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Flex manual II

1. 54 25 1 Wavelenpths au ga e uoc in 0 a eda eae BALD UR i eee eh eee 55 3192 XSeHislt VIL cones Xe selec wish dr ue e Ree tud ede ut e et te de eee tos 57 Sooo TIMING reederi a todo Gee ul e ed eani de P a anta abi dad bee du 58 2 5 AUOT ech eh M Asc ad o Red ot 4 RE Not Mot ce A dd 59 2 20 AOC AN brate tess pata dio Quee ceca ur ain De d po Severe E E E Greens 59 3 5 0 A say Plate Type reri torrens dreet ae a e EMILE 60 Fe Wellsto Rau ca Do ed ebd wie a a e e dod dd weed 61 3 5 8 Compound Source t sera Laaa aaan 63 3 5 9 Compound Transfer a5 ih oe dod ae Boa we shat udi eue RED 64 3 520 Trit rate Selection ce ied oh tk OF ob Pali rA MINER IR ON ee Rag 67 2 511 Pipette Tips Layout tss oa go de wea toed tt whe baa d Salona we oda 68 3 5 12 Compound and Tip Columts z 02 0 222245 229 0a oe E23 23S ou e Z1 69 9 519 AO USD So cuc eet ortu Bias ue ead eo EA atu Au C qu eee iMd dL EL E 72 2 90 4d Pip tte TIDA Gan ins qas QR ORIOL GES ades eode Rood Ge shh ud Re 72 3 5 15 Settings Displayed in Plate Sections eme Sep Re tae e Ei 73 3 6 Other Software Settings llli lilii ls 74 3 7 Reading the Microplate iiio ll lle esee 75 3 8 Shutdown eoe perte oe Tende ve ee ete o No n Md re nep rds 78 3 9 Other Read Modes 00 00002 ee 78 3 10 Optimizing Fluorescence Assays in Spectrum Mode 79 4 Applications 22 tebe eer be S tuy Rae hed Mocs be 85 5 Service an
2. 596 CV at 1 uL 52 uL sec FlexStation Il Operator s Manual Rev D Environmental Turn On Time Operating Conditions Electrical Power Consumption Line Voltage and Frequency Physical Size h x w x d Weight FlexStation Operator s Manual Rev D 7 2 Performance Specifications lt 5 minutes for rated accuracy 15 35 C 450 VA 90 240 VAC 50 60 Hz 19 3 x 22 8 x 15 7 in 49 cm x 58 cm x 40 cm 65 Ib 29 5 kg 121 7 Appendices 7 3 Warranty Molecular Devices Corporation Instrument Warranty Molecular Devices Corporation warrants this product against defects in material or workmanship as follows 1 All parts of the FlexStation Il instrument are warranted for a period of one 1 year from the original date of delivery 2 Alllabor charges to repair the product for a period of one 1 year from the original date of delivery will be paid by Molecular Devices Corporation 3 This warranty covers the FlexStation Il instrument only and does not extend to any computer printer software reagents or disposables used with the instrument Labor and Parts To obtain warranty service during the applicable warranty period you must take the product or deliver the product properly packaged in the original shipping materials and carton to an authorized Molecular Devices Corporation service facility You must call or write to the nearest Molecular Devices Corporation service facility to schedule warranty service Yo
3. 96 FlexStation Il Operator s Manual Rev D 6 Troubleshooting Procedures Part 6 provides instructions for troubleshooting problems that may occur with the FlexStation II instrument e 6 1 Problems During Startup e 6 2 Opening a Drawer Manually e 6 3 Understanding Potential Mechanical Problems e 6 4 Recovering from Mechanical Problems in Flex Mode e 6 5 General Error Messages e 6 6 Tilting or Removing the Fluidics Module Note These troubleshooting instructions only address problems that can occur with the fluidics operation of the instrument They do not address problems with the flash lamp or the detector or error messages related to missing data or saturated data points Contact Technical Support Note If you need to contact Technical Support at Molecular Devices see section 5 1 Technical Support 6 1 Problems During Startup Important Be sure the instrument is turned on before starting SOFTmax PRO After you start up SOFTmax PRO the Instrument Status bar should appear as in the illustration to the left with a temperature displaying in the temperature box Status Bar Detail If you see a red X in front of the instrument icon or if no temperature is displayed in the temperature box as in the illustration to the right there is either a communication problem between the instrument and the software or the instrument has experienced a fatal error 1 Check instrument power Check to be sure the FlexStation II
4. The fluidics module for is designed to aspirate fluids from a compound source plate and dispense them into an assay plate Fluid transfer is made possible with an eight channel pipettor that is fully automated including changing the tips from a tip rack In Flex mode one to eight or one to 16 wells in one column of the assay plate are read repeatedly for a selected total experimental time At a preselected point or points during that time sequence the pipettor may transfer up to three reagents from the compound plate to the assay plate The instrument continues to read at the preselected time intervals before and after fluid transfer After completion of reading the column or partial column for a preselected time the instrument can repeat this cycle with other columns All the data is collected in one data file represented as a 96 or 384 well plate For example an experiment with a two minute run time will accommodate a 96 well plate in about 24 minutes Run time x Number of columns Plate time 2 minutes x 12 columns 24 minutes The source for transferred reagents can be either a compound plate or reservoir trough located in the fluidics module 24 FlexStation Il Operator s Manual Rev D Operation Modes Operation Type Read Types System Settings Top read Bottom read Wavelength Automix before Automix between Timing Wells to read AutoCalibrate Compound source Compound transfer Triturate Pipette tips layout Com
5. Transfers You can enable up to three compound transfers within a single well during a run time The default value for the number of transfers is one 1 As you enable transfers color coded transfer buttons appear next to the Transfers box n E 3E viv Transfer 1 afe 2 Transfer 3 When you select one of the Transfer buttons that button will appear with a darker gray background You can then enter the parameters for that particular transfer in the Transfer Settings portion of this dialog box Important There is nothing else in the Transfer Settings portion of the box to indicate which transfer you are configuring Pay close attention to which transfer settings you are modifying Transfer Settings ee Transfer Settings Z ae i Time Point secs i Rate Minimum time 16 secs e Pipette Height 1 to 999 nL This setting determines the volume of fluid in microliters measured from the bottom of the assay plate microplate well above which the tip of the pipette will remain during the dispensing portion of the transfer event This setting helps ensure that the tip of the pipette is below the surface of the liquid at the end of the transfer thus minimizing the possibility that undispensed drops remain on the tips Important As you configure subsequent transfers you must calculate the amount of fluid added and set
6. are also both coaxial The instrument s electrical firmware and optical designs incorporate many features that work together to virtually eliminate instrument based day to day and instrument to instrument variations in fluorescence values measured For more detail of the optical design and an illustration of the optical system see section 1 4 4 Detection Module Bottom and Top Reading Switching to bottom or top reading capability is activated through software no manual positional switching of the read head is required Bottom reading allows for well 22 FlexStation Il Operator s Manual Rev D 1 6 Theory of Operation scanning ability maximizing the sampling area for 6 12 24 48 96 and 384 well microplates Bottom reading functions in all detection modes Important Clear bottom plates must be used when using bottom reading Bottom reading is intended for cell based assays Note RFU scale is ten times 10x larger during bottom reading FlexStation Operator s Manual Rev D 23 1 System Description 1 6 3 Modes of Operation The instrument operates in one integrated fluidics read Flex mode and four read only modes The table below compares the types of operation and features that are available in the different operating modes Note This operator s manual describes instrument behavior for Flex mode primarily Refer to the SOFTmax PRO User s Manual for instructions on other operating modes Flex Mode
7. installing the drawer adapters 42 installing the fluidics module 35 36 installing the pipettor head 37 40 setting up the computer 40 unpacking the system 32 34 Instrument Settings screen 52 54 assay plate type 60 AutoCalibrate selection 59 Automix 59 AutoRead 72 compound and tip columns 69 71 compound source 63 compound transfer 64 66 pipette tips layout 68 sensitivity 57 timing 58 triturate selection 67 wavelengths 55 FlexStation Il Operator s Manual Rev D wells to read 61 62 Instrument Status icon 29 51 introduction to system 1 2 K Kinetic mode 26 78 L lamp cover 8 M maintenance procedures See service and maintenance procedures Mask icon 30 mechanical problems avoiding 101 before using the instrument 100 power failure 101 understanding 100 101 mechanical problems Flex mode 102 12 assessing 103 evaluating the tip rack 105 expelling fluid from tips 111 inspecting inside fluidics module 106 8 opening the instrument 104 pipettor head removing 109 10 recovery procedure 112 microplate adapters See adapters microplate microplates 17 loading 44 reading 75 77 modes operational 24 26 moving the instrument 88 N Name of Plate icon 30 O operating procedures 43 84 configuring instrument settings 54 73 optimizing fluorescence assays 79 84 overview 18 44 reading microplates 75 77 FlexStation II Operator s Manual Rev
8. 1 Parts and Accessories Fuses Fuses are rated slow blow United States Canada Metric 6 3 amp time delay For further information see section 5 6 Replacing Fuses Cables Molecular Devices recommends that you use high quality double shielded cables to connect the instrument to the computer Choose cables that meet the following requirements Serial Interface Cable IBM compatible Male DB8 to Female DB9 Note Contact Molecular Devices Technical Service for specific pin out requirements Microplate Adapters The black or purple microplate adapter fits in the assay plate carriage in the reading chamber drawer to elevate standard microplates Remove the adapter when using high profile 6 12 24 or 48 well microplates Important For 96 and 384 well microplates you must use a black adapter in the reading chamber drawer when bottom reading and the purple adapter when top reading For further information see section 5 4 Using the Microplate Adapters Compound Baseplate Molecular Devices provides a metal baseplate that you must place in the compound plate drawer under the compounds plate to reduce stray light 16 FlexStation Il Operator s Manual Rev D 1 4 System Components 1 4 7 Consumables The system consumables include e Microplates e Pipette tips and racks One rack of pipette tips is shipped with the system For further information see section 7 1 Parts and Accessories Caution Do not use parts a
9. ACS i Minimum Run Time 20secs Cancel 52 Instrument Settings Screen Notice that the FLEX button on the right is highlighted Note You can select a different read mode from the Instrument Settings screen by clicking on one of the other four buttons at the top of the window The rest of these instructions assume you are remaining in Flex mode FlexStation Operator s Manual Rev D 3 4 Setting Up the Software 4 Return to the Untitled window Click OK to return to the Untitled window ws SOFTmax Pro BM xl File Edi View Experiment Control Assays Plate Window Help v Experiment v B notes 1 24 f VMax units per sec Plate 1 1 2 3 4 5 6 7 8 9 10 11 12 v 3 Plate 1 FLEX Time 300 secs Interval 9 secs Reads 34 Fluorescence Bottom read Ex Em Cutoff Lm1 485 538 530 amp uto Cutoff I Omm ootozT Wavelength Combination ILm1 Automix Off Trans H2100pi R21 Y 30p C010 Calibrate On PMT High ReadsVell 6 Lag Time 0 End Time 300 RFU Min 0 RFU Max 20000 Vmax Pts 34 34 Plate Last Read No Data Untitled Window Flex Mode 5 Create or edit Plate section s Before continuing with other software settings create or edit the Plate section s you will need Use New Plate from the Experiment menu in the Menu bar to create more Plate sections If you wish to create a plate wit
10. Control Assays Notes Window Help I Untitled v E notes E4 If You Are Familiar with SOFTmax PRO Customize This Default Protocol 1 Delete any sections you do not want through the Edit menu 2 Make any changes you wish to the Instrument Settings by pressing Setup it is assured you have an instrument connected 3 Click the Template button and assign any default or new groups as necessary you can also select replicates there 4 Select the desired Reduction and Display Raw vs Reduced parameters 5 Save this default protocol with the name Default Protocol in the same folder as the SOFTmax PRO application you will be asked if you wish to replace the existing default protocol choose Yes This modified protocol will now launch each time you open SOFTmax PRO A copy of the original default protocol can be found in the Tutorial folder if needed Plate 1 EC50 6 interpX Piot 1 Graph 1 50 b Graphitl a Fi i Es stl SOFTmax PRO New Untitled Window The first window comes up in Flex mode with a default template selected 46 FlexStation Operator s Manual Rev D 3 2 Starting Up the System gt Users new to SOFTmax PRO If you are new to SOFTmax PRO familiarize yourself with the software by reading about the default protocol and running the tutorial described in this window Note Refer to Chapter 10 of your SOFTmax PRO User s Manual gt Users familiar with SOFTmax PRO If you a
11. Do not flex twist bend or stretch the optical cables Emission 2 Collimation Monochromator Excitation Mirror Monochromator Photomultiplier Tube lt gt N Microplate Flash Lamp i Flash Lamp Elliptical Focusing Mirrors Optical System gt Overview of Optical System 1 The excitation light source is a xenon arc flash lamp Note that the lamp is off when luminescence mode is selected 2 The light passes through a band pass filter which reduces the amount of stray light to the excitation monochromator 3 The holographic diffraction grating monochromator selects the desired excitation wavelength FlexStation Il Operator s Manual Rev D 1 4 System Components 4 The excitation beam is collimated by a mirror to a 1 5mm diameter beam before entering the sample in the microplate well in bottom read mode This focusing helps to prevent part of the beam from striking adjacent wells 5 The light beam enters the well and if fluorescent molecules are present light of the emission wavelength is emitted back out to mirrors that focus it and send it to an optical bundle 6 The emission monochromator also a holographic diffraction grating monochromator allows light of the chosen emission wavelength to pass to the emission filter wheel 7 Long pass filters in the emission filter wheel further condition the light prior to detection by the photo multiplier tube PMT Using SOFTmax PRO you can choose whic
12. Figure 1 Set excitation scan to start stop approximately 50 nm below above the tentative excitation value obtained from the literature or the customary excitation filter Set step increment to 1 or 2 nm You may choose to do a preliminary scan with a 10 nm increment to determine the approximate peak location and then repeat the scan over a narrower wavelength range with a 1 or 2 nm increment Perform scan and view the results as a plot of emission fluorescence vs excitation wavelength Note the excitation wavelength at the emission peak and the maximum RFU value Note If an error message reporting missing data points occurs it may be due to possible saturation reported by SOFTmax PRO at the end of the spectral scan Reset the PMT to low and rescan the sample scan the buffer blank with the PMT set to medium or high If the error occurs after scanning with the PMT set to low it may be necessary to dilute the sample Note If the excitation scan shows no apparent peak change the PMT setting to high and rescan the sample If the spectral scan still shows no apparent peak adjust the Y scale of the zoom plot so that the plot fills the graph Select the optimal excitation wavelength If the excitation peak wavelength and emission wavelength are separated by more than 30 nm use the excitation peak wavelength value If the excitation and emission wavelengths are less than 30 nm apart use the shortest excitatio
13. Spectrum Mode The alternative cutoff wavelength choices are listed in the following table The cutoff value should be near the maximum emission wavelength preferably between the excitation wavelength and the maximal emission wavelength Automatic Cutoff Selection Endpoint and Kinetic Modes Lambda nm Emission nm 1 None lt 415 2 420 415 434 3 435 435 454 4 455 455 474 5 475 475 494 6 495 495 514 7 515 515 529 8 530 530 549 9 550 550 569 10 570 570 589 11 590 590 609 12 610 610 629 13 630 630 664 14 665 665 694 15 695 695 850 Emission Cutoff Filter Default Settings Note For Spectrum mode the default is Manual that is no automatic cutoff FlexStation Operator s Manual Rev D 83 3 Operating Procedures 4 Optional Excitation Scan with both Autofilters Scan Figure 4 In some Assays a final excitation scan with the desired emission filter and the automatic excitation filter will show additional background reduction In the example Scan Figure 84 4 note that the 445 nm peak occurring in all but an empty well is the Raman spectrum for water Note that the optimal excitation wavelength is 485 nm I Final EX scan with both filters Well Graph Raw olx 1400 1200 1000 800 irs H 400 J 200 spe i i H 350 360 370 380 380 400 410 420 430 440 450 460 470 480 490 500 Ex Wavelength in nm Em 525nm Well o B6 n c6 A D6 9 E6 F6 G6 Lambda at Maximum 499 00 49
14. User Interface This section briefly presents the basic features of the SOFT max PRO user interface More instructions regarding how to use the interface will appear throughout these instructions during relevant steps You can control the instrument by using either buttons and icons in the windows and along the tool bars or by using the pull down menus You can use either your mouse or keystrokes to make selections Note For complete details about the SOFTmax PRO application and user interface refer to your SOFTmax PRO Operator s manual Refer specifically to the following chapters 28 Chapter 6 for a discussion of Flex mode setup Chapter 8 for information about data analysis in Flex mode Chapter 10 Tutorial 2 for experience with a typical Flex mode template gt E notes B9 sw v C Plates es Setup iE Template X Reduction Eg Display ma ZA Max Min Plate 1 FLEX Time 120 secs j Interval 2secs j Reads 61 Fluorescence j Bottom read E Ex Em Cutoff Lmi485 525 515 uio Culott Automix Off Calibrate On PMT High ReadsVell 6 Wavelength Combination Lm1 Trans1 H 180p R 6 100p 15 y net Plate Last Read No Data b m Control uen 2 fa gt ff vata ue E4 f b Z crapha e Fi Untitled Window Flex Mode III FlexStation Operator s Manual Rev D 1 7 Introduction to SOFTmax PRO Application Software The Status bar all
15. Volume hoo oo jul Volume o Jul Cycles Cycles Height ioo JH Triturate Settings If you choose dispense times that are incompatible with the settings in this window an error message will appear A Fluid dispense time is too short Check fluid time points Return to the Transfer Settings part of the Compound Transfer settings window to modify the dispensing time FlexStation II Operator s Manual Rev D 67 3 Operating Procedures 3 5 11 Pipette Tips Layout 68 Note The Assay Plate setting takes precedence over all other fluidics module settings If you change the assay plate after specifying Pipette Tips Layout your tip settings will be canceled and the layout will revert to default Molecular Devices recommends that you always use a full rack of tips the default setting If you use less than a full rack of tips it is imperative that you configure this setting to correctly match the number and positions of the actual tips inserted in the tip rack drawer because the instrument cannot determine how many tips are actually present While it is possible to use a partial rack of tips and to choose which tips are available in the Pipette Tips Layout setting do so with caution Be sure that the layout described in the software matches the actual tips inserted in the tip drawer Caution Molecular Devices recommends using a full rack of tips each time you perform a transfer of fluid in Flex mode If you mistakenly ena
16. after completing all settings to obtain the minimum read interval Timing for readings can be adjusted in this window You can enter the total run time and the time interval between readings Timing Setting Dialog Box e To change the default values click in the appropriate box e Type in the desired run time or interval The acceptable run time range is from zero to a thousand 0 1000 seconds When you have entered the values you desire the total Number of Reads calculated by the instrument is displayed automatically Depending on what sensitivity you have selected in the previous setting box the Minimum Interval and the Minimum Run Time will automatically adjust The minimum interval is also dependent on the Compound Transfer settings and the number of wells to be read per column Errors If you select a time out of range an error message appears mi SOFT max Pro If you select an inappropriate interval an error message appears at the bottom of the window 58 FlexStation Operator s Manual Rev D 3 5 Configuring Instrument Settings in Flex Mode 3 5 4 Automix The Automix function is a patented feature that allows you to set automated shaking of the microplate before and or in between readings Select Before First Read to shake the plate before the first wavelength reading only You can also set Automix to shake the plate Between Reads e Click in the box next to the type of shaking you want e
17. cutoff filter at a wavelength below the emission lambda Em X maximum of the fluorophore The goal is to block residual excitation light and increase the signal to background ratio Sometimes it is appropriate FlexStation Operator s Manual Rev D 55 3 Operating Procedures 56 to select a cutoff filter at or even above the lambda maximum For example this is a good idea with fluorophores with small Stokes shift or when multiple fluorophores must be discriminated For further information see section 3 10 Optimizing Fluorescence Assays in Spectrum Mode Important If you select an emission wavelength that is close to two times the excitation wavelength if EM A 2 x EX A always be sure to use an emission cutoff filter to ensure you are blocking monochromator second order light If you run an experiment like this without an emission filter you will always observe an emission peak at two times the excitation wavelength but it is not fluorescence emission FlexStation Il Operator s Manual Rev D 3 5 Configuring Instrument Settings in Flex Mode 3 5 2 Sensitivity This setting determines the number of readings that will be performed on each well of the microplate readings are averaged and the average reading is displayed The default setting is six readings per well for fluorescence and can be adjusted between Fast and Precise Increase reading precision by moving the bar toward the right and increasing the number of rea
