FACSCalibur and FACScan

1FACSCalibur and FACScanInstructions for RunningTroubleshootingTips & Tricks3.12.2010Perttu Terho

2Instructions for running the samples with FACSCalibur .......................................................... 3Instrument startup .................................................................................................................. 3Setting up the Cytometer ....................................................................................................... 3Setting up the Computer ........................................................................................................ 4Setting up the settings ........................................................................................................... 4Before the run ........................................................................................................................ 5When you run ......................................................................................................................... 6Shut down procedure and washes ........................................................................................ 7Instructions for FACScan ............................................................................................................. 8Setting up the Cytometer ....................................................................................................... 8Troubleshooting Checklist for FACSCalibur .............................................................................. 9Problems and solutions for FACSCalibur ................................................................................. 10The connection between cytometer and computer is broken .............................................. 10Pressures are not on ........................................................................................................... 10Needle has been clogged .................................................................................................... 11Cap of the sheath flow tank is not tightly closed / it’s broken .............................................. 11Malfunction of the system fluidics ........................................................................................ 12The black metal cover of sheath flow tank is not locked ..................................................... 12Crack on the sample tube .................................................................................................... 13SET-button has been forgotten to push ............................................................................... 13The O-ring on the top of the needle has worn out ............................................................... 13Air bubbles inside the flow filter ........................................................................................... 14Air bubble in the filter tube ................................................................................................... 14Dust in the tube coming from the sheath flow tank .............................................................. 15“CellQuest Pro has unexpectedly quit” ................................................................................ 15The life span of the laser is near it’s end ............................................................................. 15The life span of the power supply of the laser is near it’s end ............................................. 15The backslash connectors of the fluidics system are not connected / broken .................... 16The metal tube of sheath flow “buoy” is loose / dropped ..................................................... 16The malfunction of FACScan when started ......................................................................... 16The laser doesn’t ignite ........................................................................................................ 17Which channel was which in CellQuest Pro? ........................................................................... 18The channels that are excitated with blue laser .................................................................. 18The channel that is excitated with red laser ........................................................................ 18Shortcut keys for CellQuest Pro ................................................................................................ 19If CellQuest Pro jams… ............................................................................................................... 20If the whole computer jams… ..................................................................................................... 21

3Instructions for running the samples withFACSCaliburHere it is assumed, that you have already the acquisition sheet and settings (fromprevious experiments).Instrument startupTurn on the CytometerIt takes about 5 minutes to warm up. However, you can set up the computer andcytometer, while the instrument is warming up.Turn on the ComputerIf computer is already on but cytometer is not, turn off the computer. Then startcytometer, and after that computer. This is the way they can establish the connection.Setting up the CytometerCheck that- Sheath fluid tank is full- Waste-tank is empty- The caps of the tanks aretightly closedPut the pressure on from thepressure buttonPressure buttonSheath fluidWasteTurn the arm to right (or left),remove the water tubeLeave the arm turned to the side. Never turn the arm tothe middle if there is no tube.Press PrimePrime empties the whole liquid system and re-fills it.This protocol removes the possible air bubbles. Primetakes about 25 seconds. After that, the instrumentturns automatically back to Standby.Press Prime againJust in case.

4Put the water tube back to the instrument, and turn the arm to the middle positionPress RUNNow instrument starts to take water. It’s only good that it rinses itself while you set upthe computer.Setting up the ComputerStart CellQuestFrom top menu ‘Acquire’ – ‘Connect to Cytometer’Acquisition-window and Browser-window are opened.From top menu ‘Acquire’ – ‘Counters’Counters-window is opened. You can expand it by pressing the green balloon on thecounter-window’s left top corner.Push Apple-button down and press 1,2,3 and 4Four boxes appear- Detectors / Amps- Threshold- Compensation- StatusClose “Untitled”-sheed by pressing red balloon from the left top corner of the sheetAnswer “Don’t save” when the program asks whether you want to save the changes.Setting up the settingsAcquisition sheet:From Top menu: File – Open document…Find and open your acquisition sheet. By moving the blue horizontal bar to the left you’llfind USERDATA-folder.If there are two USERDATA-folders, choose the upper one.

