Tools menu Luminometry Luminescence in the sample is detected The parameters that can be set for luminometry are show in the picture and described below Luminometry Label Properties Factory preset No filter Luminescence Slot A ha fe Tonia E Dam Sigel J SUTEMEN A ilter Luminescence Slot A ha Protocol name This is the name by which the p
77. on 87 Filters init o 88 Emission Olesa it dee hee aa es 89 CW LampTllters a tada dass selena oes Riesen BnsH HD Setase soe i n 90 Filter SUGGS currado ob 91 Emission filter de ito aiii candente E KE r E ee TESS 92 Physically changing the emission filter slide onnonccnncninninnnnonnnoncnancnnnranonancnancnnnnn 93 Contents CW Lamp filter wheel ooooonconnccnccnocnconoconoconoconoconocnnonononnnonnnrnn ron nnnn rn nrnnnnn cnn ncnnecnnes 94 Physically changing the CW Lamp filter wheel ooonoonicnnnnncnnccnnccnonnconnconcnancnnncnnoss 95 Changing the CW L Mp roren oreet 8 aoaea Eraro eiS Era TE EIEEE p san 95 Changing filters in the software esseeesseeeeesesseerrseerrsrrereserrrsreresesreeesrenreseenenserees 96 Filter properties cunas ti 97 EuSm Dual label normalization uurssuessessnesnnesnnesnnennesnnennnennnennonsnonsnensnensennnnnnn 98 Plate type selecto siese a reer He LI SSR ER 98 Preparing the plate ss cin iii iii 99 Measurement status page eee eeceescesecesecesecsaecsaecseecseseaecaaecaeseaeseaeeseeeereneeaees 100 Measurement warning page uurunersnensensennnnnnnnnnesnnennnennnesnnnsnensnensnensennnnnnnnnnnrnernnenn 100 Normalization confirmation page uuessessnesnnesnnesnnesnensnensensennnnnnnnnnnnsnnennnennnennne nn 101 BEANCE normaliz tion 2 212er te dias ista nen 102 Using the LANCE normalization Wizard oooonccnonnconncononononononnnonnconnonnnonn cra cnn nonccnn
78. on Aperture Normal Damp Band Counting Time fil 0 1 600 s G factor fi Cancel Help Protocol name This is the name by which the protocol is identified A password can be associated with it to prevent parameters being changed by an unauthorized person 80 Tools menu CW Lamp energy In fluorescence polarization constant voltage mode is used and the light source is driven with high power The software corrects for changes in energy of the CW lamp to ensure that results are calculated for a constant excitation energy For fluorescence polarization you should always use maximum power 65535 CW Lamp filter You can select the CW lamp excitation filter from the drop down list of available filters See the chapter on Filters for more information about how to define filters Factory pre set CW lamp filter names begin with a letter indicating the technology for which they are to be used F is for fluorometry After the letter comes the wavelength Polarizer aperture There are two sizes for the aperture of the S plane polarizer in the excitation light path You can select by option buttons whether this is Small for high density plates or Normal 96 or 384 well plates Emission filter You can select the emission filter from the drop down list of available filters See the chapter on Filters for more information about how to define filters Factory preset emission filter names begin with a letter indicating
79. onmoour 4 fs List Plates 1 1 A Plate A Protocol A Notes Ready Plate of F2 The cells in the two first rows displayed include information about the currently displayed plate The information included is Plate The number of the physical plate currently being displayed Repeat The number of the repeat of the physical plate currently being displayed Measurement end time Date and time when the measurement of the result plate was finished Start temperature Plate holder temperature when the measurement of the plate was Started End temperature Plate holder temperature when the measurement of the plate ended Barcode Barcode of the plate if used if not used it displays the text N A 60 Result viewing The cells displayed in the rows directly above each result plate gives information directly relating to the label or result type displayed in the plate grid below it The information included is Name of the label or result type displayed in the result plate Unit for the results from the result plate Instrument Background value measured for the label or result type Protocol Assay 3 2 11 1999 12 39 59 Wallac 1420 Result Viewer Me E File View Plate Help ala aafe He AAAA froox A Protocol description Protocol name Fluorescein 485nm 535nm Protocol number a Name of the plate type Generic 8x12 size plate Number of r