18. desired settings configurations before starting the reading You cannot change settings during reading or after reading and data collection are complete You can start reading at any time after defining instrument settings To read the microplates click on the Read button on the SOFT max PRO tool bar or select Read from the Control menu You can also type Ctrl R on the keyboard gt Selecting a Plate If you have created more than one Plate section and no Plate section is active choosing the Read command will display a dialog box requiring you to choose which section to read mj Select Section B N Cancel Select the correct section and click OK Ifa Plate section is active starting a read will read the active plate gt Replacing Data in a Plate If you select a Plate section that already contains data and select the Read command an alert appears requiring you to confirm that you want to replace the data with data from the new reading w SOFT max Pro Replace data in Platett1 Replace e Click Replace to proceed with the reading and replace previous data with new data e Click Cancel to stop the reading from beginning 76 FlexStation Operator s Manual Rev D 3 7 Reading the Microplate View Experiment Progress During the reading the Status bar will display information about the current reading w SOFT max Pro olx File Edit View Experiment Control Assays Plate Wi
19. exactly and any instruction labels on the instrument TO REMOVE PIPETTOR HEAD 1 Disconnect Power 2 Open access door 3 Loosen red retainer nut 4 Partially remove pipettor head 5 Pull spiral cord from mount 6 Rotate electrical connector lock and pull WHEN REINSTALLING INSURE PIPETTE RETAIN NUT IS FIRMLY TIGHTENED AS TIGHT AS POSSIBLE Biohazard Use proper handling precautions This procedure may include steps involving potentially infectious biological agents Follow your institution s protocol 1 Confirm that all power to the instrument is turned off 2 Unplug the power cord and the computer cable from the rear of the instrument 3 Open the instrument top cover Opening Instrument Cover 4 Open the inside top panel of the fluidics module Turn the quarter turn fastener on the inside top panel and unfold the panel off the fluidics module to the right 5 Inside the open chamber loosen the red mounting knob called the red retainer nut on the label FlexStation Operator s Manual Rev D 109 6 Troubleshooting Procedures Inside Top Panel Opened Eight or Sixteen Channel Pipettor Head Location of Pipettor Head Inside Fluidics Module 6 Slide the pipettor head part way out from under the metal mounting plate and of the top panel opening supporting it from underneath 7 Pull the white spiral cord off the coil hook behind the pipettor head This
20. for more than one day it is best to turn off the instrument 3 9 Other Read Modes In addition to operating the FlexStation II instrument in Flex mode fluidics read the instrument can operate in four read only modes Endpoint Mode Kinetic Mode Spectrum Mode Well Scan Mode gm Instrument Settings x Endpoint Kinetic Spectrum Well Scan FLEX Instrument Mode Icons Refer to Chapter 5 of your SOFTmax PRO User s Manual for general information on how to use the FlexStation II instrument to read microplates in one of these four read modes 78 FlexStation Operator s Manual Rev D 3 10 Optimizing Fluorescence Assays in Spectrum Mode 3 10 Optimizing Fluorescence Assays in Spectrum Mode Introduction The optimum instrument settings for detection of a particular fluorophore depend on a number of different factors Settings that can be adjusted for assay optimization include the excitation and emission wavelengths emission cutoff filter readings per well the PMT voltage the temperature of the reading chamber and the length of delay time for time resolved fluorescence e The excitation and emission wavelengths may be set in 1 nm increments within the range of the instrument 250 850 nm for excitation and 375 850 nm for emission A procedure to optimize excitation and emission wavelengths for a given assay is outlined below e The 14 emission cutoff filters assist in reducing background Sources of back ground i
21. he e i epe PES wei 19 16 2 Instrument Designe ot ad cosepo d dee dud hue ooes ho ADEOGS QU ted eee dud 21 136 3 Modes OF Operation iui ihe aeter s um dantes et ded ace e te eh 24 1 7 Introduction to SOFTmax PRO Application Software 27 2 Installation Procedures ooo 31 2 1 General Precautionary Information iiilll lll lll ll llle 31 22 Unpacking the System liiiliiiilll ll lll lll ees 32 23 Installing the Fluidics Module 0 2 000000000 2 eee 35 24 Installing the Pipettor Head 0 000000 0c ee 37 25 Setting Up the Computer 0 020 00000 00 ee 40 2 6 Connecting the Cables 00 0 00000 ee Lee ee 41 27 Installing the Drawer Adapters ll es 42 28 Installing SOFTmax PRO 0 00000000000 42 3 Operating Procedures 0 eee 43 Sil OVerVIe wo vo NE LE S e Iv a oe t d etd es 44 3 2 Starting Up the System iiiiiioiiill ll lll ll ee 45 3 3 Setting the Temperature llli lll lll len 49 FlexStation Operator s Manual Rev D iii Contents 3 3 1 Displaying Temperature c2 vis NS DAL en rte D de Pure m A Med io 49 3 9 2 Setting the Temperature with the Control Panel 0 49 3 3 3 Setting the Temperature with SOPTmax PRO 2 2446 ncn o th 50 3 4 Setting Up the Software l l llli ll lll len 51 3 5 Configuring Instrument Settings in Flex Mode
22. instrument and the cables connected to the computer 2 Liftup instrument cover Press in the handle at the bottom of the top cover and lift the cover back off the detection module revealing the fluidics module Inside Top Panel Inside Front Panel he Tip Rack Drawer Fluidics Module Detection Wo Location of Inside Panels and Tip Rack Drawer 3 Remove front panel of fluidics module DO NOT REMOVE COVER UNTIL POWER IS DISCONNECTED FOR TRAINED SERVICE PERSONNEL ONLY Do not operate instrument unless all covers are in place Turn the four quarter turn screws and lift the front panel out You many need to inspect the interior of the module to determine if the pipettor was in the process of a fluid transfer when the power was interrupted The pipettor head should be at rest in the uppermost home position If not there may be fluid in the tips that needs to be removed Instructions for these procedures follow 104 FlexStation Il Operator s Manual Rev D 6 4 Recovering from Mechanical Problems in Flex Mode 6 4 3 Evaluating the Tip Rack 1 Inspect the tip rack drawer The problem may be obvious and visible from the outside if the tip rack drawer is partially open and jammed Possible scenarios include 2 Tip rack cover was not removed e If the cover was not removed from the tip rack gently pull the tip rack carriage out of the fluidics module to the right Remove the tip rack
23. on the distance that you will be moving the instrument you may wish to repackage the FlexStation II instrument in its original shipping cartons Otherwise carry the instrument or place it on a rolling cart to transport it 5 3 Cleaning the Instrument 88 Biohazard Wear gloves during any cleaning procedure that could involve contact with either hazardous or biohazardous materials or fluids Periodically you should clean the outside surfaces of the FlexStation II instrument using a cloth or sponge that has been dampened with water Do not use abrasive cleaners If required clean the surfaces using a mild soap solution diluted with water or a glass cleaner and then wipe with a damp cloth or sponge to remove any residue Caution Do not spray cleaner directly onto the instrument or into any openings If needed clean the reading chamber drawer using a cloth or sponge that has been dampened with water Caution Never clean the inside of the instrument Do not allow excess water or other fluids to drip inside the instrument Cleaning Up Spills Caution Be sure the power is off to the instrument and the power cord is detached from the rear panel before opening any covers or panels If fluids spill into the bottom of the reading chamber when the reading chamber drawer is outside the instrument they will be directed to a tray at the bottom of the instrument from which they will exit to the bench or counter beneath the instrument Wipe u
24. pipettor head manually Reach into the fluidics module and gently pull the tips off the cones e Gently push the tip rack and compound plate or trough drawers all the way outside to the right 6 Close the instrument and reboot Once you have secured all tips and returned the tip rack and compound plate drawers to their outside rightmost position you can close the instrument and return to operation e Replace the inside front panel and lower the top cover e Reattach the power cord to the instrument and reconnect the instrument to the computer e Turn the power on the to instrument It will take about two minutes for the instrument to complete its startup sequence e Save your SOFTmax PRO file to prevent loss of data then close and restart the software Important Failure to restart SOFTmax PRO may result in communication problems between the instrument and computer FlexStation Operator s Manual Rev D 107 6 Troubleshooting Procedures 108 7 Confirm instrument to computer communication When communication between the instrument and computer is interrupted as with a power failure a red X appears over the FLEX icon on the SOFT max PRO status bar Once you correct the problem and the instrument has completed its startup cycle again the red X over the instrument icon in the status bar should disappear indicating that communication has been successfully reestablished FLEX Icon Ready FLEX Icon No Communicati
25. rack possibly due to imperfectly molded tips e After transfer an unreleased tip may remain on the nose cone and be jammed against one of the lower drawers or into another tip during a subsequent transfer operation e Ifthe tips are accidentally released from the nose cones during operation they may fall outside of the rack e f the power fails during a pipetting step serious damage can result to the instrument if appropriate corrective steps are not taken As can be seen there are several opportunities for mechanical problems within the fluidics module Fortunately these problems will be rare if you understand how to avoid them Furthermore the design of the FlexStation II instrument provides the means for successfully recovering from most problems 6 3 2 Before Using the Instrument Molecular Devices recommends that you disconnect the power and open up the instrument to become familiar with the mechanical parts of the fluidics module Observe that it is possible to manually and always gently move the drawers doors and pipettor head Moving the parts manually is important for recovering from some mechanical problems For further information see 6 2 Opening a Drawer Manually FlexStation Il Operator s Manual Rev D 6 3 Understanding Potential Mechanical Problems 6 3 3 Avoiding Mechanical Problems Following a few simple steps will help avoid many potential problems Make sure the red knob that holds the pipettor he
26. selection of a full rack of tips From those settings SOFT max PRO selects tip and compound columns 1 through 4 for the first transfer The second transfer uses the next four columns 5 through 8 and the third transfer employs the remaining four unused columns 9 through 12 When the number of columns configured in the Wells to Read setting is the same or greater than the number of columns of both Tips and Columns the algorithm fills the Tips and Columns targets as before that is each assay plate column is targeted with a new tip column and new compound column until all tips and columns have been targeted SOFTmax PRO does not fill beyond the limitations of the available Tips and Compound columns Once all available tips and compound columns have been assigned SOFTmax PRO simply stops assigning targets This setting window displays one two or three transfers with blue pink and green color coded tips to match the setting made in the Compound Transfer window and two pop up menus Tips Column and Compound Column e The Tips Column menu displays a sequence of numbers that match the selected columns in the Pipette Tips tab e The Compound Column menu displays numbers from 1 through the number of columns of the compound plate or the number of troughs selected in the Compound Source tab A Tips or Compound row displays 12 columns to represent the number of columns in a 96 well assay plate The actual number of columns and their placemen
27. the pipette height accordingly e Rate 1 8 This setting determines the rate at which the fluid is dispensed into the well of the assay microplate A setting of 1 is equal to 16 pL second and each subsequent number increases in increments of 16 microliters Therefore a setting of 2 is equal to 32 pL second etc A setting of 3 or 4 may help minimize cell damage or dislodgment Note For non contact dispensing use a rate of 8 to ensure all liquid is dispensed from pipette tip e Volume This setting determines the volume of material to be dispensed from the source to each individual well chosen to receive that transfer For a 96 well microplate the range is 1 200 pL For a 384 well microplate the range is 1 30 pL Important Keep in mind the maximum total volume each well can hold as you accumulate volumes with multiple transfers FlexStation Operator s Manual Rev D 65 3 Operating Procedures 66 e Time Point Minimum Time to 9999 seconds This setting determines the time after the start of the reading when the fluid is scheduled to begin being dispensed Your time point will eventually be your baseline time This is not the time interval between transfers Important The Time Point cannot be smaller than the Minimum Time identified by the software for each transfer Minimum Time This information line automatically displays the minimum time required before a pipetting event can occur This minimum time value is cumu
28. the power switch and disconnect the power cord from the main power source before performing any maintenance procedure that requires removal of any panel cover or disassembly of any interior instrument component FlexStation Operator s Manual Rev D 87 5 Service and Maintenance Procedures 5 2 Moving the Instrument If you need to relocate the FlexStation II instrument follow these steps Warning The FlexStation ll instrument weighs approximately 55 pounds To avoid injury it is recommended that two people lift the instrument together using proper lifting techniques Ensure that the new location meets the proper specifications as described in section 2 Installation Remove any microplates and adapters if any reservoirs and any tips from all three drawers and then close the drawers Caution Remove all plates and adapters before moving the instrument Anything left in the instrument could come loose and jam inside the instrument causing damage Be sure reading chamber screws are still in place Contact Technical Service if reading chamber screws have been removed Be sure all covers are closed and securely fastened Turn off the power switch and unplug the power cord from the source and from the receptacle on the back of the instrument Disconnect the instrument from the computer and any printer Remove the fluidics module For related instructions see section 2 3 Installing the Fluidics Module Depending
29. the temperature 49 50 starting up the 45 48 system components See components system system description 1 30 components system 6 17 introduction 1 2 overview of operation 18 overview of operator s manual 3 safety information 4 5 theory of operation 19 20 FlexStation Il Operator s Manual Rev D T technical support 87 Temp on off key 48 49 temperature display on control panel 49 indicator 48 regulation 21 setting 48 49 50 Temperature display field 29 51 Template Editor 74 Template icon 30 theory of operation 19 20 time elapsed in current run 77 time tagged data 22 timing 54 for each operational mode 25 setting 58 tip rack drawer 9 10 68 101 inspecting 105 opening and closing 48 tip rack key 48 top reading 17 22 23 25 Transfer button 65 triturate 25 54 selecting 67 troubleshooting procedures 97 117 assessing mechanical problems 103 expelling fluid from tips 111 fluidics module inspecting inside 106 8 fluidics module tilting or removing 115 17 FlexStation Operator s Manual Rev D 7 4 Index general error messages 113 14 mechanical problems Flex mode 102 12 opening drawers manually 99 opening the instrument 104 pipettor head removing 109 10 problems during startup 97 117 recovery procedure 112 tip rack evaluating problems with 105 understanding mechanical problems 100 101 U Untitled window 28 Flex mode 53 new
30. to replace the fuses Spare fuses are shipped with the instrument If you no longer have spare fuses you may obtain new ones from Molecular Devices part number 4600 0029 or from a local hardware store Make sure fuses are rated slowblow 6 3 amp Use the following procedure for replacing the fuses Disconnect power to the instrument a Turn off power to the instrument Remove the power cord from the outlet and from the instrument power cord receptacle b Remove the printer cable and computer cable if connected from the back of the instrument 2 Locate the fuse box a Turn the instrument if necessary to access the rear panel b On the left hand side of the rear panel viewed from the back is the power switch fuse box and power cord receptacle Fuse Box Cover WM WOM WM WMTW Fuse Box Location 3 Remove fuse box a Press to the right of the black plastic cover of the fuse box to release it Pull the fuse box cover away from the instrument The fuse box will begin to slide forward b Continue gently pulling the fuse box forward until it is free of the instrument FlexStation II Operator s Manual Rev D 5 6 Replacing Fuses 4 Replace fuse s a Once the fuse holder is out of the instrument you can see that it contains two fuses b Itis possible that only one of the fuses may have blown Molecular Devices recommends that you replace both fuses however to ensure continued proper operation P
31. use Auto Cutoff Excitation Emission M Auto Cuttoff m Bes v Ese v Selecting more than one wavelength expands the settings box to a maximum of four wavelengths Wavelengths BiNumber of Wavelengths i Excitation Emissrerr Auto Cutoff om fos Tr Ee Jr Do 07 ne fs Ie fee 09 sins fos Be Sa Default settings or previously specified wavelengths will be displayed You can choose a wavelength from the common choices in the pull down menu or simply type over an existing setting You can choose any wavelength within available range Scanning monochromator wavelength ranges e Excitation A range 250 850 nm e Emission range 360 850 nm Emission Cutoff Filter Settings Select an emission cutoff filter that will block as much of the residual excitation light as possible without unduly reducing the fluorescence signal The cutoff wavelength options are 420 435 475 495 515 530 550 570 590 610 630 665 or 695 nm You can also select None if you don t want any cutoff The cutoff value should be near the maximum emission wavelength preferably between the excitation wavelength and the maximal emission wavelength but at least 10 nm greater than the excitation wavelength Flex mode offers 14 emission cutoff filters The nominal value of a cutoff filter is the wavelength at which the filter transmission is 50 Above that wavelength transmission is nearly 0 In general you should choose a
32. 1 4 6 Accessories e 1 4 7 Consumables Instrument Rear View 6 FlexStation Il Operator s Manual Rev D 1 4 System Components 1 4 1 Covers and Instrument Panels Top Cover The instrument is protected by a molded plastic housing The large top cover protects the fluidics module and the exposed portions of the detection module Note The top cover can be lifted back as shown in the figure below for certain limited troubleshooting procedures See section 6 4 2 Opening the Instrument Important To achieve optimal performance during readings you must operate the system with the top cover in place Lifting Off the Top Cover Control Panel The control panel consists of an LCD and six pressure sensitive membrane keys which can be used to initiate and regulate the temperature and to open close the drawers A 2 x 3 character liquid crystal display LCD shows the current instrument temperature at all times and the set point temperature when the incubator is on Note See sections 3 2 Starting Up the System and 3 3 Setting the Temperature for information about using the control panel actual C Set Temp Drawers compounds reading chamber Control Panel FlexStation Operator s Manual Rev D 7 1 System Description Input Output Panels There are two input output panels on the rear of the instrument e The upper input output panel on the back co
33. 4 4 Inspecting Inside the Fluidics Module 00 000 000 106 6 4 5 Removing the Pipettor Head 2s don bu above et arb eR URS 109 6 4 6 Expelling Undispensed Fluid from Tips 0 a aaan lesse 111 OUT Recovery Proced EE cusa cl ar ton d Sas t d adea Sa pet c dodo 112 6 5 General Error Messages lll les 113 6 6 Tilting or Removing the Fluidics Module 115 6 6 1 Tilting the Fluidics Module cu s 8 a9 09 at V rod atre tee bed ad 116 6 6 2 Removing the Fluidics Modules ua ads cute ep ote erae Rok x a 117 7 Appentdiees ooo eode Se ole Wied db sce eue eme e dee 119 7 1 Parts and Accessories illl il eA 119 7 2 Performance Specifications 0000000 2 ll llle 120 Fas Warranty ena ev Um re x dene er o e p us de Ae orb dg en 122 A4 Index urs bet ed bbe LES dled AA Ard As dut dri eue dt 123 FlexStation Operator s Manual Rev D v FlexStation Operator s Manual Rev D vi 1 System Description Part 1 of this manual provides background information on the system including descriptions of the principal components and overviews of how the system functions It is divided into the following sections e 1 1 Introduction e 1 2 Operator s Manual Overview e 1 3 Safety Information e 1 4 System Components e 1 5 Overview of Operation e 1 6 Theory of Operation e 1 7 Introduction to SOFTmax PRO Application Software 1 1 Introduction FlexSta
34. 46 Plate Section 47 user interface 28 30 WwW watranty statement 122 wavelengths 25 54 selecting 55 well scan editor 25 Well Scan mode 26 78 wells to read 54 for each operational mode 25 selecting 61 62 127 7 Appendices 128 FlexStation Il Operator s Manual Rev D
35. 8 00 497 00 493 00 434 00 499 00 Scan Figure 4 5 Comments a In endpoint or kinetic fluorescence modes the Autofilter feature will generally select the same cutoff filter wavelength as will the above optimization method If desired however you may specify the cutoff filters manually For emission wavelengths where there is no cutoff filter less than 420 nm or greater than 695 nm experimental iteration is usually the best method of determining the optimal emission and excitation wavelengths Begin optimization by performing steps 1 3 above For excitation wavelengths greater than 695 nm use the 695 emission cutoff filter Note that the emission optics block all wavelengths below 360 nm FlexStation Operator s Manual Rev D 4 Applications Keep your Application Notes and Protocols in this part of your Operator s Manual for handy reference Protocols Stay up to date with Molecular Devices FlexStation II Application Notes and Protocols You can locate these on the company s web site www moldev com Data Management See the SOFTmax PRO User s Manual for all information regarding data management FlexStation Operator s Manual Rev D 85 4 Applications 86 FlexStation Il Operator s Manual Rev D 5 Service and Maintenance Procedures Part 5 provides procedures for regular maintenance that the FlexStation II instrument will need as well as instructions for contacting Technical Support e 5 1 Technic