5Cytometer settings:From top menu: Cytometer – Instrument settings- Choose open- Find the tube, which has been run with the samesettings you would to use now, press Open- Press SET. You should see the settings valueschanging.- Press DONE.Browser for setting up the data storage location:From top menu: Acquire – Parameter descriptionThe new browser window should be opened, with thename of your acquisition sheet.From the browser window press “Change…” from DirectoryGo to your folder and create a new folder by pressing ”New folder”. Select the folderyou just created and press “Choose”.From the browser window press “Change…” from FileDefine the file name:- For the first row (custom prefix) write the file name prefix. It is recommended to usethe date as file name (e.g. 2010-06-23)- Next drop-down menu should be “Custom prefix”- Next drop down menu should be “File count”- The number in the File count box should be 1. If it’s not, change it to 1.Choose the directoryby clicking thisChange the file nameby clicking thisBefore the runNow the system should be ready to run. Before running, check the Status-box thateverything is alright. Cytometer status must be “Ready”.If it’s “Not ready”

6- Most probably the lasers haven’t warmed up yet. Wait for couple of minutesIt it’s “Standby”- Check that you have put the pressure on from the pressure button- Check that the status of the instrument is RUN- Check that there are no cracks in the sample tube. If there are (even very tiny),throw the tube away and take a new one.If it still doesn’t workCheck the troubleshooting from this folder.If you can’t solve the problem, call system administrator.When you runVortex every sample wellWhen you put the sample into the instrument, the pressure chooser should be “low”Remember to uncheck “Setup”, otherwise nothing will be savedDoesn’t saveSavesRun your sampleThe running speed with LO should be less than 1000 cells/sec. If it is higher, yoursample might be too concentrated and the risk of clogging increases. You should diluteyour sample a bit.If you want to stop the run before the set cell number has been achievedPress PAUSE and SAVE. The run will stop and the cells run so far will be saved. Thefile count will increase by one.If you want to cancel the run (e.g. for setting the instrument)Press PAUSE and ABORT. The run will stop. The tube is not saved. File count don’tincrease.

7Shut down procedure and washesPut the instrument run speed to HI.Water- Put a little bit over half tube of DI water into the instrument- Let it take water about 30 sec when the arm is on left or right side- Put the arm to the middle- Let it take water about 3 minutesDeconRepeat the procedure described above, but now with decon.Water once moreRepeat the procedure, now with water.Press StandbyRelease the pressure from the pressure button- You should hear pfffffff –soundRemove the waste tank, pour the waste into the sink. Pour about 20 ml 5%hypochlorite (green liquid) into the waste tank.Open the cap of the sheath fluid tank, fill it with FACSFlow. There is a line drawn tothe side of the tank, don’t fill over that line.Close CellQuest- For every open sheet, the program asks whether you would like to save them. If youhave saved them already, just select “Don’t save”.Log your usage- Go to the reservation book: http://services.btk.fi/reservation- Find your reservation- Press “I’m done”-button and follow the instructionsCheck whether you were the last user of the dayIf you were the last user, or next user is coming after 2 hours, wait for 5 minutes andshut down the instrument.If someone is coming during next 2 hours, you can let the instrument on. However, if thenext reservation is after 5 pm, shut down the instrument.

8Instructions for FACScanRunning the samples with FACScan is 90% similar to FACSCalibur. The only bigdifference is “Setting up the Cytometer” (page 3), which can be found here.Setting up the CytometerCheck that- Sheath fluid tank is full- Waste-tank is empty- The caps of the tanks aretightly closedPut the pressure on from thepressure buttonFluid tankPressurebuttonWaste-tankTurn the arm to right (or left), remove the water tubeLeave the arm turned to the side. Never turn the arm to themiddle if there is no tube.Turn the status selector to “DRAIN”, wait for 10 secondsThis empties the fluidics systemTurn the status selector to “FILL”, wait for 10 secondsThis fills it again.Repeat “DRAIN” and “FILL”Turn the status selector to “RUN”Put the water tube back, and move the arm to the middleNow instrument starts to take water. It’s only good that it rinses itself while you set upthe computer.