80. p to 1536 well plates can be counted depending on the technology and model as well as Petri dishes slide filters and Terasaki plates Plates can be loaded manually or with stackers for automatic operation Output can be to a file on the PC and or to a laser printer The following figure shows the Wallac 1420 system with its PC and laser printer Introduction Software installation Note the following instructions assume that the counter PC and printer have been connected up and switched on see the installation instructions in the Instrument manual 1 Load the software CD Ned Load CD 2 The setup loader will start automatically and give you a choice of either selecting the Installation Wizard to guide you through the installation or reading files from the CD 3 When you have completed the installation you should remove the CD from the PC otherwise the setup loader will start again next time you boot the PC 10 Introduction Operation overview Parameters must first be set using the protocol editor This is activated by selecting a protocol in the Explorer A sample plate can then be loaded and measured using the protocol set for it Other parameters affecting the whole system e g labels filters etc can be set in the Tools menu 11 Introduction 12 Operating Wallac 1420 14 Operating Wallac 1420 Starting up Wallac 1420 When you boot the system the following window appears
81. pty and if not to empty it proceed as follows Open the top and front cover of the dispenser and then lift off the blue plate at the back of the dispenser 140 Appendix 3 Dispenser operation Pull out the holder for the waste vial Empty out any liquid following the disposal procedure recommended by the manufacturer of the reagent you have been using Then replace the waste vial on its holder and slide it back into place Replace the blue plate This is important because it prevents light leakage to Wallac 1420 141 Appendix 3 Dispenser operation Preparing the tubing Put the tubing into a container of water 142 Appendix 3 Dispenser operation Dispenser maintenance Wallac 1420 Manager Clicking thDispenser maintenance icon or the item in the Tools menu invokes the Dispenser Maintenance Setup page to perform common dispenser maintenance operations This is enabled only when working with a system that has the dispenser installed The current state of the instrument server is shown at the top of the page Dispenser Maintenance App The setup page presents you with a graphical representation of the number of pumps 1 4 available when performing an operation You can select the pump s which are going to perform the operation by checking boxes under the respective pump pictures The operation to be performed can be either Fill Empty or Flush and you can select which of these you wish
82. r Wallac 1420 TRF LANCE Normalization Wizard Pi 105 Tools menu The Next button becomes available when the measurement has been completed If the measurement was completed without any warnings the window shows the text Assay is ready Click Next to move to the Normalization confirmation page otherwise the wizard moves to the Measurement warning page Measurement warning page This is used to inform you that something out of the ordinary has happened during the measurement of the normalization assay This page gives you enough information to decide whether the results can be accepted regardless of the problem that caused the warnings Clicking Next on this page advances the wizard to the normalization confirmation page Clicking Cancel closes the wizard and the normalization factors are not recorded in the database 106 Tools menu Normalization confirmation page Wallac 1420 TRF LANCE Normalization Wizard The normalization measurement is now completed Check the results and press Finish to accept the new normalization parameters or Cancel to ignore them Normalization parameters Blank 74 Cross talk ratio 0 0510 High A 58 High D 243 This is the last page of the LANCE normalization wizard and appears after normalization has been successfully completed It shows you the measured normalization values and requires you to confirm that you accept these values before they are stored in the d
83. r information about results see the chapter on Result viewing 37 Protocol editing Protocol editor Note changes to protocols should only be made by authorized persons The protocol editor window contains seven tabs Samples shows a plate map to allow you to edit wells and plates ID you can identify the protocol Measurement you can select the labels used for measurement Plate you can select the plate type and other parameters Outputs you can define the output Events you can give a command to be followed after an assay has been run with this protocol General information about the protocol You can set parameters for up to 40 plates in one protocol Protocol entries are checked for validity by the software and invalid entries are informed when you try to switch from one view to another When you have edited a protocol you can use the command line or the button bar to save or print the protocol Note if you want to change the plate type do this before setting other plate parameters This is because when the plate type is changed all the previous plates will be deleted and one plate of the selected type will appear 38 Protocol editing Samples if Fluorescein 485nm 535nm Protocol editor alel ep El A e 000000 Selecting this tab causes a plate map to be displayed to allow you to edit samples Buttons allow you to select the first plate previous plate next plate or last plate