36. 85 assay plate 25 54 loading 75 selecting 60 AutoCalibrate 25 54 59 Automix 25 54 button 29 function 21 setting 59 AutoRead 25 48 54 settings for 72 B bottom reading 15 17 22 23 25 buttons Automix 29 Compounds 42 89 Drawer 29 FLEX 52 Incubator 29 50 Read 29 76 77 Reading Chamber 42 89 Setup 30 52 Stop 29 77 Transfer 65 C cables 16 90 109 119 cleaning the instrument 88 components system 6 17 accessories 16 computer 15 FlexStation Operator s Manual Rev D 7 4 Index consumables 17 covers 7 8 detection module 13 14 drawers 9 10 fluidics module 11 12 optical system 14 15 panels 7 8 pipettor head 12 compound and tip columns 25 54 69 71 compound baseplate 16 installing 42 use of 89 compound plate 89 for each operational mode 25 loading 75 selecting 63 compound plate drawer 9 10 89 opening and closing 48 compound source 54 compound transfer 54 58 for each operational mode 25 settings 64 66 settings window 67 69 71 Compounds button 42 89 compounds key 48 computer 1 32 minimum configuration 15 setting up 40 starting up 46 computer cable 16 51 90 109 119 connecting 41 control panel 7 Compounds button 89 temperature display 45 using 48 49 using to load microplates 75 using to turn on incubator 44 covers 7 8 fuse 90 123 7 Appendices 124 lamp 8 92 93 95 removing shipping screw
37. Compound Transfer setting works in conjunction with the Compound amp Tip Columns setting described below Until well assignments are made in the Compounds amp Columns setting the Compound Transfer setting will show No targets assigned FlexStation Operator s Manual Rev D 3 5 Configuring Instrument Settings in Flex Mode 3 5 10 Triturate Selection Note The choices shown in this section are based on the number of compound transfers you specified in the previous settings window If no transfers are enabled no triturate settings are applicable Trituration is mixing of the contents of the wells in either or both the compound source and assay plates Trituration is accomplished by fluid being alternately aspirated from and dispensed back into a well e First select the transfer if more than one is enabled during which you wish to perform trituration e Then click in the box to select either Compound Source or Assay Plate or both e You can set the Volume of fluid to be withdrawn from the well and the number of times Cycles it will be aspirated and dispensed back into that well e In addition for assay plates you must also enter a value for the Height at which the trituration occurs The height setting should take into account the volume selected so that the tips remain below the liquid surface and do not draw air BN vi Y Transfer 1 Transfer 2 Transfer 3 pM Compound Source pV Assay Plate vuns
38. D 7 4 Index setting the temperature 49 50 setting up the software 51 53 shutdown 78 starting up the system 45 48 optical system 14 15 22 overview of operation 18 44 overview of operator s manual 3 P panels 7 8 104 parallel port 8 parts replaceable 119 performance specifications 121 photomultiplier tube 57 sensitivity 25 setting voltage 79 pipette tip rack 16 17 loading 44 pipette tips 17 air gap 54 72 expelling fluid from 111 layout 54 68 setting layout for each operational mode 25 pipettor head 12 16 installing 37 40 removal 109 10 Plate icon 30 77 Plate Section screen 62 73 Plate Section tool bar 30 Plate sections 77 creating and editing 53 selecting 76 power cord 8 16 109 119 connecting 41 power failure 101 power switch 8 45 Preferences dialog window 51 printer cable 90 Printer icon 30 procedures installation See installation procedures maintenance See service and maintenance procedures operating See operating procedures service See service and maintenance procedures 125 7 Appendices 126 troubleshooting See troubleshooting procedures protocols 85 R Read button 29 76 77 read modes 78 reading chamber 13 Reading Chamber button 42 89 reading chamber drawer 9 10 13 cleaning 88 installing microplate adapter 89 opening and closing 48 reading chamber key 48 readings per well for fluorescence assays 79 recovery procedur
39. FlexStation Il and FlexStation II Benchtop Scanning Fluorometer and Integrated Fluid Transfer Workstation Operator s Manual LA Molecular Devices PN 0112 0077 Rev D Revision History Date Revision Version Description 03 01 A 1 0 Initial release 05 01 B 1 1 Text changes 08 01 C 1 2 Text changes 03 03 D 1 3 Text changes Molecular Devices Corporation Sales Offices 1311 Orleans Drive USA Canada 800 635 5577 Sunnyvale CA 94089 USA UK 44 118 944 8000 Tel 408 747 1700 Germany 49 89 9620 2340 Online www moldev com Japan 81 797 32 2877 Copyright Copyright 2003 Molecular Devices Corporation All rights reserved No part of this publication may be reproduced transmitted transcribed stored in a retrieval system or translated into any language or computer language in any form or by any means electronic mechanical magnetic optical chemical manual or otherwise without the prior written permission of Molecular Devices Corporation 1311 Orleans Drive Sunnyvale California 94089 United States of America Patents The FlexStation instrument and methods have US and International patents pending Trademarks SPECTRAplate and AUTOMIX are trademarks and FlexStation and SOFTmax are registered trademarks of Molecular Devices Corporation Cliniplate is a registered trademark of Labsystems DELFIA is a registered trademark of Wallac Oy Emerald II is a trademark of Tropix Inc All other company and pr
40. Installing the Pipettor Head for a more complete version of these next steps with illustrations 1 Slide the pipettor head back under its mounting plate inside the fluidics module making sure to secure the white spiral cord back on its retaining hook 2 Screw the red knob retainer nut to secure the pipettor head Make sure the knob is as tight as possible 3 Replace the inner front panel and lower the cover back onto the instrument 4 Reattach the power cord to the instrument and reconnect the instrument to the computer 5 Turn the power on the to instrument It will take about two minutes for the instrument to complete its startup sequence 6 Save your SOFTmax PRO file if necessary to prevent loss of data then close and restart the software FlexStation Operator s Manual Rev D 111 6 Troubleshooting Procedures 112 6 4 7 Recovery Procedure When there is an instrument malfunction in which fluid was left in the tips rebooting the instrument does not automatically reset the fluidics module An additional recover cycle is necessary to restore successful communication between the instrument and the computer This procedure is a precautionary step designed to minimize the potentially very serious consequences of liquid spillage in the instrument Observe instrument display After the instrument completes the restart sequence booting up you will observe that the temperature display on the front panel of the inst
41. Layout Aspirated fluid will then be dispensed to the first available column indicated in the Wells to Read selection The software assumes e All columns in the Wells to Read selection should receive fluid e The fluids are transferred from left to right the read transfer read sequence in each column is initiated only after the previous column s read transfer read event is completed the total read time for that column e The fluid transfer targets are cumulative from transfer to transfer that is the second transfer s targets start with the next available clean tip and untargeted compound column rather than reusing tips and compound columns targeted by the preceding fluid transfer and e Each fluid transfer will use a new tip The software assumes that all of the columns in the Wells to Read area will receive fluid during the initial transfer The Tips target grid will be filled left to right starting with the first available tip column and incrementing the Tip target by one until all columns in the Wells to Read selection have been filled FlexStation Operator s Manual Rev D 69 3 Operating Procedures 70 Compound amp Tip Columns Compound amp Tips Columns Window with Three Transfers In the illustration above the instrument is configured for three fluid transfers as follows e Wells to Read selection of columns 1 through 4 e Compound Source selection of a 12 column compound plate e Pipette Tips Layout
42. OVER UNTIL POWER IS DISCONNECTED FOR TRAINED SERVICE PERSONNEL ONLY Do not operate instrument unless all covers are in place 1 Remove pipettor head from its corrugated plastic carton 2 Open the inside top panel of the instrument Turn the quarter turn fastener on the inside top cover and unfold the cover off the fluidics module to the right Opening Inside Top Cover 3 Attach round connector a Hold the pipettor head in your left hand and the round black 14 pin connector in your right hand b Move the pipettor head into its approximate position under the z stage plate and red mounting knob c Maneuver the cable down toward the back of the cavity and align the connector over the receptacle d Press the connector in place and screw down the black outer collar over the pins FlexStation Operator s Manual Rev D 37 2 Installation Procedures Pipettor Head Attaching Connector e Press the first four loops of white spiral cord onto the coil hook in the upper left corner of the top panel opening This secures the cable up out of the way of the pipettor head when it moves about in the fluidics module during operation Securing Spiral Cord to Hook 38 FlexStation Il Operator s Manual Rev D 2 4 Installing the Pipettor Head 4 Attach the pipettor head a With one hand pull up on the red knob b With the other hand align the metal plate at the back of th
43. Switch on instrument power a Locate the power switch for the instrument as shown in the figure below b Press the rocker switch to the ON position I Fuse Box Cover Power Switch WM WOM WMI WM Parallel Port not currently in use di ees Power Cord RS 222 Serial Cable Power Cord Receptacle RS 232 Serial Port Power Switch Location The instrument automatically performs diagnostic checks to ensure that it is functioning correctly All three drawers open and shut After about four minutes the control panel should display the temperature inside the reading chamber The reading chamber drawer automatically opens After about five minutes the instrument will be warmed up and ready Note There will be no set point temperature displayed at this point because the incubator has not been turned on actual C Set Temp Drawers tip rack compounds reading set pt C chamber Control Panel Ready FlexStation Operator s Manual Rev D 45 3 Operating Procedures 2 Switch on host computer and click on the SOFTmax PRO icon gue a Turn on the host computer and allow Windows to start up Ws b Click on the SOFTmax PRO icon to start the program 3 View Untitled SOFTmax PRO window Note If you get an error message while the software is starting up see part 6 Troubleshooting Procedures After the SOFT max PRO screen appears an Untitled window appears File Edi View Experiment
44. Typea value in the box on the right to indicate how long shaking should last Automix time can last from 1 to 999 seconds TAY Grp i V Before First Read secs Between Reads secs 1 Automix Settings 3 5 5 AutoCalibrate This checkbox allows you to disable or enable automatic calibration The default is enabled Turn autocalibration off to allow the instrument to begin or complete readings more quickly Important Allow the instrument to perform an autocalibration of at least one plate before you disable this function Note The instrument maintains the most recent autocalibration settings in memory NVRAM until another autocalibration is performed IV AutoCalibrate AutoCalibrate Selection FlexStation Operator s Manual Rev D 59 3 Operating Procedures 60 3 5 6 Assay Plate Type You can select the type of assay plate you want to use from the following menu Select an assay plate type 96 well plates 96 Well Standard 96 Vell Greiner blk clrbtm 96 Well Greiner clear 96 Well Costar blk clrbtm 96 Well Costar clear 96 Well Falcon clear Assay Plate Types Be sure to select a plate type that matches the type and well configuration of the actual microplate you will be using Plates do vary particularly with regard to well bottom height Note If you change the Assay Plate Type setting within a Plate section the we
45. ad 96 wells Bottom Read 384 wells Top Read 384 wells Bottom Read Time Resolved Fluorescence 384 wells Calibration Luminescence Photometric Sensitivity Wavelength Range General Photometric Microplate Formats Light Source Lamp Life Detector Read Time Single Column Read Frequency 96 wells Single Well Read Time Frequency Detection Spot Size Top Read Detection Spot Size Bottom Read Shaker Time Temperature control reading chamber SOFTmax PRO Software System Validation Fluidics 8 channel pipettor Volume Range Dispensing Precision Maximum Dispense Rate Fluidics 16 channel pipettor Volume Range Dispensing Precision Maximum Dispense Rate 250 850 nm 360 850 nm 9 nm 18 nm Scanning monochromator tunable in 1 nm increments 2nm 3 fmol well FITC 8 fmol well FITC 3 fmol well FITC 6 fmol well FITC 0 5 fmol well Eu Ch Self calibrating with built in fluorescence calibrators 10 amol ALKPhos 360 850 nm 6 12 24 48 96 384 Xenon flash lamp 1 joule flash user replaceable Two years normal operation one year warranty Photomultiplier R 3896 9 sec 96 wells 29 sec 384 wells measurement type may extend read time 1Hz 20Hz 3 5mm 1 5mm 0 999 seconds From 1 C above ambient to 37 C in 30 min Windows 95 98 NT 2000 compliant Internal standards for fluorescence and wavelength 1 200 uL 8 CV at 5 uL 2 CV at 50 uL 208 uL sec 1 30 uL 396 CV at 10 uL
46. ad in place is securely tightened Only use tips specified for the FlexStation II instrument in a 96 or 384 rack configuration 96 Rack Configuration 384 Rack Configuration Clear tips MDC PN 9000 0623 Clear tips MDC PN 9000 0512 Make sure you maintain appropriate well formats i e 384 rack tips with 384 compound plate and 384 assay microplate Remove the lid of the tip rack before placing it in the tip rack drawer Make sure you seat the tip rack plates and troughs if applicable securely in the correct drawers Make sure that the layout of the tips that you specify in your SOFTmax PRO protocol accurately represents what you have in the in the tip rack Make sure that the compound and assay plates that you specify in your SOFT max PRO protocol accurately represent microplates you are actually using Note The software has no way of confirming the presence absence or configuration of tips or plates in the instrument drawers 6 3 4 In Case of Power Failure Interacting properly with the instrument following a power failure is very important If there is a power failure while the instrument is running you must manually turn off the power switch at the back of the instrument Follow the instructions in the next section to avoid instrument damage FlexStation Operator s Manual Rev D 101 6 Troubleshooting Procedures 6 4 Recovering from Mechanical Problems in Flex Mode 102 The procedures in this section
47. al Support e 5 2 Moving the Instrument e 5 3 Cleaning the Instrument e 5 4 Using the Microplate Adapters e 5 5 Using the Compound Baseplate e 5 6 Replacing Fuses e 5 7 Replacing the Flash Lamp e 5 8 Long Term Shutdown Service Trained Users Whenever the following warning message appears a service trained user must perform the procedure to ensure user safety and to prevent instrument damage Example Warning The following procedures must be completed by a service trained user Do not attempt the following procedures if you have not been trained properly by appropriate Molecular Devices Personnel 5 1 Technical Support Molecular Devices Corporation is a leading worldwide manufacturer and distributor of analytical instrumentation We are committed to the quality of our products and to fully supporting our customers with the highest possible level of technical service In order to fully benefit from our technical services please complete the registration warranty card and return it to the address printed on the card If you have any problems using the FlexStation II instrument in the United States contact the Technical Services group at 1 800 635 5577 elsewhere contact your local representative Warning All maintenance procedures described in this manual can be safely performed by qualified personnel Maintenance not covered in this manual should be performed only by a Molecular Devices representative Warning Turn off
48. al volume in the Compound Transfer settings window that will be shown as a gray fill As the liquid volumes are cumulative the first event s volume is shown above the initial liquid volume if any the second event s volume will be shown above the first and the third event s volume above the second To deselect a tip or compound assignment first select the appropriate cell s and then press Backspace on the computer keyboard To change an assignment select the well s and choose new values or type a value on the keyboard Important If you have multiple wells selected and you type a value that value will be shown in the first selected well and subsequent wells will increment to the next higher value Again this does not take into account the actual pipette tips shown to be available in the Pipette Tips Layout selection FlexStation Operator s Manual Rev D 71 3 Operating Procedures 3 5 13 AutoRead The AutoRead feature allows automatic readings of subsequent Plate sections in the order in which they appear in the experiment Click in the check box to enable AutoRead You can set an interval between automatic plate readings by typing a number in the Delay box v Turn autoread on Delay 5 sees Setting AutoRead Delay 3 5 14 Pipette Tip Air Gap To set the pipette tip air gap the volume between the end of the pipette tip and the bottom of the liquid in the tip select Select Air Gap from the Control menu to re
49. are intended for cases in which tips have jammed or the power fails while you are using the instrument in Flex mode and you see an error message on the computer screen indicating a problem with the instrument You may need to open the instrument in order to locate and correct the problem The procedures in this section include e 6 4 1 Assessing a Mechanical Problem e 6 42 Opening the Instrument e 6 4 3 Evaluating the Tip Rack e 6 4 4 Inspecting Inside the Fluidics Module e 6 4 5 Removing the Pipettor Head e 6 4 6 Expelling Undispensed Fluid from Tips Warning Always make sure the power switch on the instrument is in the OFF position and remove the power cord from the back of the instrument prior to removing any interior panels Caution Do not touch or loosen any screws or parts other than those specifically designated in these instructions Doing so may cause misalignment and will void the instrument warranty FlexStation Il Operator s Manual Rev D 6 4 Recovering from Mechanical Problems in Flex Mode 6 4 1 Assessing a Mechanical Problem Depending on when the problem occurs during the fluidics operation one of several error messages may appear in SOFT max PRO The error message may not provide complete information about what has occurred The software cannot confirm presence absence or exact position of tips and or plates Whenever you experience a mechanical failure of the fluidics operation follow the entire
50. at Maximum 505 00 508 00 506 00 506 00 Reduction J Show Reduced ScaleTo pata Scan Figure 2 FlexStation Operator s Manual Rev D 81 3 Operating Procedures 3 Emission Scan with Emission Autofilter Scan Figure 3 When you use Autofilter in Endpoint mode for the emission cutoff filter a filter is automatically selected that will block as much of the residual excitation light as possible without unduly reducing the fluorescence signal In Spectrum mode the default is no filter You can determine the optimum filter as follows a 82 First select an emission filter that is at least 20 nm larger than the selected excitation wavelength Complete emission scan from the filter wavelength over a range of 50 nm to 100 nm larger Compare signal to buffer background in the scan and choose the wavelength with the maximum ration You may want to repeat the scan with the next larger emission filter and compare results An example is shown in the Scan Figure 3 Note that the optimal emission wavelength is 525 nm 1 EM scan with 515 cutoff Well Graph Raw Oy x 1400 1200 1000 7 RFU 570 580 590 500 500 510 520 530 540 550 560 Em Wavelength in nm Ex 485nm E6 501 00 523 00 o c6 A DB 500 00 500 00 Well o B6 Lambda at Maximum Show Reduced Scale To Data Scan Figure 3 FlexStation Operator s Manual Rev D 3 10 Optimizing Fluorescence Assays in
51. ation scan to find peak excitation wavelength A e Preliminary emission scan to find peak emission wavelength A e Emission scan with Autofilter function selected to optimize signal to background ratio e Optional excitation scan with both Autofilters functioning to further optimize signal to background ratio This process requires samples in the microplate that contain moderate concentrations of the fluorophore and samples representative of the background i e buffer without fluorophore FlexStation Operator s Manual Rev D 79 3 Operating Procedures 80 1 Preliminary Excitation Scan Scan Figure 1 a Using SOFTmax PRO set up a Plate section for a fluorescence read spectrum mode Em Fixed Ex Scan with no cutoff filter default and medium PMT Set emission wavelength based on the tentative value from the literature or from a customary filter set used to measure your fluorophore and add 30 nm If the emission wavelength is not known select a tentative emission wavelength about 50 nanometers greater than the absorbance maximum of the fluorophore If necessary the absorbance maximum can be determined by performing a spectral scan in a UV Vis spectrophotometer i Prelim EX scan Well Graph Raw ioj x 14000 12000 10000 250 270 290 310 330 350 370 390 410 430 450 470 490 Ex Wavelength in nm Em 525nm Well o D6 n E6 A FB o GB Lambda at Maximum 500 00 500 00 500 00 286 00 eo reb Scan
52. atistical data analysis The system can perform a variety of fluorescence applications as well as some limited time resolved fluorescence and luminescence operations The system s extreme flexibility and high sensitivity make it appropriate for applications in biochemistry cell biology immunology molecular biology and microbiology The instrument uses two holographic diffraction grating monochromators that allow for individual optimization of both excitation and emission wavelengths The dual scanning capability can also be used to determine excitation and emission settings for new fluorescent probes Mirrored FlexStation Operator s Manual Rev D 1 1 System Description optics focus the light into the sample and cutoff filters reduce stray light and minimize background interference The light source is a high power xenon flash lamp The system provides additional flexibility by allowing a variable number of lamp flashes per reading The temperature in the microplate reading chamber is isothermal both at ambient and when the incubator is turned on When the incubator is on the system controls the incubator temperature from 1 C above ambient to 45 C The system operates in one integrated fluidics read mode and four read only modes Using the Automix feature the contents of the wells in a microplate can be mixed automatically by shaking which makes it possible to perform kinetic analysis of solid phase enzyme mediated rea