9Troubleshooting Checklist for FACSCaliburThere are three most common problems with FACSCalibur:- You can’t see the cells- The instrument is on “Standby”, although it shouldn’t be- The cells on the screen look different than before (the populations are not therewhere they should be)When you confront the problem, please go this checklist through. For every list item,there is a page number, where you can find the more specific instructions fortroubleshooting.If you still can’t get the instrument working, call:Perttu 050-5265 710Markku 041-5072 510

10Problems and solutions for FACSCaliburThe connection between cytometer and computer is brokenSymptomsIf the computer attached to cytometer has been started before the cytometer, or thecomputer has left on after the shut down of cytometer and cytometer has then restarted,the connection between the cytometer and computer is broken.Although CellQuest behaves normally (you can connect to cytometer and run) andStatus-box says that everything is normal, no cells can be seen.DiagnosticsChange FSC voltage multiplier to E03 fromDetectors/Amps-box (threshold must be FSC, valueabout 50, no sec threshold).Then start the run (by pressing “Acquire”). If theconnection is ok, you should see something on FCS-SSCdot plot (this is detector noise, nothing “real”). If you stillcan’t see anything, the connection is broken.Here you should see the cells, andcounter should show even hundreds ofevents/secSolutionRestart the computer. During the startup, the connection between the computer andcytometer will be re-established.Pressures are not onSymptomsWhen you try to run the sample, the computer says “Cytometer status is not ready”.DiagnosticsCheck the on/off pressure button between the tanks; is it on?

11SolutionPut the pressure button “ON”, wait for 10 seconds, and then try again.Needle has been cloggedSymptomsCytometer should be ok, but no cells can be seen. You have already tested theconnection between cytometer and computer from “The connection between cytometerand computer is broken” (page 10) and saw the detector noise.DiagnosticsPut the water tube running (check that the tube is not broken), put the flow speed to HI.Draw the sharp line to the tube, marking the current level of water.Wait for couple of minutes. If the water level hasn’t decreased, most probably theneedle has been clogged.SolutionTake the tube away, and execute PRIME at least two times.Take the half tube of FACSClean solution (can be found above sink). Don’t spill on yourclothes (liquid contains bleach). Put the tube running, and mark the level of the liquid.Run the tube with HI flow speed for couple of minutes.If FACSClean is able to run through the system, throw the FACSClean tube away andrinse the needle with new water tube. Run again for couple of minutes.If you feel, that the FACSClean treatment didn’t work, repeat PRIME and FACSClean.Cap of the sheath flow tank is not tightly closed / it’s brokenSymptomsCytometer should be ok, but status is “Standby”.DiagnosticsTry to tighten the cap of sheath flow tank; was it slightly open? Check the cap: can yousee possible cracks, where the pressure could escape. Are the screws (both cap andtank) ok?SolutionIn many cases it’s enough to tighten the cap. If the cap is broken, ask the instrumentsupport to change the cap. If the screws of tank are broken, ask them to change thecank.

12Malfunction of the system fluidicsSymptomsCytometer should be ok, but no cells can be seen. You have already checked that theconnection is ok (The connection between cytometer and computer is broken, page 10).You think that the clogging of the needle isn’t probable. The instrument has been on forseveral hours.DiagnosticsWhen pressures are on and instrument isRUN, open the cap of the waste container.Raise the metal part up, still keeping itabove the hole.If the fluidics system works normally, youshould see the system dropping liquid intothe waste couple of drops / sec.If you can’t see anything dropping into thewaste, the whole fluidics system has beenstopped.SolutionMost probably some magnetic valve has jammed into wrong position because ofoverheating.The black metal cover of sheath flow tank is not lockedSymptomsPressure is on, everything should be in order, but instrument is on Standby.DiagnosticsCheck the black metal cover on the top of flow tank. Cover has holes, and you can lockit into correct position by pushing down and pulling back. The instrument knows that it islocked, when the cover pushes the switch on the right side of the cover. If the cover isnot locked, it doesn’t push the switch and instrument is on Standby.SolutionLock the cover.