84. rom the result viewer by selecting View all result data Note Each flash contributes to the total excitation energy Flashes keep on occurring until the required total excitation energy is achieved However because the energy comes in discrete flashes the last flash will cause the total excitation energy to exceed the required amount by a maximum of not more than the energy in one flash This will not be significant if there are many flashes but could be significant if there are only a few flashes E g in standard measurements the amount of flashes is about 1000 the variation in the total excitation energy is thus 1 1000 or 0 1 If the amount of flashes is about 100 the variation in excitation energies is 1 100 or 1 Light Integration Reference Level These values change the excitation energy approximately linearly Changing this value from 50 to 100 has about the same effect as changing the capacitor from 1 to 2 The number of flashes can be seen from the result viewer by selecting View all result data Emission filters You can select the emission filter from the drop down list of available filters See the chapter on Filters for more information about how to define filters Factory pre set emission filter names begin with a letter indicating the technology for which they are to be used D is for time resolved fluorometry After the letter comes the wavelength Emission aperture In front of the photomultiplier tube there is a 4 positio
85. rotocol is identified A password can be associated with it to prevent parameters being changed by an unauthorized person Emission filters You can select the emission filter from the drop down list of available filters See the chapter on Filters for more information about how to define filters Factory preset emission filter names begin with a letter indicating the technology for which they are to be used Lis for luminescence After the letter comes the wavelength Emission aperture In front of the photomultiplier tube there is a 4 position aperture slide which has three different apertures and a shutter position 78 Tools menu The normal aperture is circular with a diameter of about 4 mm and it is used for factory set labels The damped aperture is also circular but only 1 mm in diameter This allows you to shift the dynamic range of the emission signal for a particular user selected label which may otherwise exceed the linear range of the instrument This damped aperture is used for the top fluorometry factory set labels The band aperture is an approximately 1 mm x 3 mm rectangle and it reserved mainly for future applications Its size and alignment is about the same as the excitation area from the flash lamp The shutter is used at all times when no measurement is occurring to prevent stray light getting to the photomultiplier tube Counting time This parameter determines how long the luminescence is counted for Secon
86. s in the Labels window 67 Buttons in the Misc settings window 116 C Change the CW Lamp 95 Changing the CW Lamp filter wheel 95 Changing the emission filter slide 93 Column width changing 57 Columns of wells 118 Communication Settings 122 Constant voltage mode 74 Control modes Constant voltage mode 74 For fast kinetics 74 For fluorescence polarization 74 Stabilized energy mode 74 Counter position Fluorometry 75 Counting parameters LANCE 86 Counting parameters TRF 72 CW_Lamp control mode 74 CW Lamp changing 95 CW Lamp energy F Polarization 81 CW Lamp energy Fluorometry 74 CW Lamp filter wheel 94 151 Index CW Lamp filters 74 76 81 90 D Define labels 67 Delay button Measurement tab 44 Delete button Measurement tab 43 Demo 16 Demo mode 119 Dimensions of a plate 118 Dispense button Measurement tab 43 Dispenser information 139 Dispenser maintenance 65 143 147 Needles 147 Dispenser unit 9 Display problems 130 Down button Measurement tab 43 Dual label 37 Dual label normalization 98 Dual label normalization wizard 98 E Eject slide button 93 Emission aperture 75 81 Emission filter slide 92 Emission filters 71 74 76 78 81 85 89 Empty 143 End button 30 Error Result Viewer 62 Eu Sm dual label selection 37 EuSm Dual label normalization 65 98 EuSm Dual label normalization wizard 98 Events Protocol editor 50 Events
87. s visible or a linear scale in which every part of the range is treated equally 18 Operating Wallac 1420 Temperature Wallac 1420 Manager Sl aj e In the Temperature tab of the main window you select plate heating and the target temperature The latter can be set with 0 1 C precision within the interval 15 45 C The minimum temperature is 15 C or room temperature plus 2 C depending on which is higher If the target is changed the new value is only effective when the Apply button has been clicked The figure shows the temperature rising rapidly to its new level and then stabilizing there If the plate heating option is not installed these controls are disabled The current temperature is shown in the bottom right hand corner of the window irrespective of which tab is selected Tf the plate heating option is not installed these controls are disabled 19 Operating Wallac 1420 Barcode options An optional barcode reader can be included This allows you to load barcode labelled plates which are identified by the barcode reader Codabar Code39 Interleaved 2 of 5 Code 128 UPC and EAN barcodes can be read There are two sorts of identification that can be used This identification can be by plate number in which case you must select the protocol as part of the start up procedure Alternatively the barcode can be the number of the protocol to be used for measuring that plate This system is espec