53. ble a pipetting function from a tip that is not present or if you enable more or fewer tips than are actually available the instrument can malfunction potentially causing serious damage Pipette Tips Layout Settings The dimension of this layout will be determined by the Assay Plate Type you select Highlight the tips you want use with the cursor Tips must be contiguous but do not need to start in the 1 column You cannot choose a partial row or column or a single well Important Selected tips must be contiguous FlexStation Operator s Manual Rev D 3 5 Configuring Instrument Settings in Flex Mode 3 5 12 Compound and Tip Columns The choices in this window depend upon the number of transfers chosen in the Compound Transfer window When one or more transfers are enabled these settings allow you to choose the tips and compounds that will be used for transfers The settings in this window are either automatically assigned by the software or you can manually assign them into any configuration you require gt Compound amp Tip Columns Automatically Assigned SOFTmax PRO automatically enters information in the Compound amp Tip Columns window taking into consideration the number of transfers the Wells to Read section and the Pipette Tips Layout selected area already selected The software assumes fluid will be aspirated from the compound plate starting with the first available column indicated in the Pipette Tip
54. ck the temperature or turn on the incubator View the Control Panel and note the temperature inside the reading chamber Use the control panel to turn on the incubator if it is required by your experiment It may take a while for the temperature to stabilize so do this before configuring other instrument parameters Note Incubator settings can also be set using SOFTmax PRO Configure instrument settings using SOFTmax PRO Configure the read mode type of analysis template etc as desired Create sections Notes and Plates as needed in the Experiment Section of the Software window Load prepared pipette tip rack and microplates into drawers Load prepared racks and microplates into the drawers Use drawer adapters as needed Begin reading Using SOFT max PRO start the reading Detailed instructions for these and other procedures are located in the following sections of part 3 FlexStation Il Operator s Manual Rev D 3 2 Starting Up the System 3 2 Starting Up the System Normally you do not need to switch off power at the end of the day If the system will not be used for more than a day it is best to turn off the instrument Use the following procedure only if the system has been switched off Note Refer to the SOFTmax PRO operator s manual for software installation and registration instructions These instructions assume the software is completely loaded on your computer and you are ready to begin an experiment 1
55. cover and reposition the tip rack in the carriage e Replace the inside front panel and top cover Reattach the power cord and computer cable to the instrument and reboot the instrument e Save your SOFTmax PRO file to prevent loss of data then close and restart the software Important Failure to restart SOFTmax PRO may result in communication problems between the instrument and computer 3 Tip rack is improperly positioned e f the tip rack is jammed because it was not positioned securely in the drawer pull the carriage out of the instrument to access the rack e fall the tips are accounted for reposition the rack in the carriage e Replace the inside front panel and top cover Reattach the power cord and computer cable to the instrument and reboot the instrument e Save your SOFTmax PRO file to prevent loss of data then close and restart the software Important Failure to restart SOFTmax PRO may result in communication problems between the instrument and computer 4 Tips are not seated properly e Ifthe tip rack is jammed because one or more tips are not seated properly in the rack and preventing the drawer from opening you may be able to remove the rack and problematic tips from outside the fluidics module by pulling the tip rack all the way out f not gently push the tip rack back inside the instrument Reach in from the front and remove the tip rack by lifting it up and out toward you Be careful not to spi
56. ctions such as a kinetic ELISA In Flex mode the pipettor can also be used to mix the contents of the compound plate or the assay plate a procedure called trituration FlexStation II Instrument 2 FlexStation Operator s Manual Rev D 1 2 Operator s Manual Overview 1 2 Operator s Manual Overview This manual was written to ensure safe and proper use of the system Before use read this manual carefully in order to realize the full capabilities of the system Also if something is unclear during daily use or if a problem occurs please refer to this manual This manual is organized as follows Part 1 System Description provides background information on the system including descriptions of the principal components and overviews of how the system functions Part 2 Installation Procedures provide instructions for assembling the instrument and installing the SOFTmax PRO application on your Windows based computer Part 3 Operating Procedures provides instructions for operating the system with the SOFTmax PRO application software in Flex fluidics read mode Part 4 Applications provides a space for detailed protocols for several FlexStation II mode experiments Part 5 Service and Maintenance Procedures provide instructions for routine mainte nance including replacing the lamp changing the pipettor head moving the system to a new location and minor cleaning Part 6 Troubleshooting Procedure
57. d Maintenance Procedures 87 5 1 Technical Support liiiiiisllllll lll ll ee 87 5 2 Moving the Instrument liii lll e e es 88 5 3 Cleaning the Instrument illl lll ese 88 5 4 Using the Microplate Adapters lll ll es 89 5 5 Using the Compound Baseplate lll less 89 5 6 Replacing Fuses lil ll ee ees 90 5 7 Replacing the FlashLamp 0 lees 92 5 8 Long Term Shutdown 00 ol ll LLL LL ses 95 FlexStation Il Operator s Manual Rev D Contents 6 Troubleshooting Procedures 0 0 97 6 1 Problems During Startup 0 0 000000 lll les 97 6 2 Opening a Drawer Manually 0 0 0 0 0 0 0000002 ee 99 6 3 Understanding Potential Mechanical Problems 100 Ooi Backgr nd o2 c ieee ARE RA CI eye ede s eh a eea 100 6 3 2 Before Using the Instrument 2m eed ete pro een sess Ama ida 100 6 3 3 Avoiding Mechanical Problems eror oe Saws vs 101 6 3 4 In Case of Power EAllute ciis 8 der a bgahAvddPan onze e pedes eda dus 101 6 4 Recovering from Mechanical Problems in Flex Mode 102 6 4 1 Assessing a Mechanical Problem i o iov ee eae cem b eet br 103 6 4 2 Opening the Instrument 3553 e ye eT POIL ooh 2peRPIT 104 6 4 3 Evaluating the Tip Rack to tete tee esce Nu be a Led d edd 105 6
58. d the back Row A 4 Lift the front Row H of the adapter and remove it from the drawer 5 5 Using the Compound Baseplate To use the compound baseplate place the metal baseplate into the bottom of the compound plate drawer under the compound plate 1 Turn power to the instrument on 2 Press the Compounds button on the control panel or select Open Compound Drawer from the Control menu 3 Lower the compound baseplate into the compound drawer with its cutout corner facing the front left corner of the drawer Remove the compound baseplate in the same way Caution Always remove any plates and adapters from the instrument drawers before moving the instrument or before any service or maintenance procedures Plates and adapters can easily become jammed inside the instrument causing damage FlexStation II Operator s Manual Rev D 89 5 Service and Maintenance Procedures 5 6 Replacing Fuses Fuses burn out occasionally and must be replaced If the instrument does not seem to be getting power after switching it on the LCD shows no display first check to see whether the power cord is securely plugged in to a functioning power outlet and to the receptacle at the rear of the instrument If power failed while the instrument was already on check that the power cord is not loose or disconnected and that power to the power outlet is functioning properly If these checks fail to remedy the loss of power follow the steps listed below
59. de the instrument causing damage For instructions on removing adapters see section 5 4 Using the Microplate Adapters 2 8 Installing SOFTmax PRO Install the SOFTmax PRO software application onto the personal computer according to the instructions in the SOFTmax PRO User s Manual 42 FlexStation Il Operator s Manual Rev D 3 Operating Procedures Part 3 explains how to start up the system and how to use the control panel and the SOFTmax PRO application software to configure instrument settings define experiment protocols and run the analysis as described in the following sections e 3 1 Overview e 3 2 Starting Up the System e 3 3 Setting the Temperature e 3 4 Setting Up the Software e 3 5 Configuring Instrument Settings in Flex Mode e 3 6 Other Software Settings e 3 7 Reading the Microplate e 3 8 Shutdown e 3 9 Other Read Modes e 3 10 Optimizing Fluorescence Assays in Spectrum Mode Note Information in part 3 assumes the instrument and computer are installed and connected properly For further information see part 2 Installation Procedures FlexStation Operator s Manual Rev D 43 3 Operating Procedures 3 1 Overview 44 The following list provides an overview of the basic operating procedures required before using the system Start up the system Turn on the power to the instrument and then the computer if they are not already on Launch the SOFT max PRO application software Che
60. dics module do so now If you need to remove the fluidics module continue with the instructions in the section below Replace the fluidics module Using the handle lower the fluidics module carefully back over the detection module Make sure it is firmly seated Replace cover and reattach cables Lower the top cover over the instrument Reattach the power cord and computer cables Fluidics Module Electrical Connection to Fluidics Module Excitation Fiber and Restraint Emission Fiber and Restraint Tilting the Fluidics Module FlexStation Il Operator s Manual Rev D 6 6 Tilting or Removing the Fluidics Module 6 6 2 Removing the Fluidics Module Caution Use caution during this procedure Follow these instructions exactly and any instruction labels on the instrument TO REMOVE FLUIDICS MODULE 1 Using handle rotate module up 2 Disconnect electrical connection Disconnect restraint 3 4 Loosen 1 4 turn fasteners on bottom of module WHEN REINSTALLING INSURE MODULE IS FIRMLY SEATED Follow the steps in procedure 6 7 1 to step 4 1 Disconnect the fluidics module a Locate the electrical connector between the bottom of the fluidics module and the housing b Unscrew the retaining screws and unplug the connector c Locate the fluidics module restraint a green cable Disconnect the restraint from the housing 2 Release fluidics module from plate a Locate and loosen the quarte
61. dings per well Decrease the reading precision by moving the bar to the left and reducing the number of readings per well Readings per well range 1 to 30 readings per well Typically the default setting is adequate Only in wells with very low signal will you get improved precision by increasing the number of reads per well Note Increasing the number of reads also increases the total read time Ta Readings Normal Precise PMT Sensitivity Sensitivity Settings The photomultiplier tube PMT is a photon detector that detects light through the use of photoemission and successive instances of secondary emission to produce enough electrons to generate a strong signal p7 Readings Fast Normal Precise 22 Automatic i v High m YI PMT Settings Click in the PMT Sensitivity box to display the menu choices Settings are manual and limited to High Medium or Low Set the voltage of the PMT to Low for samples having a higher concentration Choose High PMT sensitivity for samples with lower concentration Note An additional setting Automatic is available only in for Endpoint reads and Spectral scans Automatic is not available for Kinetic or Flex modes FlexStation Operator s Manual Rev D 57 3 Operating Procedures 3 5 3 Timing Important The Timing setting depends on several settings which appear after it in the list You will need to return to Timing
62. e 112 Reduction icon 30 reduction parameters 74 RS 232 port 8 S safety information 4 5 conventions used in manual 4 screens Instrument Settings 52 54 Plate Section 62 73 Preferences dialog 51 Untitled window 28 Untitled window Flex mode 53 Untitled new 46 Untitled Plate Section 47 sensitivity 54 setting 57 serial port 8 51 service and maintenance procedures 87 95 cleaning the instrument 88 long term shutdown 95 moving the instrument 88 replacing the flash lamp 92 95 technical support 87 using microplate adapters 89 using the compound baseplate 89 90 91 set point temperature indicator 48 settings instrument 54 73 assay plate 60 AutoCalibrate 59 Automix 59 AutoRead 72 compound and tip columns 69 71 compound source selection 63 compound transfer 64 66 finishing 54 modifying 54 on Plate Section screen 73 pipette tip air gap 72 pipette tips layout 68 sensitivity 57 timing 58 triturate selection 67 wavelengths 55 56 wells to read 61 62 Setup button 30 52 shipping containers 32 shutdown procedures 78 long term 95 SOFTmax PRO 15 27 installing 42 introduction to 27 30 software settings 51 53 74 specifications performance 121 Spectrum mode 26 78 optimizing fluorescence assays 79 84 starting up the system 45 48 Status bar 29 51 during readings 77 Stop button 29 77 system installing the 31 42 operating procedures 43 84 setting
63. e detection module and can be tilted off to the side to provide access to the optical system for troubleshooting or maintenance The detection module is contained in a molded plastic housing to which the top cover is attached at the back of the instrument Fluidics Module Detection Module Reading Chamber Reading Chamber Drawer with Assay Plate Detection Module Detail Reading Chamber The reading chamber includes the assay plate carriage which holds the assay microplate in the reading chamber during read cycles The reading chamber may be maintained at an elevated temperature It contains both top and bottom read heads which can be selected via software command The instrument utilizes a plate sensor to assure that an assay plate is present in the reading chamber before a reading begins FlexStation Operator s Manual Rev D 13 14 1 System Description Optical System The optical system includes a xenon flash lamp the monochromators excitation bandpass filters emission cut off filters and a PMT detector There are a number of cables and fibers that exit the optics bench and enter the read chamber They are the excitation fiber thin and black has a collar and pins emission fiber fatter with attached electrical cord electrical connector to the read head black with brass fitting Caution Optical fibers are very fragile especially the excitation fiber Handle cables with extreme care
64. e pipettor head with the screw hole and the two locating pins underneath the red knob Slide the plate up into place Alignment Pins Positioning Pipettor Head Screw down the red knob securing the pipettor head so that it hangs in place from the black bar Caution Ensure the red knob the pipettor retaining nut is tightened as firmly as possible The pipettor head is now installed 5 Close the fluidics module Fold the inside top panel back over the pipettor head and lock the quarter turn screw in place FlexStation Operator s Manual Rev D 39 2 Installation Procedures 6 Close the top cover Bring the top cover back over the fluidics module and snap it into place at the handle on the detection module Make sure the latch clicks shut Inside Top Panel Opened Eight or Sixteen Channel Pipettor Head Pipettor Head Installed 2 5 Setting Up the Computer Set up the computer and monitor according to the instructions that come in their P P g packaging Place them close to the instrument on the bench The power cords for the computer and monitor are provided in the computer packaging Connect them to the computer hardware see next section but not to the power outlet at the wall Caution Do not attach the computer to a power outlet until the computer and the instrument are connected 40 FlexStation II Operator s Manual Rev D 2 6 Con
65. e sure all items are present Unpacking the Fluidics Module and Accessories 1 2 3 4 Remove the fluidics module from the protective foam Set in a safe place Remove the box containing the pipettor head and the bags of accessories Open the accessories bags remove cables and the Allen wrench You will need them later in the assembly procedure Set packaging aside gt Tools Needed e Allen wrench hex 3 32 ball drive L provided e Phillips screwdriver not provided Unpacking the Detection Module Important The system should be kept in a location that is dedicated to its use on a level surface away from direct sun light dust drafts vibration and moisture Locate the largest carton which contains the main instrument body detection module It weighs approximately 55 pounds 25 kilograms Place the box either on the floor next to the bench where you intend to install it or on the bench itself Remove shipping clips a Locate the four plastic clips holding the top of the carton in place There is a clip in the bottom center of each box face b First press inward on the two vertical tabs middle of the clip recess FlexStation Il Operator s Manual Rev D 2 2 Unpacking the System c Squeeze the tabs together and pull outward The tabs should pivot up and out and the whole clip should come out 4 Remove the upper shipping carton and the upper foam 5 Open the twist tie at the top of the pla
66. ed window a Observe the Instrument Status icon in the left corner of the Status bar The icon is purple when the SOFT max PRO application correctly recognizes the instrument b Observe the temperature displayed in the Temperature display field File Edi View Experiment Control Assays Notes Window Help EB paz Cet 4 bol EA Instrument Status Bar U D D Important If there is a red X in front of the Instrument Status icon if there is no XD c temperature in the Temperature display field or if you see other problems you may need to adjust instrument preferences See next step 2 Confirm Flex preferences Confirm that the instrument is communicating properly with the computer as part of routine software setup a Select Preferences from the Edit menu The Preferences dialog box appears ma Preferences x Serial Port COM1 v Serial Comm Speed 9600 v i Exp be Autosave File Ee Time ae Plate C Aquarius Spt3 2b1a i IV Include Labels J Time Interpolate File Prefix Data fu Append Date SUFlimay PHO File Text File Autoprint Cancel Preferences Dialog Window b Make sure that the serial port setting agrees with the actual port the computer cable RS 232 cable is connected to This is usually Com1 The serial comm speed should be 9600 Baud Note Once you read an assay plate in Flex mode the serial communication speed will change to 57600 Ba
67. edures 2 7 Installing the Drawer Adapters The drawer adapters include the microplate adapters and compound baseplate Microplate Adapter Installation In order to bottom read a standard 96 or 384 well microplate you need to install the black microplate adapter in the reading chamber drawer The black adapter elevates the plate in the drawer Similarly use the purple microplate adapter for top reading when using 96 or 384 well plates Caution Incorrect insertion or removal of the adapter may cause damage to the microplate drawer 1 Turn power to the instrument on 2 Press the Reading Chamber button on the front panel The reading chamber drawer opens 3 Hold the adapter so that the label is on the left side facing up 4 Place the top back Row A portion of the adapter into the drawer first While pushing against the back edge of the adapter lower the front of the adapter into the drawer Compound Baseplate Installation Place the metal compound baseplate into the bottom of the compound plate drawer 1 Turn on power to the instrument 2 Press the Compounds button on the front panel The compound plate drawer opens 3 Lower the baseplate into the compound drawer with its cutout corner facing the front left corner of the drawer Caution Always remove any plates and adapters from the instrument drawers before moving the instrument or before any service or maintenance procedures Plates and adapters can easily become jammed insi
68. eft hand and use your right hand to connect the 15 pin sub D electrical connector The connector fits into the communication port on the far bottom edge of the fluidics module It must be aligned properly to fit b Press connector in firmly Secure two communication port screws lightly with your fingers FLUIDES CONNECT Note There are labels on both the fluidics connector Fluidics Connector CONNECTOR FLUIDICS HERE and near the communication port Connect Fluidics Here that help identify these parts 4 Lower fluidics module into place Caution Be careful when lowering the fluidics module that you do not trap or compress any of the optical fibers coming up from the detection module a Ensure that all cables and wires are tucked out of the way b Gently lower the fluidics module by the handle down over the detection module and onto the locating pins c Check that the fluidics module sits securely on the detection module Caution When installing the fluidics module ensure module is firmly seated 36 FlexStation Il Operator s Manual Rev D 2 4 Installing the Pipettor Head 2 4 Installing the Pipettor Head The next step in installing the instrument is to place the pipettor head into the fluidics module Use the same procedure for both the 8 channel and 16 channel pipettor heads Caution Use caution during this procedure Follow these instructions and any instruction labels on the instrument exactly DO NOT REMOVE C
69. ement fuses with the required current rating and specification Improper fuses or short circuiting the fuse holders can cause fire or damage the instrument Power rating Ensure the system is connected to a power receptacle that provides voltage and current within the specified rating for the system Use of an incompatible power receptacle can create electrical shock and fire hazards Remove watches and jewelry before removing any panels from the instrument Warning labels There are several labels affixed to the instrument covers and inside panels The purpose of these labels is to alert you to use caution when servicing a component or the instrument Be aware that ignoring the instructions on any instrument label can result in a hazardous condition that can cause injury Identification Labels The following labels among others appear on the instrument T V Rheinland Product Safety FlexStation ll FlexStation I 3 100 240V 5A 50 60Hz FUSE T6 3 AH 100 240V 5A 50 60Hz FUSE T6 3 AH SLO BLO SLO BLO L4 Molecular Devices KJ Molecular Devices 1311 Orleans Drive Sunnyvale 1311 Orleans Drive Sunnyvale CA 94089 408 747 1700 CA 94089 408 747 1700 FlexStation II Operator s Manual Rev D 5 1 System Description 1 4 System Components This section describes the major system components listed below e 1 4 1 Covers and Instrument Panels e 1 42 Drawers e 1 4 3 Fluidics Module e 1 4 4 Detection Module e 1 4 5 Computer e