13Crack on the sample tubeSymptomsEverything should be ok, but instrument is on Standby.DiagnosticsCheck your tube. Can you see any cracks, even very tiny ones? If there are cracks, thepressure leaks out and instrument’s status is Standby.SolutionChange your sample into new tube.SET-button has been forgotten to pushSymptomsEverything should be ok, but the cells don’t look correct (they don’t fit into gates etc.).The settings have been loaded from some old tube.DiagnosticsChoose from the top menu Cytometer – Instrument Settings. Open the settings again.Compare the loaded settings to the instrument settings (in Detectors/Amps etc.) If thesediffer, you have forgotten to press SET-button after loading the settings.SolutionPress SET, see the settings changing.The O-ring on the top of the needle has worn outSymptomsThe cell running speed is low. Whenopening the arm holding the tube,the tube might drop.DiagnosticsIt’s hard to tell whether the O-ringhas worn out, it may look justnormal.O-ringO-rengasConeKartioSuojakuoriNeedle coverSolution

14Ask support to change the O-ring.For support persons: Unscrew the Cone, and pull the needle cover down. Now you canchange the O-ring (those having the metal spring inside). After changing, push theneedle cover back as up as it goes. Screw the cone tight.Air bubbles inside the flow filterDiagnosticsThere is a filter between Sheath flow tank and waste tank. The fluidics is running fromdown to up. Sometimes air bubbles may stick into the filter, preventing the running ofthe fluidics. One should see the possible air bubbles inside the filter.Sometimes the filter is so full of air, that no fluidics can be seen. It’s difficult to estimatewhether the filter is full of air or full of liquid. However, the case where the whole filter isfilled with air is very rare.SolutionRemove the air with bypass valve. While pressure ison, open the tube squeezer. You should see thefluidics running the bypass tube into the waste. Letthe fluidics run until all air bubbles have beenremoved. You can slightly knock the filter to get thestuck air bubbles moving.Close the tube squeezer accurately after the air removal procedure.Air bubble in the filter tubeDiagnosticsThe sheath filter between sheath tank and waste tank containstwo output tubes; thick and thin (bypass tube). Sometimes airbubble sticks into the middle of the thick tube. This bubblemight prevent the fluidics from running properly.SolutionWhen the pressure is on, disconnect the connector going to theinstrument. The backslash connector, so nothing goes through, unless it’s properlyconnected.You can make the fluidics running through the connector by pushing the head of theconnector. Gently push the head and let the air bubble come out. Connect theconnector back to the instrument.

15Dust in the tube coming from the sheath flow tankDiagnosticsYou can see the dust ball in the tube coming from sheath flow tank.SolutionDisconnect the connector of that tube. Put the pressure on. Squeeze the tube verytightly, so that you can block the flow with your fingers.Take the connector off from the tube (most probably the dust ball can’t go throughconnector but sticks there). Loose the tube slightly and let the fluidics rinse until the dustball has come out.Squeeze the tube again, put the connector back to the tube and connect the connectorto the instrument.“CellQuest Pro has unexpectedly quit”SymptomsCellQuest crashes in the middle of the usage. This might happen especially when youare opening the analysis sheet which has been created with another computer.SolutionIf you were running the samples or creating new objects, you just have to restart theprogram and try again.If you were trying to open the analysis sheet and failed, the only possibility is to recreatethe analysis sheet.The life span of the laser is near it’s endDiagnosticsThe “Laser Current” on Status Box is near 8.SolutionAsk the support to order the service to change the laser. This can’t be done withoutprofessional’s help, because the aligning requires special tools.The life span of the power supply of the laser is near it’s end