88. sh Energy Level The Flash Energy Level sets the voltage of the discharge capacitor the range is from 1 to 255 corresponding to 500 to 800 volts Note that although the lamp input energy depends on the squared power of the discharge voltage the output optical power is not linearly dependent on the input energy By changing this parameter you will fine tune the Flash Energy If you increase the number you will reduce counting time but counts from your sample will remain about the same CAUTION If software version 2 0 release 8 or higher is installed on an instrument having a serial number less than 4201396 then you should avoid the combination of settings Flash Energy Area High and Flash Energy Level high values e g 255 along with 384 well or higher plates The reason is that this high power continuous usage combination may damage the older type of flash lamp The cooled flash excitation system available for newer instruments is especially designed to cope with this Excitation filter There are two non changeable excitation filters in the flash excitation path Both are colour glass filters The main filter is D 340 which has a maximum transmittance at 340 and half bandwidth of about 35 nm This filter is used for factory set protocols The second filter D320 has a maximum transmittance at 320 but its bandwidth is much broader it includes the same excitation band as D340 but its lower edge goes down to 280nm allowing excitation o
89. st Dispense button Clicking this causes a dialogue to appear that lets you specify the parameters for a dispense operation This button is enabled when the dimensions of the plate type used in the protocol are 8x12 or less You can select the number of injectors to be used from 1 to 4 depending on how many are installed the volume to be dispensed in microlitres A slide bar helps you to select this volume 350 microlitres is the maximum the speed from 1 to 5 where 5 is the fastest and 4 the default the increment using a slide bar with a range from plus 100 microlitres to minus 100 microlitres this is the volume added to or subtracted from the previous volume dispensed before the next dispensing 43 Protocol editing the number of replicates from 1 to 3 the injection mode You can either aspirate and then dispense the same amount aspVol dispVol or you can fill the whole syringe and then dispense several times aspVol syringeVol The former is more accurate but the latter is faster At the bottom of the dialogue is a check box Perform on first plate repeat only If you only want dispensing for the first of the plate repeats check this box If you want dispensing for every repeat then leave it unchecked Clicking the OK button inserts a dispense operation in the measurement operations list Delay button Clicking this causes a dialogue to appear that lets you specify the duration of the delay between the complet
90. sult viewing List Assay 3 2 11 1999 12 39 59 Wallac 1420 Result Viewer _ of x File View Plate Help ajaja rel 141 fox gt A A A DA A esc Repeat Well Type 00 00 00 20 567949 00 00 00 40 201371 00 00 00 60 810653 00 00 00 80 589159 00 00 01 00 485074 00 00 01 20 356788 00 00 01 40 897002 00 00 01 60 824611 00 00 01 80 749138 00 00 02 00 182367 00 00 02 20 860354 00 00 02 40 713396 00 00 02 60 518399 00 00 02 80 310954 00 00 03 00 24834 00 00 03 20 100488 00 00 03 40 370807 00 00 03 60 155839 00 00 03 80 174239 00 00 04 00 988639 00 00 04 20 451235 00 00 04 40 127892 00 00 04 60 14622 00 00 04 80 18822 00 00 05 00 384101 00 00 05 20 536346 00 00 05 40 575472 00 00 05 60 605746 00 00 05 860 611094 00 00 06 00 174571 ZE E j 23 23 2323232323 22 23 223233333233 2323232232332 The following information appears Plate ID Number of repeats Well number and Sample type abbreviation e g M Measured In addition there are pairs of columns The number of pairs depends on what you have set in protocol editing parameters The first column of each pair is the measurement time and the other the result value You can change column widths to get more information In the case of high density plates results can be split over several tabs View all result data This item in the View menu is only active if List view is selected It causes an extra column to appear in the result list giving the follow