70. for the compound plate The pipettor head moves vertically between the drawers The fluidics module can be opened by service trained users from the inside front panel if necessary for maintenance or from the inside top panel to install or remove the pipettor head The entire fluidics module can be removed for maintenance or to transport the system to another location For further information see 2 3 Installing the Fluidics Module See part 6 Troubleshooting Procedures for related service procedures Inside Top Panel Inside Front Panel Fluidics Module Detection Module Fluidics Module FlexStation Operator s Manual Rev D 11 1 System Description Pipettor Head The instrument is configured with an eight channel pipettor head for use with 96 well microplates In addition the FlexStation 11584 includes a 16 channel pipettor head for use with 384 well microplates Note For further information see 2 4 Installing the Pipettor Head Inside Top Panel Opened Eight or Sixteen Channel Pipettor Head Pipettor Head 12 FlexStation II Operator s Manual Rev D 1 4 System Components 1 4 4 Detection Module The detection module is the lower portion of the instrument This module houses the reading chamber the optics bench several cables and optic fibers the power supply the flash lamp and other hardware The fluidics module attaches to th
71. g such product The Seller also makes no warranty that the products sold under this agreement are delivered free of the rightful claim of any third party by way of patent infringement or the like If the Buyer furnishes specifications to the Seller the Buyer agrees to hold the Seller harmless against any claim which arises out of compliance with the specifications Any description of the products contained in this agreement is for the sole purpose of identifying them Any such description is not part of the basis of the bargain and does not constitute a warranty that the products shall conform to that description Any sample or model used in connection with this agreement is for illustrative purposes only is not part of the basis of the bargain and is not to be construed as a warranty that the products will conform to the sample or model No affirmation of fact or promise made by the Seller whether or not in this agreement shall constitute a warranty that the products will conform to the affirmation or the promise For the name of the nearest authorized Molecular Devices Corporation service facility please contact Molecular Devices at one of the following telephone numbers 800 635 5577 US and Canada 122 FlexStation Il Operator s Manual Rev D 7 4 Index A accessories 16 part numbers for 119 unpacking 32 adapters microplate 16 installing 89 removing 89 air gap pipette tip See pipette tips air gap application notes
72. g the Microplate 3 7 Reading the Microplate Loading Tips and Microplates Prepare your tip rack compound plate and the assay plate you want to analyze and load them into the instrument Caution Be sure the underside of the assay plate is dry before you place it in the reading chamber drawer Damage to the lower read head can occur from liquids that come into contact with it Biohazard lf the microplate has fluid on the underside dry it using a paper towel or equivalent before placing it in the drawer 1 Open the appropriate drawer Open the drawers by either manually pressing the appropriate drawer buttons on the control panel or by using SOFTmax PRO 2 Insert tips and plates Insert the filled tip rack and plates into the drawers placing well A1 into the upper left corner of the drawer as you look at it e Make sure the compound plate is flat against the compound baseplate e Make sure the assay plate is flat against the black adapter for 96 and 384 well microplates or the drawer bottom for 6 12 24 or 48 well microplates and non Flex applications Note For further information see sections 5 4 Using the Microplate Adapters and 5 5 Using the Compound Baseplate 3 Close drawers Press the buttons on the control panel or use SOFTmax PRO to close the drawers again FlexStation II Operator s Manual Rev D 75 3 Operating Procedures Starting the Reading Important Be sure you have completed all
73. h filter to use or use none or have a filter automatically selected 8 The PMT detects the emitted light and passes a quantitative signal to the instrument s electronics which then send the data to the computer 1 4 5 Computer The FlexStation II instrument works as a system with the SOFTmax PRO application software SOFTmax PRO must be installed on a dedicated personal computer to communicate with and control instrument functions e The instrument is equipped with an 8 pin DIN RS 232 serial port for connecting to a personal computer e SOFTmax PRO software version 4 4 or greater is required to control the FlexStation II instrument e The minimum computer configuration includes a Pentium processor with 256K RAM a 10 GB hard drive CD ROM drive and Windows 98 2000 NT For further information see sections 2 8 Installing SOFTmax PRO and 3 4 Setting Up the Software FlexStation Operator s Manual Rev D 15 1 System Description 1 4 6 Accessories The accessories included with the system are e microplate adapter for use in reading chamber drawer compound baseplate for use in the compound plate drawer computer cable power cord USA Canada power cord ECI fuses 2 each operator s manual pipettor head 8 channel pipettor head 16 channel FlexStation II554 instruments pipette tip rack Allen wrench All necessary accessories are shipped in the fluidics module box For further information see section 7
74. h settings identical to a particular existing plate highlight that plate then select Duplicate from the Edit drop down menu Double click on the Plate word in the Plate Section tool bar to open a dialog box and name your Plate sections Note Refer to SOFTmax PRO user manual for details about this procedure FlexStation Operator s Manual Rev D 53 3 Operating Procedures 3 5 Configuring Instrument Settings in Flex Mode This section explains how to use the Instrument Settings screen see page 52 to configure the protocol for your experiments Protocol parameters for Flex mode are listed below e 3 5 1 Wavelengths e 3 5 9 Compound Transfer e 3 5 2 Sensitivity e 3 5 10 Triturate Selection e 3 5 3 Timing e 3 5 11 Pipette Tips Layout e 3 5 4 Automix e 3 5 12 Compound and Tip Columns e 3 5 5 AutoCalibrate e 3 5 13 AutoRead e 3 5 6 Assay Plate Type e 3 5 14 Pipette Tip Air Gap e 3 5 7 Wells to Read e 3 5 15 Settings Displayed in Plate Sections e 3 5 8 Compound Source Note Parameters marked with an asterisk must correlate See following discussion Important You must define instrument settings before beginning a reading or the instrument will use default settings Settings cannot be changed after collecting data Modifying Settings Modify settings in the order in which they appear in the Instrument Settings screen to avoid missing any settings However the Timing setting depends on several setti
75. her than those specifically designated in the instructions Doing so might cause misalignment and will void the system warranty Caution Do not attempt to assemble or disassemble the instrument with the pipettor tips in place Spillage or damage to the pipettor tips or the instrument can occur FlexStation Operator s Manual Rev D 31 2 Installation Procedures 2 2 Unpacking the System 32 If you are reading this you have already located the Operator s Manual This part provides instructions on how to continue to unpack the system safely Warning The instrument weighs approximately 55 pounds and should be lifted with care It is recommended that two persons lift the instrument together taking the proper precautions to avoid injury Each FlexStation II instrument comes in five specially designed shipping cartons Fluidics module and accessories Detection module main instrument body in housing Personal computer Computer monitor SOFTmax PRO software package Please retain the cartons all boxes and any significant packing materials If the system needs to be moved to a different location use the original packing materials and cartons whenever possible If the cartons have been damaged in transit it is particularly important that you retain them for inspection by the carrier in case there has also been damage to the instrument As you unpack the system pieces examine the packing list that accompanies the system to b
76. ice and Maintenance Procedures 4 Open top cover Open the top cover of the instrument to provide better access and clearance to the lamp s mounting box This will make it easier to remove the lamp assembly 5 Remove flash lamp assembly Slide the lamp assembly horizontally out the side of the instrument and lay it on its heat sink fins leaving the orange electrical cord connected Mounting Block Mounting Screws Spring loaded Removing the Flash Lamp Assembly 6 Withdraw lamp from assembly a Use a 3 32 Allen wrench to remove the two screws that hold down the diamond shaped retainer Be careful not to lose screws Flashlamp Retainer Window Flashlamp Copper Coil Spring Multi pin Socket Withdraw Lamp 94 FlexStation Il Operator s Manual Rev D 5 8 Long Term Shutdown b Withdraw the silver colored lamp from its multi pin socket by gently rocking it and pulling it upward c Notice the location of the copper coil spring and keep it at hand 7 Install new lamp a Remove the new lamp from its packaging be careful not to touch the lamp window Caution Do not touch the window at any time b With the copper coil spring in place around the socket carefully align the lamp pins in the proper orientation according to the pin pattern Again do not touch the window c Install the lamp with a gentle rocking motion and steady downward pressure d Replace the diamond shaped lamp retainer over the new
77. instrument is plugged in and turned on 2 Is instrument still warming up Check to be sure the instrument has finished its warming up sequence Wait until the temperature is displayed on the front panel of the FlexStation II instrument FlexStation Operator s Manual Rev D 97 6 Troubleshooting Procedures 3 Check Preferences Go to Preferences in the Edit drop down menu of the software View the Preferences dialog box W Preferences coM1 v r A a a C aquarius Spt3 2b1a Set Iv E put ipperd Wate te SURI may Fa File po Vest hile _Caneel OK Edit Preferences Dialog Box There are two settings in this dialog box that must be correct for the instrument to operate a Make sure the Serial Port selected is the same as the one on the computer to which the instrument is physically connected It may be COMI or COM2 b Make sure the Serial Comm Speed is correctly selected The communication speed is 9600 during the instrument s initial warm up sequence while it is first communicating with the software Note The Serial Comm Speed automatically changes to 57 600 when you select Flex mode from the Settings window If you follow these steps and are still having trouble starting up the system contact Technical Support 98 FlexStation Operator s Manual Rev D 6 2 Opening a Drawer Manually 6 2 Opening a Drawer Manually Under some circumstances it may be necessary to open an instr
78. lamp and install the 2 screws using the 3 32 Allen wrench e Tighten the two screws securely and evenly compressing the coil spring until the lamp is seated 8 Replace the lamp assembly a Tilt up the lamp assembly and line up the lamp with the bore of the mounting block b Slide the lamp all the way into the block orienting the assembly to clear the base plate Slide it in until it stops c Tuck the orange power cord inside behind the assembly 9 Close the instrument top cover 10 Tighten the screws a Use the 5 32 hex ball driver to tighten the two vertical mounting screws which should still be captured Tighten them very firmly b Tighten the clamp screw snugly but not tightly Use caution not to over tighten Caution Do not over tighten the clamp screw or the lamp could crack inside 11 Replace lamp cover a Place the plastic lamp cover over the opening Be careful not to pinch the orange power cord if it is not tucked away b Replace and install the screw 12 Replace cords Plug in the power cord and the computer cord 5 8 Long Term Shutdown If you will not be using the FlexStation II instrument for an extended period of time clean the external surfaces of the instrument before storage Be sure the instrument is emptied of all plates and tips before storage Keep cables and accessories with the instrument during storage FlexStation Operator s Manual Rev D 95 5 Service and Maintenance Procedures
79. lative not an interval between pipetting events The value is the minimum number of seconds of elapsed time from the beginning of the read It takes into account the mechanical speed of the pipette head and the time needed to aspirate and dispense fluids trituration and Automixing The minimum time for the second pipetting event depends on when the first pipetting event occurred The calculation for the second event starts at the end of the first event and adds to that the total time that will be necessary to aspirate new fluids from the compound plate and dispense them into the assay plate Possible Problems Time point calculations are based on the number of wells read the tip column and compound column used during transfer If you select time points that are not long enough incompatible with the selected volumes and transfer speed you will see one or both of the following messages AX Fluid dispense time is too short Check fluid time points SOFTmax Pro E 2d O 4 Compound Transfer Time Point is too shart N If you select transfer volumes that add up to more than the maximum can be accommodated by the assay plate this overflow warning will appear Ay Possible assay plate overflow Check fluid dispense volumes Be sure to pay attention to the minimum time value displayed under the time point box for each transfer as the minimum time values are different depending on which transfer you are configuring Note The
80. ll assignments stored in the previous template will be discarded The previously created groups remain however so you can select new wells and apply existing groups to them Note The Assay Plate setting takes precedence over all other fluidics module settings and will affect the correctness of other settings Important If you go back and change this setting after you have selected settings that follow for example Pipette Tips Layout Compound Source or Compound and Tip Columns the earlier settings may be automatically reconfigured to reflect the new Assay Plate Type and well layout Be sure to check any earlier assignments to ensure they remain correct FlexStation Il Operator s Manual Rev D 3 5 Configuring Instrument Settings in Flex Mode 3 5 7 Wells to Read From this dialog box you can choose which plate wells to read in your experiment You can choose a combination of wells from one well only to all the wells in a plate Partial plate reading may significantly reduce the time required for certain types of readings because the instrument does not have to read the entire plate Wells to Read Window Highlight the wells you want read with the cursor Wells must be contiguous and in a rectangular arrangement but do not need to start in the 1 column You may choose a partial row or column or a single well Note In planning your experiment remember that the instrument makes fluid transfers and readings one c
81. ll tips inside the instrument 5 Tips are missing e Ifthe tip rack is jammed and there are tips missing you must attempt to retrieve them from inside the instrument Continue with the following procedure FlexStation Operator s Manual Rev D 105 6 Troubleshooting Procedures 106 6 4 4 Inspecting Inside the Fluidics Module If there is any chance at all that there could be fluid in the pipette tips you must remove the pipettor head from the instrument and dispel the fluid or remove the tips outside the instrument This must be done to avoid spilling fluid in the reading chamber Important Failure to dispel fluid outside the instrument may result in damage to the optical components Biohazard This procedure includes steps involving potentially infectious biological agents requiring that proper handling precautions be taken Follow your institution s protocol Inspect the interior of the fluidics module Determine the position of the pipettor head and the location of all the tips that were originally in the tip rack Things to consider include e Is the pipettor head loose Is the red knob tightened securely e Is the pipettor head up and out of the way in home position or is it down in the tip rack area the compounds plate area or the reading chamber e Are there any pipet tips on the pipettor head e s there is any chance there is liquid remaining in the tips If so remove the head and expel the liquid safe
82. lumns in a compound plate to a single column in an assay plate at different points during the total read time Automix The Automix function permits automatic shaking of the microplate at preset intervals thereby mixing of the contents within each well Automix must be selected before beginning a reading Automix settings vary with operation mode For Endpoint mode enabling Automix will shake the plate for a definable number of seconds and then read at all selected wavelengths When kinetic mode is chosen two types of Automix can be enabled you can set Automix to shake the plate for a definable number of seconds before the initial reading and or for a definable number of seconds before each subsequent reading Use of Automix is strongly recommended for ELISA and other solid phase enzyme mediated reactions to enhance accuracy Temperature Regulation The instrument regulates the temperature of the microplate chamber from 1 C above ambient to 45 C Upon power up when the incubator is off the temperature in the reading chamber is ambient and isothermal Turning on the incubator by pressing the Incubator key will cause the instrument to begin warming the reading chamber and the fluidics module The temperature set point defaults to 37 C at start up With the incubator on the temperature of the reading chamber can be set and regulated from 1 C above ambient to 45 C Accuracy of the temperature set point is only guaranteed if the set
83. ly See procedures in sections 6 4 5 Removing the Pipettor Head and 6 4 6 Expelling Undispensed Fluid from Tips Caution Take care that you do not inadvertently dispel fluid before the pipettor head is outside the instrument e Can you account for all tips or is there a possibility that some have dropped out of sight e Do all the drawers move freely to gentle pressure or is there a plate or tip rack jamming them Once you have determined the status of pipettor head and tips there are a number of procedure you may need to follow to resolve the problem Possible scenarios include 2 Pipettor head is in home position without tips e Ifthe pipettor head is up and out of the way without tips but the tip rack is jammed it may be because one or more tips are not seated properly and are blocking the drawer movement e Reach into the fluidics module from the front panel and remove the tip rack by lifting it up and out toward you If the tip rack drawer is not all the way inside the instrument in its leftmost position you may need to gently push it inside to the left in order to get the rack out 3 Pipettor head is in home position without tips but tips are missing e f the pipettor head is up and out of the way without tips but there are tips missing from the rack then you must locate and recover the missing tips before proceeding e Retrieve any tips you can easily see and reach through the front panel e It may be nece
84. mitted by certain substances resulting from the absorption of incident radiation Fluorescent materials or materials that absorb light energy of a characteristic wavelength excitation undergo an electronic state change and instantaneously emit light of a longer wavelength emission Most common fluorescent materials have well characterized excitation and emission spectra The first figure below shows an example of excitation and emission spectra for a fluorophore In general excitation and emission bands are broad with half bandwidths of approximately 40 nm and wavelength differences between the excitation and emission maxima the Stokes shift is relatively small about 30 nm Exceptions to this rule include the lanthanide dyes used in time resolved fluorescence they have a Stokes shift that is typically about 300 nm There is considerable overlap between the excitation and emission spectra gray area when a small Stokes shift is present Excitation Emission Maximum Maximum Stokes 1 0 Shift a o o c o o N o 5 2 0 5 amp om co 0 500 550 600 650 Wavelength nm Excitation and Emission Spectra Because the intensity of the excitation light is usually many tens of thousands of times greater than that of the emitted light some type of spectral separation is necessary to reduce the interference of the excitation light with detection of the emitted light The FlexStation II instrument incorporates many features designed
85. mperature will be maintained at the set point until you disable temperature control by touching the Temp on off key again When the incubator is off the temperature within the reading chamber returns to ambient Note If the power is shut off to the instrument for any reason you will need to turn on the incubator again and allow sufficient time at least 10 minutes for the control algorithm to fully stabilize the reading chamber temperature FlexStation Operator s Manual Rev D 49 50 3 Operating Procedures 3 3 3 Setting the Temperature with SOFTmax PRO ru You can turn on the incubator with software by selecting Incubator from the Control menu Or you can click on the Incubator button on the instrument Status bar The Incubator dialog window appears ws Incubator x C Off Temperature br JS On X Cancel Incubator Dialog Window You can leave the temperature setting at the default value or you can type a different value into the highlighted box Note The incubator setting is independent of the protocol being run Running an experiment does not automatically select the temperature set point After a reading the temperature set point range and average actual temperature are recorded in the saved file FlexStation Il Operator s Manual Rev D 3 4 Setting Up the Software 3 4 Setting Up the Software Use the following procedure to check instrument status and settings 1 Observe SOFTmax PRO Untitl
86. ms brand tips For clear tips use Molecular Devices PN 9000 0512 FlexStation Operator s Manual Rev D 17 1 System Description 1 5 Overview of Operation Using the FlexStation II system is a process in five stages 1 Choosing an experiment 2 Preparing the instrument 3 Preparing the software 4 Running the experiment 5 Analyzing the data gt Choosing an experiment consists of e New or repeated experiment Does protocol exist gt Preparing the instrument consists of e Turning on the power e Setting temperature if needed e Preparing and loading tips plates and compounds gt Preparing the software consists of e Entering software preferences e Selecting instrument settings e Defining templates reduction parameters and display parameters e Confirming hardware and software set up gt Running the experiment consists of e Initiating the operation detection or fluidics plus detection e Saving the data file gt Analyzing the data consists of e Modifying the template or parameters as desired e Saving the data file e Analyzing the data e Exporting data to another software application as desired 18 FlexStation Il Operator s Manual Rev D 1 6 Theory of Operation 1 6 Theory of Operation This section includes the following topics e 1 6 1 Introduction to Fluorometry e 1 62 Instrument Design e 1 6 3 Modes of Operation 1 6 1 Introduction to Fluorometry Fluorescence is the light e