16SymptomsAfter turning on the instrument it takes longer and longer, before instrument status is“Ready”SolutionAsk support to change the power supply, if there is one.For support: If you get the permission from BD service, you can change it by yourself.It’s relatively easy to unplug the wires, change the power supply and re-plug the wires.The backslash connectors of the fluidics system are notconnected / brokenSymptomsThe fluidics doesn’t run. The symptoms may be quite similar to “Malfunction of thesystem fluidics” (page 12).SolutionTry to open and close the connectors. If they are not properly connected, they mightprevent the flow of the system fluidics.This problem is very rare; the other solutions should be gone through before this.The metal tube of sheath flow “buoy” is loose / droppedSymptomsNo cells can be seen. The symptoms may bequite similar to “Malfunction of the systemfluidics” (page 12).SolutionOpen the sheath flow tank and check the metaltube. Push it up so, that it will be tightlyconnected to the buoy.In extreme cases the whole metal tube has dropped and can be found inside the sheathtank.The malfunction of FACScan when startedSymptoms

17FACScan is on, but CellQuest can’t connect to the instrument. Even if the connectionsucceeds, the status is “Not Ready”.DiagnosticsFACScan front panel should look like this:If you can’t see ”E00” and ”052” texts, it’s probable that FACScan hasn’t startedproperly.SolutionShut the instrument down, wait for 10 seconds and restart it.The laser doesn’t igniteSymptomsThe Status box of CellQuest says that “Laser Power” is 0.DiagnosticsThe information from status box is already enough to tell, that the laser hasn’t ignited.You can check the situation by opening the instrument and trying to see whether youcan see the blue gloom inside the laser (seeing might be a bit tricky). If you can’t seeanything, it’s probable that the laser is not on.SolutionShut down the cytometer and wait for 10 seconds. Restart. In most cases the problemis solved by restarting. If not, try again.If this problem is frequent, check “The life span of the laser is near it’s end” (page 15)and “The life span of the power supply of the laser is near it’s end” (page 15).

18Which channel was which in CellQuest Pro?The channels that are excitated with blue laserThe excitation laser is 488 nm.FSCSSCFL1FL2FL3Forward Scatter = Cell sizeSide Scatter = Cell granularityGreen (530 ± 15 nm)FITC, Alexa488, GFP, YFP, CFSE, Fluo-4 etc.Orange (585 ± 21 nm)R-PE (PE), Cy3, Alexa555, PI etc.Red (670 – nm)PerCP, PerCP-Cy5.5 etc.The channel that is excitated with red laserThe excitation laser is 633 nm.FL4Red (661 ± 8 nm)APC, Alexa633, Alexa647 etc.

19Shortcut keys for CellQuest ProAASelect all windows (if now window is active)Select all regions inside the window (if one window is active)D Select the new file to all selected windows} Jump to the next file in directory (affects all selected windows){ Jump to the previous file in directory (affects all selected windows)Delete (window or region). Attention! Del-button doesn’t workCVXP[click]Copy (same as CTRL-C in Windows)Paste (same as CTRL-V in Windows)Cut (same as CTRL-X in Windows)PrintSelect many windows (by clicking the border of the window)Attention! Window can be in three stages: Not selected, Selected or ActiveNot selected Selected Active(White border) (rectangles on the corners) (colorful border)You can unselect all by clicking the mouse e.g. the background of the sheet.You can select the window by clicking its border.You can activate the window by clicking it in the middle.Inspector shows the values of the window ONLY when the window is SELECTED.

20If CellQuest Pro jams…Sometimes CellQuest Pro jams and keeps rotating the colorful balloon-mouse icon.Nothing works.Start task manager2) Then pressESC1) Push thesetwo buttonsdownBy this way you can get “Force Quit Applications”-window open.Close CellQuest Pro from the menu.If you can find “Classic Environment” from the list:- Instead of selecting CellQuest, select Classic Environment and Force Quit- Try whether CellQuest works again.- If not, open “Force Quit Applications” again, select CellQuest Pro and Force Quit

21If the whole computer jams…Sometimes the whole computer jams;- Nothing happends, whatever you do- The screen goes black- Etc.If this occurs, you can force the computer to shut down.Keep the powerbutton pushed downfor few seconds.