91. sults shown see page 57 Labels for defining labels used in measurements Filters for defining filter types EuSm dual label normalization wizard requires TR FIA to be installed LANCE normalization wizard requires LANCE to be installed Plate dimension wizard for fine tuning the plate position when measuring high density plates Dispenser maintenance described in Appendix 3 Miscellaneous settings including plate types and well names User level selecting the features accessible to the user Options includes demo mode selection barcode and plate map features 65 Tools menu User level User level in the Tools menu allows you to select the user level Routine Advanced or Service If the User Level is selected to be Routine this will automatically disable any system operations To enable these operations you must select the Advanced level Service is only for qualified service specialists It gives access to e g Instrument Options User Level 66 Tools menu Labels TRF Fluorometiy Photometry Luminometry FP LANCE Europium Samarium Terbium Dysprosium Europium Fast Samarium in europium window Add Copy Remove Eset In this window there are tabs corresponding to the measurement technologies labels in the instrument Each tab includes the names of the currently defined labels Names of user defined labels can be freely defined b
92. sured remove it and load the next plate 1 Remove the measured plate 2 Load the next plate 3 Press the button briefly 6 Briefly press the button on the counter this will be blinking if there are more plates to be loaded for this assay If it is not blinking then the assay has finished Blinking light shows assay Continue until all plates have been measured 23 Operating Wallac 1420 Starting operation with the Start Wizard An alternative way to start counting is to use the Start Wizard This allows you to define how many plates you are going to use and to give the positions of samples Proceed as follows 1 Click on the Start Wizard icon and follow the instructions that appear Wallac 1420 Manager The Start Wizard will appear to guide you through the procedure for starting Wallac 1420 Start Wizard The steps are 24 Operating Wallac 1420 Introduction to the wizard You can select the introduction not to appear next time if you want Protocol selection click the protocol you want to use for measurement Definition of the sample names and number of plates See Samples tab on page 39 for a description of the principles involved in sample definition Note Errors in protocol selection or plate layout definition will lead to incorrect results Comments that you want added to the results just type in what you want Load the first plate the protocol name and co
93. the chapter on Filters for more information about how to define filters Factory pre set CW lamp filter names begin with a letter indicating the technology for which they are to be used F is for fluorometry After the letter comes the wavelength Excitation aperture Wallac 1420 040 041 042 043 only There are three sizes of excitation aperture controlling the size of the incident light beam You can select with the option buttons Small Normal or Large Normal is the default Emission filters You can select the emission filter from the drop down list of available filters See the chapter on Filters for more information about how to define filters Factory preset emission filter names begin with a letter indicating the technology for which they are to be used F is for fluorometry After the letter comes the wavelength 74 Tools menu Emission aperture In front of the photomultiplier tube there is a 4 position aperture slide which has three different apertures and a shutter position The normal aperture is circular with a diameter of about 4 mm and it is used for factory set labels The damped aperture is also circular but only 1 mm in diameter This allows you to shift the dynamic range of the emission signal for a particular user selected label that may otherwise exceed the linear range of the instrument This damped aperture is used for the top fluorometry factory set labels The band aperture is an approximately 1 mm x 3 mm
94. the size of the results If you move the cursor on to a well that has been measured the numerical value will appear At the bottom of the window there is information about the sample being measured including its current value Note in multiple label scanning and kinetics measurements only results from the first measurement of the label are visible in the Live display You can see results from all labels in the Latest results display which you can get by clicking the Latest assay run icon Wallac 1420 Manager e er o er _ NN ass apjajsiaiojeje IP jaojejel IIIS S WWVOOOQQO005 WVOOWOO0 PIPPI AMINO A E E L E F is H QUUOYUuUDdOS SIDE OWVOOOVOO OOVOO80 You can choose the result range you want used to determine the colour code of the wells The closer a result is to the upper limit of the range the more red it will appear The closer to the lower limit it is the more blue it will appear These colours will correspond to the 17 Operating Wallac 1420 values shown in the legend appearing beside the plate map Choose the range that is appropriate for the samples you are measuring so that you get the greatest variation in sample colours control Live display Te Logarithm Line You can choose whether you want the scale to be logarithmic which means that the difference between smaller values will be more visible whereas the difference between higher values will be les