87. mum surface area detection for whole cell assays No plate adapter is used for tissue culture plates of 24 wells or less In Well Scan mode the fluidics module is not enabled For more information on this mode please review the appropriate section in the SOFTmax PRO User s Manual FlexStation Il Operator s Manual Rev D 1 7 Introduction to SOFTmax PRO Application Software 1 7 Introduction to SOFTmax PRO Application Software The SOFTmax PRO application software controls all aspects and operations of the system You will set up and run experiments using this software as well as use it to perform data analysis The SOFT max PRO application also allows you to store experiment files data and protocols on your PC or to export files for further manipulation The software uses standard Windows conventions for menus dialog boxes windows and mouse control These instructions assume that you are familiar with the basic operation of your computer This FlexStation II Operator s Manual describes using the SOFTmax PRO application to run FlexStation II experiments There is limited information about using the software for the other read modes For further details about the software including how to install SOFTmax PRO on your PC and to fully understand its multifaceted design including customization of the software refer to your SOFT max PRO operator s manual FlexStation Operator s Manual Rev D 27 1 System Description
88. n provided to ensure correct results and optimal performance An instruction that if not followed could result in loss of data Warning An instruction that if not followed can result in potential injury to a person working with the system Biohazard Indicates a condition involving potentially infectious biological agents requiring that proper handling precautions be taken Electrical Safety Warning Follow all instructions in this manual and on system labels If you use the system in a manner not specified by Molecular Devices any protections provided by the system may be impaired Service Trained Users There are two types of users described in this manual Most procedures required for operating and troubleshooting can be performed by any user who has read the instructions in this manual and is familiar with the system However all installation procedures and some more complex service and troubleshooting procedures require the expertise of a service trained user Whenever the following warning message appears a service trained user must perform the procedure to ensure user safety and to prevent instrument damage Example Warning The following procedures must be completed by a service trained user Do not attempt the following procedures if you have not been trained properly by appropriate Molecular Devices personnel Avoiding Mechanical Problems In Flex Mode Because of the complex mechanical nature of the FlexStation II in
89. n the instrument reading chamber Use these keys to adjust the temperature up or down starting at the previous temperature setting or the default of 37 C if no setting had been made Press either arrow once to increase or decrease the temperature shown in the display by an increment of 0 1 C press and hold to scroll Open or close the tip rack drawer Open or close the compound plate drawer Open or close the reading chamber drawer Whether or not the drawer will remain open depends on the incubator setting If the incubator is off the drawer will remain open if the incubator is on the drawer will close after approximately 10 seconds to assist in maintaining temperature control within the microplate chamber Note If the AutoRead feature is selected in Instrument Setup the drawer remains closed after the assay plate reading Provides The actual temperature inside the reading chamber at any given time the number displayed in the top of the LCD The set point temperature you select for the current experiment the number displayed in the lower portion of the LCD This number is only displayed when the incubator is on FlexStation Ill Operator s Manual Rev D 3 3 Setting the Temperature 3 3 Setting the Temperature If you want an elevated temperature within the instrument for your experiment turn on the incubator at least 20 minutes before you plan to start plate reading Up to 20 minutes may be required for the tem
90. n wave length that gives 9096 maximal emission FlexStation Il Operator s Manual Rev D 3 10 Optimizing Fluorescence Assays in Spectrum Mode Follow the plot to the left of the peak until the RFU value falls to approximately 90 of the maximum and then drop a line from the 90 point on the plot to the x axis refer to the figure below RFU at 9096 of max X max 90 of max A Wavelength Plot of RFU vs Wavelength 2 Preliminary Emission Scan Scan Figure 2 The preliminary emission scan without filters allows you to see the actual emission spectrum peak a In SOFTmax PRO set up a second Plate section for a fluorescence read spectrum mode Ex Fixed Em Scan with no cutoff filter default and medium PMT b Set excitation wavelength to the value determined in 1f above c Set emission scan to start stop approximately 50 nm below above the tentative emission value obtained from the literature or existing filter pair d Set step increment to 1 2 nm or do a preliminary scan with a 10 nm increment to determine the approximate peak location and then repeat the scan over a narrower wavelength range using a 1 2 nm increment e Perform scan and view results as a plot of fluorescence vs emission wavelength DRED EM scan no filter Well Graph Raw iof x 400000 300000 ff pons rd 2000 4 500 510 520 530 540 550 560 570 580 590 600 Em Wavelength in nm Ex 485nm Well oc o C2 A C3 o C4 Lambda
91. nclude stray excitation light and native fluorescence of plate materials sample constituents and solvents SOFTmax PRO has default settings see table following for Endpoint and Kinetic modes which you can override if desired The spectral scan mode default uses no cutoff filter e The number of readings per well may vary between 1 used for a quick estimate and 30 for more precise measurements The default number of readings per well varies with the read mode for fluorescence the default is 6 for luminescence the default is 30 for time resolved fluorescence the default is 20 e The voltage of the photomultiplier tube may be set to low for higher concentration samples medium or high for lower concentration samples in all read modes In Endpoint and Spectrum mode there is an additional setting automatic in which the instrument will automatically adjust the PMT voltage for varying concentrations of sample in the plate Other important factors that are independent of the instrument but which affect assay optimization include the Stokes shift When the Stokes shift is very small optimizing the excitation and emission wavelengths and correct cutoff filter choices are very important gt Optimizing Excitation and Emission Wavelengths with Spectrum Scanning Optimizing excitation and emission wavelengths is a simple three step procedure that maximizes the fluorescence signal and minimizes background noise e Preliminary excit
92. nd accessories that are not authorized specified by or provided by Molecular Devices Using unauthorized parts can damage the instrument Microplates The instrument can accommodate standard 6 12 24 48 96 and 384 well microplates In Flex mode however you can only use 96 or 384 well formatted assay plates A1 A12 OOOOOOOOOOOO sOODOOOOOOOOOO0 OOOOOOOOOO0O00 00000000000 OODOOOOOOOOO0 OOOOOOOOOOO0 sOOOOOOOO0O0O0O00 QOOOOOOOOOOO0 H1 H12 Top View of a 96 Well Microplate For fluorescence Molecular Devices generally recommends black walled clear bottom microplates for bottom reading and all black microplates for top reading because they have lower backgrounds than clear plates For luminescence white microplates may optimize light collection Note Not all microplates are made with the same materials Some plastics most notably polystyrene have significant native fluorescence and can cause moderate to sever background fluorescence especially in the UV range If your fluorescence experiments require high sensitivity it may be appropriate to use microplates designed and designated by the manufacturer to reduce background fluorescence Pipette Tips and Racks e For 96 well assays Molecular Devices specifies 200 pL Robbins brand new generation Super Seal Autotips For clear tips use Molecular Devices PN 9000 0623 e For 384 well assays Molecular Devices specifies 45 pL Lab Syste
93. ndow Help EB 257 6 C3 Plates Readng of34 O o00 00 The Read button changes to Stop Clicking on the Stop button will halt the reading The plate name and icon appear to indicate which Plate Plates uc section is currently being read This box displays the amount of time elapsed since the o 01 42 beginning of the reading This box displays the portion of the reading currently under Reading 13 f 34 ea nal o n gt Data Display You can observe the gradual accumulation of data in the Plate section of the SOFT max PRO window The values read by the instrument will appear in the data display of the Plate section as they are received from the instrument to the software in real time See SOFT max PRO User s Manual for information about how to change the data display FlexStation II Operator s Manual Rev D 77 3 Operating Procedures 3 8 Shutdown When a reading is completed you can perform more readings or shut down the instrument Shutting down the FlexStation II instrument includes the following steps e Make sure all three drawers are empty of pipette tips and trays e Clean up any spills that may have occurred during the day s experiments e Save any data files desired in SOFTmax PRO e Turn off the instrument and or the computer according to your laboratory s practice Note Normally you do not need to switch off the power at the end of the day If the system will not be used
94. necting the Cables 2 6 Connecting the Cables After the instrument is assembled and the computer is set up proceed to connect the power cords and computer cables Caution Make sure that all assembly is completed before connecting power cord Fuse Box Cover Power Switch WM WHOM WWMM WWW Parallel Port not currently in use i Power Cord RS 232 Serial Cable Power Cord Receptacle RS 232 Serial Port Power Cord Locations 1 Locate the instrument power cord P N 4400 0002 and the computer serial cable P N 9000 0149 in the FlexStation II accessories box 2 Insert the 8 pin DIN round end of the serial cable into the RS 232 serial port receptacle on the back panel of the instrument Attach the other end to the COM serial port on the back of the computer 3 Insert the female end of the power cord into the power receptacle at the rear of the instrument 4 Connect the instrument power cord a grounded power outlet of the appropriate voltage Molecular Devices recommends that you use a surge protector between the power cord and the grounded power outlet 5 Connect the computer hardware power cords to similarly grounded power outlets Caution Be sure no cables run beneath the instrument Leave at least three inches between the back of the instrument and the nearest objects or surfaces to ensure proper ventilation and cooling FlexStation Operator s Manual Rev D 41 2 Installation Proc
95. ng or Removing the Fluidics Module If you need to tilt or remove the fluidics module to clean up after a large spill or to recover tips follow these instructions Warning The following procedures must be completed by a service trained user Do not attempt the following procedures if you have not been trained properly by appropriate Molecular Devices Personnel Warning Do not remove cover until power is disconnected Do not operate instrument unless all covers are in place Biohazard This procedure includes steps involving potentially infectious biological agents requiring that proper handling precautions be taken Follow your institution s biohazard protocol Opening the Top Cover FlexStation Operator s Manual Rev D 115 6 Troubleshooting Procedures 116 6 6 1 Tilting the Fluidics Module 1 Power off Turn the power off to the instrument Disconnect the power cords from the instrument and the cables connected to the computer Caution Never operate the instrument with the covers removed Damage to the instrument can occur Read and obey all warning labels Lift up instrument cover Press in the handle at the bottom of the top cover and lift the cover back off the detection module revealing the fluidics module Tilt fluidics module Using the handle tilt the fluidics module to the left Clean spill If you can clean up the spill or remove the tips without removing the flui
96. ngs which appear after it in the list You will need to return to Timing after completing all settings to obtain the minimum read interval It is essential that settings for the fluidics operations are correct and correlate with one another In particular layouts and settings for Assay Plate Type Wells to Read Compound Source Compound Transfer Pipette Tips Layout and Compound amp Tip Columns must correlate These settings are marked with an asterix in the list above SOFTmax PRO software will cross check these settings as you move through the configuration windows and when you click OK to close the Instrument Settings screen If settings do not correlate an error message will appear and you must correct settings before continuing Finishing Settings Cancel 54 After you configure all Flex instrument settings to your specifications click OK at the bottom of the window to return to the Experiment Window You must click OK to capture the new settings If you click Cancel the instrument returns to the Experiment Window without making any modifications FlexStation Operator s Manual Rev D 3 5 Configuring Instrument Settings in Flex Mode 3 5 1 Wavelengths You can select the number of wavelengths wavelength pairs to be read in the Wavelengths options dialog box You can select as many as four excitation and emission wavelength pairs You can select an emission cutoff filter for each pair or you can
97. nterfering fluorescent species are present you may be able to find excitation and emission wavelengths that avoid this interference For this reason it is desirable to scan emission for both an intermediate concentration of labeled sample as well as unlabeled sample background The settings that yield the maximal ratio of sample emission to background emission are optimal To measure fluorescence accurately it is necessary to reduce light scatter The governing equation for fluorescence is Fluorescence extinction coefficient x concentration x quantum yield x excitation intensity x path length x emission collection efficiency For more information regarding optimizing excitation and emission wavelengths using the spectral scanning capabilities of the instrument see 3 10 Optimizing Fluorescence Assays in Spectrum Mode 20 FlexStation Operator s Manual Rev D 1 6 Theory of Operation 1 6 2 Instrument Design Fluidics The instrument is designed with a fluidics module that transfers liquids to the assay plate during the kinetic reading The fluidics module incorporates an eight channel pipettor that automatically changes tips and transfers reagents to the plate that is read in the fluorometer Pipette height and dispensing rate are adjustable The instrument can add reagents within milliseconds of a column being read enabling fast kinetic assays of transient responses As many as three compounds can be transferred from co
98. nty e 7 4 Index 7 1 Parts and Accessories Caution Do not use parts and accessories not authorized specified by or provided by Molecular Devices Using unauthorized parts can damage the instrument Contact Molecular Devices Customer Service or your authorized service representative to obtain parts and accessories Molecular Devices Corporation 1311 Orleans Drive Sunnyvale California 94089 Tel 408 747 1700 Fax 408 747 3601 US Canada Toll Free 800 635 5577 Online www moldev com Part Number Accessory 4400 0002 Power cord US 4400 0036 Power cord European 9000 0149 Computer cable 2300 0800 Top read microplate adapter reading chamber 0310 9336 Bottom read microplate adapter reading chamber 2500 1029 Compound Baseplate compound plate drawer 4600 0029 Fuse 0310 3992 Pipettor head 8 channel 0310 4034 Pipettor head 16 channel 0112 0077 Operator s manual 9100 0051 Allen wrench hex 3 32 ball L shape 9000 0512 Tips clear 384 wells 30 ul capacity 10 racks case 9000 0623 Tips clear 96 wells 200 ul capacity 10 racks case Robbins PN 1043 04 5MD FlexStation Operator s Manual Rev D 119 7 Appendices 7 2 Performance Specifications 120 Fluorescence Photometric Excitation Wavelength Range Emission Wavelength Range Excitation Wavelength Bandwidth Emission Wavelength Bandwidth Wavelength Selection Wavelength Accuracy Sensitivity signal 3xSD of baseline 96 wells Top Re
99. o select a plate type that matches the type and well configuration of the actual compound plate you will be using and the number of wells selected in the Assay Plate Type setting In particular the well bottom height is different for different types of plates and selecting plate type correctly is important to prevent jamming pipette tips into the bottom of the well The instrument assumes a 20 pL pipette height when aspirating from a compound source plate See illustration below Pipette Tip Aspirate Height 20 uL Default 10 uL for 384 wells Well Bottom Height A Compound Plate Well Bottom Height Caution Selecting an incorrect compound plate type can result in pipette tips jamming into the wells and damaging the plate and the instrument Select a compound plate type Beckman 96 2 3mL Costar 96 opaque 3mL Costar 96 Ubtm clear 3mL Costar 96 Vbtm 3mL Greiner 96 Vbtm 3mL Numc 96 Ybtm clear 3mL Polyfiltronics 96 2mL 4 Twin Troughs Compound Source Settings Dialog Window FlexStation Operator s Manual Rev D 63 3 Operating Procedures 3 5 9 Compound Transfer Compound Transfer is an important setting to configure correctly In addition to configuring precise fluid transfers for your experiments this dialog box also helps prevent flooding of the assay plate The fields in this dialog box will allow you to set volumes for up to three transfers However you must be careful to keep in mind the ac
100. oduct names are trademarks or registered trademarks of their respective owners Disclaimer Molecular Devices Corporation reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Molecular Devices Corporation assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information FlexStation Il Operator s Manual Rev D Contents 1 System Description o ee 1 11 Introduction duis ult RR ed paw hho T ee aaah Seeks ve ew aaa ed 1 12 Operator s Manual Overview 0 ils 3 1 3 Safety Information conor po er ee o ope e ng edente 4 14 System Components 2 22 05 0262 2 cba ede ede iade did d REA 6 1 4 1 Covers and Instrument Panels lilii lessen 7 MAZ WD a WLS EE ETE DC LOU DELLI M 9 1 3 Fluidics Module lliiiiiiseseleseeese e 11 1 4 4 Detection Module o sisse e ns 13 14 9 Computer sore aure Stute otn e a mart eo tuse us sabre etse dea 15 1 4 6 Acc ssOrlessz oed ter Bou win dad etd waa bo Sip tals e dpa ae wiped GOA 16 1 7 Consumables 7 45 ces eh hee DELE OI E Ae EA 17 15 Overview of Operation 0 eee 18 1 66 Theory of Operation 223 45 52 2 Ye 2s AAs Ea IA TABBIiTU Wei 19 1 6 1 Introduction to Fluorometry v Ai oa See PhS
101. olumn at a time You may want to use partial rows A H rather than partial columns 1 12 or 1 24 for most situations Important Selected wells must be contiguous and in a rectangular arrangement FlexStation Ill Operator s Manual Rev D 61 62 3 Operating Procedures If you select a partial plate for reading only those wells selected will be visible in the data display In the figure below the Plate section reflects that wells A1 through H2 have been selected for reading mi SOFT max Pro File Edit View Experiment Control Assays Plate Window Help v C3 Piter V Max units per sec Plate 1 1 2 o M A FLEX B j Time 300 secs j Interval 9 secs C j Reads 34 B Fluorescence E j Bottom read F Ex Em Cutoff G j Lm1 485 538 530 M j Wavelength Combination ILm1 Automix Off Trans H 100p R21 v230p01 7 Calibrate On PMT High Reads Well 6 Lag Time 0 End Time 300 RFU Min 0 RFU Max 20000 Vimax Pts 34 34 Plate Last Read No Data mls Plate Section with Selected Wells to Read FlexStation Il Operator s Manual Rev D 3 5 Configuring Instrument Settings in Flex Mode 3 5 8 Compound Source This setting allows you to select a compound source plate Compound plates store fluids that are aspirated withdrawn from the compound plate and then injected dispensed into the assay plate during the run Be sure t
102. on If the red X remains after the warm up sequence select Preferences from the Edit menu of SOFT max PRO and view the Preferences dialog box ma Preferences x Serial Port COM1 v Serial Comm Speed 9600 v Memes nen mut pu i Aquarius Spt3 2b1 4 t Ee Time ae Plate ie i j j i IV Include Labels i T Time Interpolate File Prefix bata fu Append Date e SUFiimay PHO File Text File Filters Autoprint Cancel Preferences Dialog Box Make sure the Serial Comm Speed the communication rate or baud rate is set to 9600 Note When the FlexStation ll instrument starts up it begins with a serial comm rate of 9600 Baud Upon initiation of a microplate reading SOFTmax PRO issues a command to change the rate to 57600 Baud where it remains until power is shut off to the instrument If you continue to see the red X after confirming the Serial Comm Speed see section 6 4 7 Recovery Procedure FlexStation Il Operator s Manual Rev D 6 4 Recovering from Mechanical Problems in Flex Mode 6 4 5 Removing the Pipettor Head You may need to remove the pipettor head to remove empty tips or to dispel liquid remaining in tips after a mechanical failure To remove the pipettor head follow the instructions in this procedure To reinstall the pipettor head see instructions in section 2 4 Installing the Pipettor Head Caution Use caution during this procedure Follow these instructions
103. otes on the experiment in the Notes section Groups are also contained in experiments when you define a template You may create Graph sections as desired Groups are automatic Graphs are optional See your SOFT max PRO User s Manual for details 30 FlexStation Il Operator s Manual Rev D 2 Installation Procedures Part 2 provides information about how to install the FlexStation II system in your laboratory Installation must be done by qualified Molecular Devices personnel or a service trained user Caution The following procedures must be completed by a service trained user Do not attempt the installation procedures if you have not been trained properly by appropriate Molecular Devices Personnel The following sections describe the installation procedure e 2 1 General Precautionary Information e 2 2 Unpacking the System e 2 3 Installing the Fluidics Module e 2 4 Installing the Pipettor Head e 2 5 Setting Up the Computer e 2 6 Connecting the e 2 7 Installing the Drawer Adapters e 2 8 Installing SOFTmax PRO 2 1 General Precautionary Information Warning Always make sure the power switch on the instrument is in the OFF position and remove the power cord from the back of the instrument prior to any installation or relocation of the system Warning Do not install or operate the system in an environment where potentially damaging liquids or gases are present Caution Do not touch or loosen any screws or parts ot