95. the technology for which they are to be used F is for fluorometry After the letter comes the wavelength Emission aperture In front of the photomultiplier tube there is a 4 position aperture slide which has three different apertures and a shutter position The normal aperture which is circular with a diameter of about 4 mm should be used for fluorescence polarization Counting time This parameter determines how long the excitation continues The following times are recommended for 96 and 384 well plates Concentration nM Measuring time s 2 or more 0 1 1 0 2 0 5 0 3 0 1 1 0 81 Tools menu G factor In fluorescence polarization assays a G factor is used to correct for the bias in polarization values caused by differences in instruments and assay conditions To calculate the G factor prepare buffer and 1 nM fluorescein samples replicates should be at least 24 The formula used is ea G CorrectedS 1000 CorrectedP L d 1000 where the corrected parallel and perpendicular S and P counts are background subtracted fluorescence count rates obtained from a pure fluorophore solution e g CorrectedS averageS 1nM averageS buffer and CorrectedP averageP 1nM averageP buffer L is the literature polarization value for the fluorophore in mP E g the value for 1 nM fluorescein is 27 mP in aqueous buffer at room temperature The G factor is typically between 0 8 and 1 2 The G f
96. the variation in the total excitation energy is thus 1 1000 or 0 1 If the amount of flashes is about 100 the variation in excitation energies is 1 100 or 1 Light Integration Reference Level These values change the excitation energy approximately linearly Changing this value from 50 to 100 has about the same effect as changing the capacitor from 1 to 2 The number of flashes can be seen from the result viewer by selecting View all result data Emission filter You can select the emission filter from the drop down list of available filters See the chapter on Filters for more information about how to define filters Factory pre set emission filter names begin with a letter indicating the technology for which they are to be used D is for time resolved fluorometry After the letter comes the wavelength Emission aperture In front of the photomultiplier tube there is a 4 position aperture slide which has three different apertures and a shutter position The normal aperture is circular with a diameter of about 4 mm and it is used for factory set labels The damped aperture is also circular but only 1 mm in diameter This allows you to shift the dynamic range of the emission signal for a particular user selected label which may otherwise exceed the linear range of the instrument This damped aperture is used for the top fluorometry factory set labels The band aperture is an approximately 1 mm x 3 mm rectangle and it reserved mainly for
97. tion Appendix 3 Dispenser operation Introduction The optional dispenser allows you to make measurements that require e g the addition of reagent to start the process as in the case of flash luminescence or start and stop an enzyme reaction It can also be used for dispensing Enhancement Solution for a DELFIA time resolved fluorometry measurement It can be used with any of the technologies supported by Wallac 1420 The dispenser can have up to four pumps fitted This allows you to have separate pumps dedicated for specific purposes All the needles are directed to the same well allowing dispensing of more than one reagent to the same well The number of pumps fitted and the total volume of each pump are set during installation This information then appears when dispenser maintenance is selected The default setting for the dispensed volume can be between 5 to 350 microlitres Note dispensing to 384 well plates is possible if just one pump is installed The optional dispenser is a separate unit from Wallac 1420 itself making for convenient maintenance and flexibility in syringe selection The information about installing the dispenser unit will be found in the Instrument manual as part of the installation instructions The following instructions tell how to use the dispenser 139 Appendix 3 Dispenser operation Emptying the waste vial Note the waste vial must be empty when operation starts To check that the waste vial is em
98. to perform The operation is then performed on all the selected pumps Note before performing this operations make sure the dispenser has been prepared as described earlier 143 Appendix 3 Dispenser operation Fill is used for filling the syringe and tubing before operation begins Empty is used after operation ends to empty the contents of the syringe and tubing back into the bottle Flush is used for cleaning the syringe and tubing after it has been emptied Click Flush Dispenser Maintenance 144 Appendix 3 Dispenser operation Put a reagent bottle into the holder Fit the tubing into the grip 145 Appendix 3 Dispenser operation Measurement Close the covers of the dispenser The system should now be ready for operation Proceed with the measurements 146 Appendix 3 Dispenser operation Maintenance After measurement has been completed perform the following maintenance operations Click the Empty command This will cause the reagent to be sucked back into the reagent bottle so that nape is wasted Dispenser Maintenance Open the covers Removz the tubing from the reagent bottle and put it into a container of water and select the Flush command again Note it may be necessary to use a solution of 70 ethanol rather than water for rinsing the tubing See the reagent manufacturer s instructions Needle maintenance Every time the dispenser has been used the tubing must be flush
99. tuations increase the software performance drastically You can find this program by clicking the Wallac 1420 icon in your Windows 95 Start menu It is called Wallac 1420 Database maintenance 130 Troubleshooting Check that no other major resource consuming programs are running while using the Wallac 1420 software Check that there is at least 20 MB available on your C drive Defragment your hard disk You can find this function in your Windows 95 Start menu under Programs Acessories System tools Disk Defragmenter Processing error This dialogue will be displayed if the Instrument server goes to an error state or if the Manager cannot communicate with the Instrument server Right after the header there is text telling to user that Instrument server has stopped processing of the Workstation software because of an error After that there is a description about the error given by Instrument server or explanation that Manager cannot communicate with the Instrument server if that is the case When you click the OK button the reboot sequence is activated and the PC will be restarted System requirements not met This dialogue will be displayed during Manager startup if NoSystemCheckAtStartup command line option was not used and the minimum system requirements for Manager were not fulfilled The Current System panel is used to display information about the current system parameters The System requirements panel is
100. ture should be when a plate is measured The plate heating controls are only enabled when used with a system that supports the plate heating option Note Setting this option will not activate the plate heating automatically but 1t will cause a warning to appear if the temperature differs significantly from what has been specified in the protocol 47 Protocol editing Outputs f Fluorescein 485nm 535nm Protocol editor MProtocol description MError description List output MPlate MProtocol description MError description MNotes The Outputs tab in the Protocol editor allows you to select where results will be sent at the end of a run This can be to a printer and or file Click the check box of the output device s you want If you have selected Printer output you must select the format either plate format results are in a table format with rows and columns in the order shown in the plate map and or list format results are in a single column starting with those from the first well When this option is not set the Include in output checked list box is disabled This list box defines which parts of the assay run data should be included in the printed output List Plate Protocol description Error description Notes 48 Protocol editing If you have selected File output you must specify the file type from the drop down menu of possible types The types you can save files in are Excel 5 or later
101. ues before they are stored in the database The two values produced by the normalization are the background and the europium contribution to the samarium window During actual dual label measurements the europium counts in the europium windows are first calculated by subtracting the background from the measured counts Knowing the europium counts and the normalization ratio the actual samarium counts in the samarium window can be calculated by subtracting the europium contribution to the measured counts Additional information about the reliability of the factors is also shown so that you can be sure that the results of the measurement are correct Note to avoid accidental clicking of the Finish button it only becomes visible after five seconds have elapsed since the page was opened During the time the Finish button is disabled the text of the button is Wait t where t is the remaining waiting time and it is updated every second When you click Finish the new values replace the old values in the database The Back button is enabled only if the wizard moved to this page from the Measurement warning page 101 Tools menu LANCE normalization Note LANCE must be installed for this to be enabled Using the LANCE normalization wizard Before LANCE time resolved measurements can be made the instrument must be normalized for the protocol to be used This procedure corrects for the following effects in LANCE measurements background fro