104. ows you to monitor instrument status and access several functions with the click of a button You can verify communication with SOFT max Pro and monitor the reading chamber temperature The Status bar also provides buttons used to begin a reading open the Incubator dialog box shake the microplate Automix and open or close the instrument drawers The status bar can be hidden by selecting Hide Status from the View menu M The following icons are present in the Status bar and are used to set up the instrument or interact with it during operation Note Different Molecular Devices systems have different icons This Icon Has the Following Function FB The Instrument Status icon provides visual confirmation that SOFTmax PRO is communicating with the instrument Double click this icon to display Preferences dialog box Displays the current temperature inside the instrument Click this icon to display the Preferences dialog box Click to begin reading It changes to Stop during a reading Clicking this button also closes any open drawers Click the Incubator button to open the Incubator dialog box to change temperature settings Click the Automix button to manually shake the assay plate Note The manual shaking that occurs when you click this button differs from the Automixing that can be selected as an instrument setting Click the Drawer button to open or close the Reading chamber drawer FlexStation Operato
105. p spills immediately Clean the exterior of the unit and the drawer as necessary FlexStation Il Operator s Manual Rev D 5 4 Using the Microplate Adapters 5 4 Using the Microplate Adapters Microplate Adapter Installation If you are reading standard 96 or 384 well microplates you need to install a microplate adapter in the reading chamber drawer The black adapter elevates the plate in the drawer for optimum performance with standard 96 or 384 well microplates in bottom read mode The purple adapter optimizes performance with standard 96 or 384 well microplates in top read mode Caution Incorrect insertion or removal of the adapter may cause damage to the microplate drawer 1 Turn power to the instrument on 2 Press the Reading Chamber button on the front panel or select Open Drawer from the Control menu 3 Hold the adapter so that the label is on the left side facing up 4 Place the top back Row A portion of the adapter into the drawer first While pushing against the back edge of the adapter lower the front of the adapter into the drawer Removing the Microplate Adapter If you are reading high profile 6 12 24 or 48 well plates and the adapter is in the drawer you will need to remove the adapter 1 Turn power to the instrument on 2 Press the Reading Chamber button on the front panel or select Open Drawer from the Control menu 3 Hold the adapter at the front long side of the drawer and push towar
106. pecified by or provided by Molecular Devices Using unauthorized parts can damage the instrument gt Compound Plate Drawer The compound plate drawer holds a 96 and 384 well microplate or reservoir trough The instrument can simultaneously transfer a column of either e Eight fluids from a 96 well compound plate to a 96 well assay plate or e Sixteen fluids from a 384 well compound plate to a 384 well assay plate Important Be sure to install the compound baseplate before placing a compound plate in the drawer gt Reading Chamber Drawer 10 The reading chamber drawer opens to accept a 96 and 384 well microplate for analysis in the reading chamber It is the lowest of the three drawers The reading chamber drawer operation varies depending on the incubator status When the incubator is off the reading chamber drawer is open at power up and after a read When the incubator is on the drawer closes automatically to maintain the temperature of the reading chamber Important Be sure to install a microplate adapter before placing an assay plate in the drawer for standard microplates For further information see section 2 7 Installing the Drawer Adapters FlexStation Operator s Manual Rev D 1 4 System Components 1 4 3 Fluidics Module The fluidics module houses the pipettor head several motors and all the fluidics components There are two horizontally moving carriers one for the pipette tip rack and the other
107. perature within the chamber to reach the set point Turning on the incubator and choosing a temperature set point can be done using the software or the front panel of the instrument Range The instrument will operate with a chamber temperature between 1 C above ambient and 45 C Temperature cannot be regulated at a set point that is outside this range Note It is possible to enter a temperature setting that is outside the operational range either through software or with the control panel However the instrument will not respond with a temperature outside the allowable range 3 3 1 Displaying Temperature Two temperatures are displayed in the LCD on the control panel The upper reading is the temperature measured inside the reading chamber When the incubator is off this upper number will be the ambient temperature The lower reading is the set point that is the temperature you desire for the current experiment and it is displayed when the incubator is enabled 3 3 2 Setting the Temperature with the Control Panel To enable the incubator press the Temp on off key on the control panel The display will indicate that temperature control is on by displaying numbers in the lower half of the LCD The instrument will set the instrument to the default temperature 37 C To change the temperature set point press the up or down arrow keys until the desired temperature set point appears on the display The reading chamber te
108. plastic pushers located in the front left corner of each drawer hold the plates racks or reservoirs securely in place when the drawers are closed Pusher Carrier Drawer Detail Caution Do not obstruct the movement of any of the drawers If you must retrieve a plate after an error condition or power outage and if the drawer will not open it is possible to open the drawer manually See section 6 2 Opening a Drawer Manually FlexStation Operator s Manual Rev D 9 1 System Description Assays Window Help You can open and close the drawers using either the SOFTmax PRO Instrument Setup application or by manually pressing the drawer keys on the instrument control Read Ref Open Drawer Incubator Calibration Ctri A panel Using SOFTmax PRO go the Control menu and select Tip drawer for the tip rack drawer Compound Drawer for the compound plate drawer or Open Drawer for the reading chamber drawer Open Tip Drawer Set Air Gap be Open Compound Drawer You can also open or close the reading chamber drawer with the Drawer icon on the Status Bar gt Tip Rack Drawer The top drawer holds the pipette tip rack Only tips specified by Molecular Devices for use with the FlexStation II System can be safely used For further information see section 7 1 Parts and Accessories Caution Do not use parts and accessories that are not authorized s
109. point is at least 1 C above ambient If the temperature set point is lower than the ambient temperature the chamber temperature will remain at ambient Temperature regulation is controlled by heaters only and therefore cannot cool the temperature to a setting lower than ambient Temperature regulation and control of the reading chamber is achieved through electric heaters a fan efficient insulation and temperature sensors The heaters are located within the instrument which is insulated to maintain the temperature set point The seven sensors are mounted inside the chamber and measure the air temperature and chamber temperature The temperature feedback closed loop control algorithms measure the chamber air temperature compares it to the temperature set point and use the difference to calculate the regulation of the heating cycles This technique results in accurate precise control of the reading chamber temperature with a temperature variation of the air across the entire assay plate of less than 0 5 C Temperature uniformity within the assay plate itself will depend upon its design materials and or configuration FlexStation Operator s Manual Rev D 21 1 System Description Important Temperature of samples in all assay plates will be affected by evaporation Time Tagged Data The FlexStation II platform is a single channel reading system Although the scan time is very fast 8 wells in about 1 0 seconds 1 well in abou
110. pound amp tips columns AutoRead Well Scan Editor PMT sensitivity Assay plate type Endpoint Detection Fluorescence Luminescence TRF Yes or No Yes or No Yes Yes No No Yes Yes No No No No No Yes No Yes Yes 1 6 Theory of Operation Kinetic Spectrum Well Scan Flex Detection Detection Detection Fluidics Detection Fluorescence Fluorescence Fluorescence Fluorescence Luminescence Luminescence Luminescence only TRF TRF TRF Yes or No Yes or No Yes or No No Yes or No Yes or No Yes or No Yes always on Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No Yes No No No Yes No No No Yes No No No Yes No No No Yes Yes Yes Yes Yes No No Yes No Yes Yes Yes Yes Yes Yes Yes Yes Operation Modes and Features Note The system performs limited luminescence and TRF readings Endpoint Mode In Endpoint mode as well as in Kinetic and Flex modes you may select from one to four excitation emission pairs to obtain Relative Fluorescence Units RFU readings for each well of a microplate In Endpoint mode the fluidics module is not enabled For more information on this mode please review the appropriate section in the SOFTmax PRO User s Manual Kinetic Mode FlexStation Operator s Manual Rev D Kinetic analysis can be performed for a total run time of up to 99 hours The kinetic read interval depends upon the instrument set
111. procedure until you can pinpoint the source of the problem Fluidics Error 1 Fluidics module not detected This message may occur if the fluidics module is not correctly installed e Fluidics module not detected The current plate setup has a pipetting event To read with this setup the fluidics module must be attached Please follow troubleshooting procedures in the FLEXstation manual Fluidics Error 2 Pipetter failure 1 This message may occur if the tip rack is jammed Tips may be missing e Pipetter failure There may be undispensed fluid in the pipette tips Please follow recovery procedures in the FLEXstation manual Failure to do so may cause serious damage Fluidics Error 3 Pipetter failure 2 This message occurs if fluid remains in the tips The instrument needs to be manually recovered at this point e Pipetter failure Undispensed fluid is in the tips Please follow recovery procedures in the FLEXstation manual Failure to do so may cause serious damage Note If you need to contact Technical Support during the following procedures be sure to note which error message you observed FlexStation Operator s Manual Rev D 103 6 Troubleshooting Procedures 6 4 2 Opening the Instrument Caution Use caution during this procedure Follow these instructions and any instruction labels on the instrument exactly 1 Power off Turn the power off to the instrument Disconnect the power cords from the
112. r s Manual Rev D 29 1 System Description One SOFTmax PRO file contains at least one experiment but may contain more than one Each experiment can contain a section for Notes and one or more Plates You can enter Notes and edit Plates using the tool bars shown below IN Untitled Torx p E notes E4 Ae 7 Plate GB Setup E Template Reduction EzjDisplau kz The following icons appear on the Plate Section tool bar This Icon Has the Following Function Double click the Plate icon to open the Plate section in a new window Plates Double click the Name of Plate icon to open the Section dialog box Click the Setup button to open the Instrument Settings dialog box for this plate E Template Click the Template icon to open the Template dialog box where you can create or edit the template This is used to group table setup for defining areas of the assay plate 5 Reduction Click the Reduction icon to configure settings for data analysis and graph reduction E Display Click the Display icon to open the Display dialog box and change your display properties L Click the Graph icon to enlarge sections of the display into graphic form Z Click the Mask icon to mask selected wells n Click the Printer icon to include or exclude an section from a printed report SOFTmax PRO provides other icons and tool bars For example you can keepN
113. r turn fasteners in the metal plate where the bottom of the fluidics module is attached to the detection chamber b Lift the fluidics module carefully off the detection chamber and set it on the bench 3 Clean up spill and or retrieve tips a Clean the spill If you are attempting to remove tips remove any obvious ones on top or around the sides of the reading chamber Look through the openings in the read head you may be able to see tips inside b Ifyou see tips all the way down in the bottom tray of the instrument you can safely leave them there They will be out of the way of the operation of the read heads c Reattach the fluidics module by following the steps above in the reverse order Note For detailed instructions and illustrations see section 2 3 Installing the Fluidics Module d Follow steps 5 and 6 in the above procedure to reassemble the instrument 4 Contact Technical Support If you see tips lodged inside the reading chamber contact Technical Support Do not attempt to tilt or remove the read heads on your own Caution Do not attempt to tilt or remove the read heads Failure to do so can damage fragile optical components and may void your warranty FlexStation Operator s Manual Rev D 117 6 Troubleshooting Procedures 118 FlexStation Il Operator s Manual Rev D 7 Appendices The Appendices include the following sections e 7 1 Parts and Accessories e 7 2 Performance Specifications e 7 3 Warra
114. re already familiar with SOFT max PRO software you can close the Notes section and open the Plate section You are now ready to begin setting up your experiment protocols Bi Untitled v E Plate Ee a ee ES Gg Setup Ed Template X Reduction E Display l Z4 Max Min Plate 1 FLEX Time 120 secs j Interval 2secs Reads 61 Fluorescence B Bottom read 1 Ex Em Cutoff i Lm1 485 525 515 Auto Cutoff Automix Off Calibrate On i PMT High ReadsivVell 6 Lag Time 0 j End Time 120 RFU Min 0 RFU Max 6000 Wavelength Combination ILm1 Trans1 H 180p R 6 100p 15 upper e Last Read No Data gt ff contra Mott Z4 sf Ey gt ff vata Us 24 a gt Z Crap in Ft uu ial Untitled Window Plate Section of Default Template Note For instructions on adjusting software settings see section 3 4 Setting Up the Software FlexStation Ill Operator s Manual Rev D 47 3 Operating Procedures 48 Using the Control Panel actual C Set Temp reading chamber This button Temp on off tip rack compounds reading chamber WU amp This indicator actual C set pt C Detail of Control Panel with LCD Allows you to Enable or disable the incubator When the incubator is on the set temperature and measured internal temperature are shown on the front panel LCD display Enter a set point for the temperature i
115. rocedure Follow these instructions and any instruction labels on the instrument exactly Warning Do not remove cover until power is disconnected Do not operate instrument unless all covers are in place 1 Locate fluidics module hardware a With the cover open you can see the hinged flange metal plate to the left side of the exposed reading chamber b Notice also the two quarter turn fasteners Zeus screws attached to the flange and two locating pins near the middle of the reading chamber Handle Holes for Locating Pins Fluidics Module Electrical Connector Hinged Flange with Quarter Turn Fasteners 2 Detection Module Read Chamber Positioning the Fluidics Module 2 Liftfluidics module into position Warning You may need two people to continue with this procedure The fluidics module is heavy and it may be difficult and awkward to both hold it in place and attach it to the base a Lift fluidics module using the handle into position over the detection module just to the left of the metal flange b Tilt module up at about 90 align the quarter turn fasteners in the flange to the holes in the bottom of the fluidics module FlexStation Operator s Manual Rev D 35 2 Installation Procedures c Connect the quarter turn fasteners to the bottom of the fluidics module Lock the fasteners into place 3 Attach electrical connector a Hold fluidics module tilted back with your l
116. ror message you observed Note See section 6 4 1 Assessing a Mechanical Problem for error messages specific to fluidics operation failures General Error 1 No assay plate If you get this message check to be sure there is an assay plate correctly placed in the reading chamber drawer Start the reading again Q There is no assay plate Please insert a plate before reading If you get any one of the following messages attempt your reading again before continuing with troubleshooting General Error 2 Unexpected response result code 105 Could not complete the last command because Instrument Problem Unexpected response Result Code 105 General Error 3 Communication problem response to late Instrument communication problem Response too late General Error 4 Unexpected response e Instrument Problem Unexpected response General Error 5 Measurement aborted Instrument Problem Measurement aborted Instrument will be temporarily unavailable during reset FlexStation II Operator s Manual Rev D 113 6 Troubleshooting Procedures General Error 6 Result code x p General Error 7 Fatal error If you observe the following message try to determine if there are tips jamming the read head follow procedure in section 6 7 1 If this is not the case call Technical Support X 114 FlexStation Il Operator s Manual Rev D 6 6 Tilting or Removing the Fluidics Module 6 6 Tilti
117. rument displays the actual current temperature again However the red X is still covering the instrument icon on the SOFT max PRO status bar Select Recover from the Control menu In SOFT max PRO select Recover from the Control menu The recover process is essentially a second startup cycle which restores the fluidics module to operating conditions and reboots the instrument Press Continue to recover the software wa SOFT max Pro tips before resetting Failure to do so before continuing may lead to Recover will reset FLEXstation You must manually remove pipette serious damage Please refer to the FLEXstation manual before proceeding Tu Continue Recover Dialog Box 3 Double check preferences If the red X still remains after the Recover procedure return to the Preferences dialog box in the software and recheck that the serial comm rate is set to 9600 For instructions see section 6 1 Problems During Startup If you cannot establish communication between the instrument and the computer with the recover procedure contact Technical Support FlexStation Il Operator s Manual Rev D 6 5 General Error Messages 6 5 General Error Messages Error messages can appear during operation of the instrument Follow any instructions present in the message Anytime a message persists or if you cannot return the instrument and software to normal operation contact Technical Support and be sure to note which er
118. s 33 tip rack 105 top 7 34 37 40 94 95 104 109 115 116 D data appearing in Plate section 77 display parameters 74 management 85 replacing with new 76 design instrument 21 23 detection module 11 13 14 unpacking 32 34 Display icon 30 drawer adapters installing 42 Drawer button 29 drawers 9 10 42 44 45 See also reading chamber drawer tip rack drawer compound plate drawer closing 29 emptying 78 opening 75 opening manually 99 E Endpoint mode 25 78 error messages fluidics module 103 general 113 14 F filters emission cutoff 55 56 flash lamp 2 8 13 14 120 precautionary statement 5 replacing 92 95 FLEX button 52 Flex mode 24 46 53 avoiding mechanical problems 4 default at startup 52 instrument settings in 54 73 recovering from mechanical problems 102 12 serial communication speed 51 fluidics module 11 12 design 21 inspecting inside of 106 8 installing 35 36 tilting or removing 115 17 unpacking 32 fluorescence assays 79 84 fuses 16 replacing 90 91 G Graph icon 30 I icons Display 30 Graph 30 Instrument Status 29 51 Mask 30 Name of Plate 30 Plate 30 77 Printer 30 Reduction 30 Template 30 incubator 2 7 10 21 setting temperature 49 50 switching on 44 48 Incubator button 29 50 input output panels 8 installation procedures 31 42 connecting the power cords 41 general precautions 31 installing SOFTmax PRO 42
119. s provide instructions for diagnosing and solving common problems as well as a list of error conditions Part 7 Appendices provides technical specifications a list of spare parts the index and warranty statement Conventions Used in this Manual The names of keys that appear on the FlexStation II control panel and icons that appear in the SOFTmax PRO software are shown in boxed type Example Start The term instrument refers generally refers to the fluidics and detection modules System refers generally to all the components detection module fluidics module computer software accessories and consumables FlexStation II Operator s Manual Rev D 3 1 System Description 1 3 Safety Information When operated properly in a safe environment and according to the instructions in this manual there are no known hazards associated with the FlexStation II System However proper use requires an understanding of situations that are potentially dangerous and can result in serious injury All users must be familiar with the guidelines in this section before working with the system Conventions Used for Precautionary Information This manual uses the following conventions to provide technical and safety information of special interest Note Background information provided to clarify a particular step or procedure Caution An instruction that if not followed can result in damage to the system Important An instructio
120. ssary to raise the fluidics module to gain access to the reading chamber See section 6 6 Tilting or Removing the Fluidics Module FlexStation Il Operator s Manual Rev D 6 4 Recovering from Mechanical Problems in Flex Mode 4 Pipettor head is in lowest position without tips all tips are accounted for e Ifthe pipettor head is all the way down its carrier bar not in home position without tips on the nose cones and all tips are safely in the rack you can remove the tip rack easily The compound plate may also be stuck inside the fluidics module e Gently push the tip rack and compound plate or trough drawers all the way outside to the right 5 Pipettor head is not in home position with tips e If the pipettor head is somewhere other than home position and has tips on it you must proceed carefully The pipettor head may or may not be obviously jammed Tips could be jammed in the tip rack in the compound plate or in the assay plate e Carefully raise the pipettor head to home position by manually moving the belt e f there is any chance there is liquid in the tips you must then remove the pipettor head as described in sections 6 4 2 and 6 4 3 and dispel the liquid safely outside the instrument Caution Do not attempt to remove the pipettor head through the front inside panel See instructions in section 6 4 5 Removing the Pipettor Head e Ifyou are certain there is no liquid in the tips remove the tips from the