102. used to display the minimum system parameters for the Manager to start The Suggestions panel will list the reasons why the Manager cannot be started and a description about what must be done to fix the situation When the OK button is clicked the dialogue together with the manager is closed The minimum system requirements are Pentium processor 16 MB or more memory Screen size 800x600 or more 256 or more screen colours 131 Troubleshooting 132 Appendices 133 134 Appendix 1 Connecting MultiCalc to Wallac 1420 Appendix 1 Connecting MultiCalc to Wallac 1420 Introduction MultiCalc can read assay text files from Wallac 1420 Evaluation of assays can be fully automated Measured wells are read sequentially Plate printout and re order work only for full plates MultiCalc version 2 6 or higher is required Note The Windows properties setting for MultiCalc should have Misc Background Always suspend NOT CHECKED Idle sensitivity Low To see what the situation is click the MultiCalc shortcut icon with the right mouse button and select Properties from the menu In the dialogue that appears select the Misc tab and then in the Background field make sure that Always suspend is not checked See also Installation in the Instrument Manual for how to install this option in MultiCalc Wallac 1420 MultiCalc assay protocols Create or edit a protocol using the Explorer and Protocol Editor In the protocol editor d
103. ver and screw it in place then click OK on the dialogue on the screen After this you can operate the instrument 93 Tools menu CW Lamp filter wheel Cw lamp filter wheel A Properties Available unused Filters Filter Positions All filters can be dragged from the Available Filters list to an empty position in the slide or from one position to an empty one or back to the list BB Factory Defined BB User Defined cn u Excitation filters fit into a wheel positioned in front of the CW lamp There are eight positions on this wheel and two are reserved by default for fluorometry and three for photometry The remaining positions can have filters added by the user Note default filters can be removed and replaced by user filters There is also a second filter wheel B which has only four positions but these are for the most common size of commercial filters 1 e round one inch and the wheel is supplied empty with locking rings 94 Tools menu Physically changing the CW Lamp filter wheel If you want to change a filter in the CW Lamp filter wheel or you want to install wheel B then you must open the cover and lift out the filter wheel as shown in the picture Either change the filters you want or take a new filter wheel B Replace the filter wheel and make sure you close the cover before starting a measurement HOW TO REPLACE THE WHEEL AND THE LAMP ATTENTION KEEP THE LID
104. want to use The button to the right of the second box again lists all the valid keyword arguments that you can choose from see Keywords below For example you can start an external spreadsheet running and have it execute the file produced by the run This file will be identified by e g protocol name or number There are also unique internal identifiers that the system gives for each protocol and assay run 50 Protocol editing Keywords The following keywords can be used for file names in the protocol editor Outputs tab and in Assay end system command in the Events tab lt ProtocolName gt the name of the protocol Specified in the ID tab in the protocol name edit box lt ProtocolNumber gt the protocol number for the protocol Specified on the ID tab in the protocol number box lt ProtocolID gt a unique numeric identifier for the protocol lt AssayID gt a unique numeric identifier for the assay run lt RunID gt a numeric value identifying the specific run of a certain protocol lt RunDate gt date when the assay was run in the format YY YYMMDD lt Plate barcode gt the barcode of the first plate of the assay If there is no barcode on a plate you should define an explicit file name for the plate lt Output file name gt the name of the output file given in the Outputs tab In some cases the numeric value of the AssayID or the RunID may not be suitable for the program handling the file You can add a function
105. y the user but names of factory pre set labels cannot be changed See the sections below for details There are four buttons at the bottom of the labels window Add add a new item to the list Give the name and edit the properties Copy select an item to activate this Make a copy of an item It will appear with a new icon showing it is different from a default item and the name will be preceded by the words Copy of You can give it a different name and edit other parameters by selecting Properties Remove select a user created item to activate this Remove the selected item from the list Preset items and labels used in protocols cannot be removed This command requires confirmation because you cannot undo Remove 67 Tools menu Properties select an item to activate this View the properties of the selected item If the item is user defined then you can edit the properties but if it is a factory pre set you cannot edit them 68 Tools menu Time resolved fluorometry Note to be enabled this requires the TR FIA option to be installed In time resolved fluorometry a flash lamp is used to illuminate the sample different lanthanides are used as labels The picture shows the parameters that can be set TR Fluorometry Label Properties fewopim D615 Europium LANCE_615nm Slot 7 Protocol name This is the name by which the protocol is identified A password can be associated wit

User manual Software version 2.00 release 9

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