121. stic bag enclosing the instrument and slide the bag down out of the way around the base of the detection module 6 Together with another person reach inside the bag and under the instrument Lift the instrument out of its shipping tray the bottom of the carton and place it on the bench Warning The FlexStation ll instrument weighs approximately 55 pounds and should be lifted with care It is recommended that two persons lift the instrument together taking proper precautions to avoid injury 7 Setshipping materials out of the way Removing Shipping Screws 1 There are two shipping screws holding down the cover of the instrument Locate these stainless steel screws under the front flange of the detection module m Location of Shipping Screws Shipping Screws captive 2 Unscrew the shipping screws with the Allen wrench It may be necessary to move the instrument to the front of the bench to reach the screws from below The screws remain captured in the base FlexStation II Operator s Manual Rev D 33 2 Installation Procedures 3 Open the top cover by depressing the latch in the handle and pivoting the cover up and back Opening the Top Cover 34 FlexStation Il Operator s Manual Rev D 2 3 Installing the Fluidics Module 2 3 Installing the Fluidics Module The next step is to install the fluidics module onto the detection module Caution Use caution during this p
122. strument including both fluidics and optical reading smooth and reliable operation of the system depends on both good design and operator knowledge In order to prevent problems of a mechanical nature be sure to read all sections of this manual prior to attempting a reading in Flex mode See especially section 6 3 Understanding Potential Mechanical Problems 4 FlexStation Il Operator s Manual Rev D 1 3 Safety Information Safety Messages Observe the following warnings and precautions High internal voltages Always turn off power switch and unplug system power cord before removing labeled covers or panels Xenon arc flash lamp Do not look directly at the flash lamp while illuminated The lamp emits ultraviolet radiation at levels that can injure the eye if viewed directly Electrical grounding Never use a two prong plug or extension cord to connect primary power to the system Use of a two prong adapter disconnects the utility ground creating a severe shock hazard Always connect the system power cord directly to a three prong receptacle with a functional ground Spilled liquids Avoid spilling liquids on the system Fluid spilled into internal com ponents creates a potential shock hazard Wipe up all spills immediately Do not operate the system if internal components have been exposed to spilled fluid Unplug instrument if there is a fluid spill in the instrument and contact Technical Support Replacement fuses Use replac
123. t 50 ms the difference in the exact time each well is read is dependent on the number of rows chosen in a column This difference is an important factor in fast kinetic assays For this reason all readings are tagged with an exact read time and when multiple well fast kinetic responses are plotted the curves overlie each other as plotted by SOFTmax PRO If kinetic data are to be exported you may choose either time interpolated data or raw time tagged data Molecular Devices recommends that you select time interpolated data This option is explained in more detail in the SOFT max PRO user s manual Fast FLEX kinetics Well Graph Raw oy x 2500 2000 1500 yn 2 E ir 4000 Hp Cr Mer T 15 16 17 18 19 20 21 22 23 24 25 Time secs Ymax Points 5 Well o A3 o B3 C3 D3 E3 m F3 a G3 e H3 Ymax Per Second 31327 343 97 28447 320 90 246 80 31441 325 74 282 51 R 2 0 981 0 968 0 979 0 975 0 967 0 981 0 986 0 987 5 Reduction J Show Reduced Scale To pata Time Tagged Data Example Optical System The instrument uses excitation and emission filter wheels to decrease interference by stray light thus augmenting the wavelength selection that is provided by the monochromators Two independent single channel reading heads can service top and bottom reading requirements The top reading head supports coaxial excitation and emission beams in the case of the bottom reading head excitation and emission beams
124. t further depends on the number of columns and their locations as selected in the Wells to Read settings window FlexStation Operator s Manual Rev D 3 5 Configuring Instrument Settings in Flex Mode Manually Assigning Tips and Compounds Alternatively to automatic assignment you may assign any tip column and any compound column to correspond to any assay plate column To assign a tip to an assay plate well highlight one or more tip cells and select one of the items available in the Tips pop up menu The tip column selected in the pop up menu will be shown as a number in the selected Tip row s Choosing the Fill option at the top of the menu fills the selected cells with the tip numbers that correspond to the same column numbers tip 3 with column 3 etc Note that Fill does not take into account the actual tips that are available if less than a full rack is used whereas the choices in the menu beneath Fill represent the actual tip numbers that are listed as being available in the Pipette Tips Layout tab Similarly to assign a compound highlight one or more compound cells and select one of the items from the Compound pop up menu The wells shown in this settings window convey graphically the volumes of liquid in the assay plate for each pipetting event Using the color associated with each event the compound settings display the dispensed compound volume as a percentage of the total assay well volume If you entered an initi
125. te Last Read Trans3 H 100p R 1 v 501 0985 No Data Plate section with wells selected for injection The Plate section also provides information on the transfer settings and the compound sources The information will be carried below the plate table FlexStation Operator s Manual Rev D 73 3 Operating Procedures 3 6 Other Software Settings 74 There are other software settings that deal with data calculation and display In contrast to Instrument Setup parameters these data settings can be configured before during or after running an experiment These include e Setting reduction parameters e Setting data display parameters e Using the Template Editor Molecular Devices strongly recommends that you define a template and set the reduction and display parameters prior to reading the assay plate because these parameters determine how data is displayed and analyzed You can set up or modify templates reduction and display parameters after collection but this may be complicated or confusing Refer to your SOFT max PRO User s Manual for information about using the software to continue to prepare for an experiment Note While it is strongly recommended that you use the Template Editor before running an experiment it is not strictly necessary The values received from the instrument are raw fluorescence and are not affected by settings in the Template Editor FlexStation Il Operator s Manual Rev D 3 7 Readin
126. tion II Systems provide integrated fluid transfer capability from a source plate or reservoir to an assay plate with concurrent kinetic fluorometric analysis of reactions Data collection speed is sufficient for running fast cell based kinetic assays The instruments feature a bottom read mode ideal for whole cell based assays and may be automatically switched to a top read mode for solution phase reactions FlexStation II Systems can be used for drug discovery research and development secondary and post screen analysis The instruments are available in two models e The FlexStation II instrument is designed specifically for 96 well microplates e The FlexStation 1384 instrument is designed for 96 and 384 well microplates The instruments offer 96 and 384 well microplate to microplate fluid transfer 8 or 16 wells at a time and can read microplates having 6 12 24 48 96 and 384 well formats Top or bottom detection is available with a simple click of a button A microplate adapter is required in the reading chamber drawer for 96 and 384 well assay plates for both bottom and top reading The black adapter allows for optimal signal for bottom readings and the purple adapter optimizes top read scenarios A different baseplate is provided for the compound plate drawer The instrument is controlled by an external personal computer running the SOFTmax PRO software which provides integrated instrument control data display and st
127. to restrict interference from reflected excitation light Among these features is a set of band pass cutoff filters that may be set automatically by the system or manually by the user In addition if the Stokes shift is small it may be advisable to choose an excitation wavelength that is as far FlexStation Operator s Manual Rev D 19 1 System Description away from the emission maximum as possible while still being able to stimulate the fluorophore This allows a more accurate quantitation of light emission Excitation Fluorophore s Fluorophore s Reading Excitation Emission Wavelength Maximum Maximum rm Emission Reading Wavelength Relative Fluorescence 500 550 600 650 Wavelength nm Optimized Excitation and Emission Reading Wavelengths The figure above shows an example of a fluorophore with small Stokes shift When the reading wavelengths for excitation and emission are separated a smaller amount of excitation light will pass through to the emission monochromator gray area and on to the PMT resulting in a purer emission signal and more accurate data The instrument allows scanning of both excitation and emission wavelengths using separate tunable monochromators One benefit of being able to scan emission spectra is that you can assess more accurately whether the emission is due to the expected fluorophore multiple fluorophores or a variety of background sources or contaminants Another benefit is that if i
128. tual maximum volume allowed in the wells you are using as you move through the settings in this dialog box The maximum cumulative volume depends on the assay plate type you select fs Assay Plate Fluid mennene testen peter eeevee serere eet vere Initial Volume ul i Transfers 1 vw A Transfer 1 i RS Transfer Settings ee RE H Time Point secs Volume ul Minimum time 16 secs Compound Transfer Dialog Window Assay Plate Fluid Initial Volume Enter a value in the Assay Plate Fluid box that equals the largest initial volume prior to compound transfers for any well in the assay plate This value can be set at 0 269 microliters pL although obviously typical values will be about 10 200 pL for a 96 well microplate SOFTmax PRO assumes all wells hold the same initial volume The software uses this value to compute the total volume in each well after all fluids have been dispensed The software makes this calculation to warn you regarding potential overflow of fluid from the wells If there is no fluid volume in the assay plate prior to compound transfers do not enter a value in this box Any value entered is saved with the file but the value is not displayed anywhere except in the Compound Transfer setting Values for a 384 well microplate can be set at 0 120 pL 64 FlexStation Operator s Manual Rev D 3 5 Configuring Instrument Settings in Flex Mode
129. u may call Molecular Devices Corporation at the telephone number or address below to locate the nearest service facility You must schedule warranty service prior to bringing or shipping the product for servicing At the time of requesting warranty service you must present proof of purchase documentation which includes the date of purchase and Molecular Devices Corporation must have the enclosed Warranty Registration form completed signed and returned by you within ten 10 working days of the date of delivery This warranty only covers defects arising under normal usage and does not cover malfunctions of failures resulting from misuse abuse neglect alteration modification or repairs by other than an authorized Molecular Devices Corporation service facility Repair or replacement as provided under this warranty is the exclusive remedy to the purchaser the Buyer Molecular Devices Corporation the Seller shall not be liable for any incidental or consequential damages for breach of any express or implied warranty on this product except to the extent required by applicable law The Seller specifically excludes all express and implied warranties including without limitation any implied warranty that the products sold under this agreement are merchantable or are fit for any particular purpose except such warranties expressly identified as warranties and set forth in the seller s current operating manual catalog or written guarantee coverin
130. ud Important If you have correctly configured the settings in the Preferences dialog box as described in the steps above and you are still observing problems a red X over the Flex icon no temperature in the temperature display box etc then you will have to take further steps to establish communication between the computer and the instrument or FlexStation Operator s Manual Rev D 51 3 Operating Procedures to resolve a different instrument problem See part 6 Troubleshooting Procedures for instructions View Flex mode setting The SOFTmax PRO application defaults to Flex mode every time you start the software You can confirm this mode if desired by selecting Instrument Setup from the Control drop down menu You can also click on the Setup button on the Plate Section tool bar to view the Instrument Settings screen Ww Instrument Settings x Endpoint Options Wavelengths 485 525 515 Sensitivity Readings 6 PMT High Timing Time 120 secs Interval 2 secs Reads 61 Automix Before Off Between Off AutoCalibrate Once Wells To Read e Ex Em Auto Cutoff On Assay Plate Type 96 Well Standard Read area amp 1 H10 Compound Source vo Kinetic Spectrum Well Scan FLEX Wavelengths Number of Wavelengths ERA Excitation Emission M Auto Cuttoff pes Jr Es Jr cs 7 Minimum Interval 1 52 secs Ret E RUNS D Pri LOSING EIN ee Re
131. ull both fuses out of the holder and discard them c Insert new slow blow rated fuses into the fuse holder Either end of the fuse may be forward 5 Replace fuse box a Insert the fuse box into the opening in the instrument making sure that the fuses are on the right side toward the power receptacle b Press the fuse box into place making sure the cover snaps closed 6 Reconnect the instrument a Plug the power cord to the back into the instrument and then into the wall outlet b Reconnect other cables previously disconnected FlexStation Operator s Manual Rev D 91 5 Service and Maintenance Procedures 5 7 Replacing the Flash Lamp The flash lamp should be replaced when an error message appears in SOFTmax PRO to indicate replacement is necessary Caution This procedure requires trained service personnel The instrument warranty may be voided unless lamp is replaced according to these Instructions Caution Use caution during this procedure Follow these instructions and any instruction labels on the instrument exactly Requires trained service personnel LAMP COVER WARRANTY MAY BE VOIDED Disconnect power cord unless lamp is replaced according to before removing instructions in the operators manual under Flash Lamp Replacement Lamp Cover Flash Lamp Location gt Tools Required e Allen and or ball driver hex wrenches 3 32 9 64 5 32 e 2 Phillips screwdriver 1 Turn off power T
132. ument drawer manually 1 Ifthe power is not already off as can occur with a power failure or if you are already troubleshooting the instrument with the covers open turn off the power Disconnect the power cord from the instrument and the computer cables from the computer Warning The power must be off for this procedure If the power is not disconnected and a jammed carriage or drawer is suddenly freed the instrument could suddenly move resulting in operator injury 2 Using your fingernail or a narrow blade inserted about V6 inch open the drawer door to its horizontal position and pull out the carriage slowly and as gently as possible Caution Never force a carriage out of the instrument if it does not come out reasonably easily Contact Technical Support FlexStation Operator s Manual Rev D 99 6 Troubleshooting Procedures 6 3 Understanding Potential Mechanical Problems 100 Important Read these instructions before attempting to use the FlexStation ll instrument in Flex mode 6 3 1 Background The FlexStation II instrument s intended function dictates that it have many moving parts Furthermore the operator interacts extensively with the instrument by introducing various types of plastic disposable plates tips and troughs Molecular Devices has made every effort to ensure smooth and reliable operation of the FlexStation II instrument However problems can occur For example e Pipette tips can jam in the
133. up parameters chosen in SOFT max PRO but is limited to 2 hours and 45 minutes 165 minutes At the end of a reading rates are reported as RFU second for each well Kinetic analysis has many advantages when 25 1 System Description 26 determining the relative activity of an enzyme in different types of assays including the purification and characterization of enzymes and enzyme conjugates In Kinetic mode the fluidics module is not enabled For more information on this mode please review the appropriate section in the SOFTmax PRO User s Manual Spectrum Mode Spectral analysis measures fluorescence across a spectrum of wavelengths excitation 250 850 nm emission 360 850 nm When reading using fluorescence you may set a fixed wavelength for excitation and scan the emission wavelengths and vice versa All spectrum readings are made using scanning monochromators In Spectrum mode the fluidics module is not enabled For more information on this mode please review the appropriate section in the SOFTmax PRO User s Manual Well Scan Mode Some applications that involve the detection of whole cells in large area tissue culture plates may require the use of Well Scan mode As many cell lines tend to grow in aggregates or in the edges of microplate wells this non confluent growth pattern may require multiple reads at different locations in a well When used with 6 12 24 48 or 96 well plates well scanning allows maxi
134. urn off the instrument power switch and unplug the power cord and the computer cable from the rear of the instrument 2 Remove lamp cover a Remove the Phillips head screw on the top of the cover and lift the cover off vertically b Set the cover aside and keep track of the screw Note There is an interlock switch which is depressed by the lamp cover which interrupts power to the flash lamp automatically when the cover is removed 92 FlexStation Il Operator s Manual Rev D 5 7 Replacing the Flash Lamp Screw Lamp Cover Assembly Removing the Lamp Cover 3 Loosen screws on flash lamp assembly a Usea 9 64 hex Allen wrench to loosen the clamp screw located on the lamp mounting block and facing the rear of the instrument Leave the clamp screw in place b Use a 5 32 hex ball end driver or Allen wrench to loosen the two vertical mounting cap screws that are holding the Litepac mounting block to the base plate They are located toward the inside of the lamp s mounting box The two cap screws will stay in a raised position spring loaded so that you can slide out the entire Litepac without the screws getting caught in the holes Note You may need a flashlight to see the cap screws Vertical Mounting Screws Spring loaded but shown raised here for identification Flash Lamp Assembly Removing Litepac Mounting Block Screws FlexStation Operator s Manual Rev D 93 5 Serv
135. veal the dialog box The value allowed is 0 200 pL for 96 wells and 0 30 pL for 384 wells Pipette Tip ws Air Gap Setting Set the pipetter air gap to a value between 0 and 200 pl p Cancel Air Gap Setting Pipette Tip Air Gap Pipette Tip Air Gap 72 FlexStation Operator s Manual Rev D 3 5 Configuring Instrument Settings in Flex Mode 3 5 15 Settings Displayed in Plate Sections The Plate section will provide visual feedback on all instrument settings in the Instrument Settings Box the gray area to the far right of the Plate section Information about wells to read and transfer settings are also displayed in the Data Display section For example the figure below shows a Plate section with three sets of transfers The wells with blue squares in their upper left corners were selected for the first transfer wells with red triangles upper right corners were selected for the second transfer and wells with a green triangle lower left corner were selected for the third transfer v Gi Pletewt l E Max Min Plate 1 1 2 3 4 FLEX Time 120 secs Interval 2secs Reads 61 Fluorescence j Bottom read i Ex Em Cutoff Lm1 485 525 515 Buto Cutott Automix Off Calibrate On PMT High ReadsMell 6 Lag Time 0 j End Time 120 RFU Min 0 RFU Max 6000 Wavelength Combination Lm1 Trans1 H 50y R 6 50p 15 Trans2 H 50y R 1 V 100y 51 Pla
136. ver consists of a power switch fuse box cover and power cord receptacle e The lower panel consists of an RS 232 serial port and parallel port not currently active There are also a number of labels Note See section 2 6 Connecting the Cables for information about attaching the computer cable and power cords to the instrument Fuse Box Cover Power Switch WM WIM WWMM WWM Parallel Port not currently in use i ie Power Cord RS 232 Serial Cable Power Cord Receptacle RS 232 Serial Port Input Output Panels Lamp Cover The plastic lamp cover provides access to the flash lamp on the right side of the instrument as viewed from the rear Note For further information see section 5 7 Replacing the Flash Lamp Caution Flash lamp access and maintenance are restricted to service trained users Lamp Cover Rear View FlexStation Il Operator s Manual Rev D 1 4 System Components 1 4 2 Drawers The instrument has three drawers that open on the right side The two drawers in the fluidics module open and close to move the pipette tip rack and compound plates or reservoirs into and out of the instrument The reading chamber drawer in the detection module transports the assay microplate into the reading chamber Tip Rack Drawer Compound Plate Drawer Reading Chamber Drawer Instrument with Drawers Open and Carriages Accessible Small
137. will free the mechanism for easier removal Alignment Pins Pipettor Head Detail Note If you want to dispense liquid from the tips skip the next two steps and see section 6 4 6 Expelling Undispensed Fluid from Tips next 8 To completely remove the pipettor head unscrew the black connector at the end of the white spiral cord from its secure position around the receptacle 9 Pull the connector out of the receptacle and take the pipettor head the rest of the way out of the fluidics module 110 FlexStation Il Operator s Manual Rev D 6 4 Recovering from Mechanical Problems in Flex Mode 6 4 6 Expelling Undispensed Fluid from Tips Biohazard This procedure includes steps involving potentially infectious biological agents requiring that proper handling precautions be taken Follow your institution s biohazard protocol This procedure assumes you have opened the instrument and accessed the pipettor head as described in the above sections 1 Lift the pipettor head out of the fluidics module carefully as it is still connected inside the instrument 2 Hold the pipettor head over a tray or something into which you can expel liquid and remove tips 3 With your other hand rotate the black pulley on the bottom of the pipettor motor clockwise This moves the plungers and any liquid will be expelled and any tips will be ejected gt Replace the Pipettor Head Note See section 2 4

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