NOTES ON FACSCalibur USE AND TROUBLESHOOTING
Contents
1. an hour turn off the computer then the FACScalibur 9o ONE mcos Trouble Shooting Here are some frequently encountered problems and suggested ways to overcome them Please attempt these before contacting Geza or Michael Pm not seeing any events on the screen Ensure that the system is pressurised check e The pressure release switch is in the correct position All tubing is plugged in properly and un kinked That there is no air bubbles in the sheath tubing That the O ring is tight enough to form a seal with your tube of cells Your tube of cells has no cracks or breaches in it Ensure that your threshold amp baseline voltages are not set too high or too low If you re still not seeing events try shutting down the computer not restart wait 10 seconds and start the computer up again This re boots the communication between the compauter and instrument There is no sheath coming through the SIP when it is on RUN Make sure the system is pressurised see the above checklist If this is still an issue there may be a blockage With no tube on the SIP and the arm to the side press PRIME twice and press run again If this has not solved the problem attempt the blockage removal listed below The best blockage prevention is proper sample preparation Refer Sample Preparation section of these notes Blockage removal Solutions e Run fresh hypochlorite at HI flow rate for 5 15 minutes there is a stock2. be much smaller o open Parameters Saved and make sure that all the parameters you are aquiring have their boxes checked Unchecking boxes for parameters that aren t being used will make files smaller FACS analysis of Human and Infectious Samples Treat ALL human samples as infectious and follow this protocol Deu ES D Virus infected samples should also be treated as follows Wear gloves a gown and protective glasses Ensure that there is at least 100 ml neat hypochlorite in the waste tank Place Glad Wrap on the computer key board and mouse On completion of acquisition of files ran 1 hypochlorite for 5 minutes followed by Milli Q water for a further 5 minutes Remove Glad Wrap and swab down the bench area and cytometer surfaces with 70 ethanol Remove the waste tank and store for 30 minutes not days Empty the waste tank into the sink and flush away the contents with copious volumes of water Setting detectors amps and compensation windows e Forward scattered light FSC proportional to cell surface area or size e Side scattered light SSC proportional to cell granularity e FLI FL4 detectors measure fluorescence emission wavelength bands Excitation Emission Frequently Used BRENNEN 488nm Blue Laser FL 530nm FITC GFP CFSE FL 3 670nm PerCP PerCP Cy5 5 PE Texas Red PI TAAD 635nm Red Laser FL 4 661nm e From Becton Dickinson FACSCalibur System User s Guide 02 61760 02 Control
3. samples Cells alone e Single stained samples for FL1 FL2 FL3 and FLA as necessary Use an antibody that will stain a large proportion of cells but not all eg CD4 or CD8 on splenocytes This allows the location of both positive and negative cells to be assessed Adjusting detector sensitivity 1 2 Qut dw 2 9o Open Detectors Amps and Compensation windows under Cytometer menu If using the FLA detector check the Four Color box at the bottom of the Detectors Amps window This will turn on the red diode laser Wait 10 min for the laser to warm up before proceeding Set all Compensation values to 0 Turn fluid control dial to RUN and place your cells alone sample on the probe Set up a FSC vs SSC dot plot Adjust FSC Voltage and Amp Gain and SSC Voltage as required so that your populations sit clearly within the dot plot and are not scrunched up against an axis Place a gate around your population of interest Set up FL1 vs FL2 FL2 vs FL3 and FL3 vs FLA dot plots and FL1 FL2 FL3 and FLA histograms as necessary and set the gates on GI RI Set Mode to Log for FL1 FL2 FL3 and FLA in the Detectors Amps window Adjust the FL1 FL2 FL3 and FLA Voltages so the negative population in the cells alone sample sits between 10 and 10 on the histograms and in the bottom left hand corner of the dot plots 10 Place your single stained FITC control sample on the probe Check that the negative population still sits between
4. the Browser and collect single stained controls Open the Acquisition and Storage window under the Acquire menu bar and fill in the number of events you wish to collect and in what region eg GI RI e A Resolution of 256 channels is usually adequate for routine analysis rather than 1024 channels and stores less data For for best quality publication plots acquire at maximum resolution and increase the number of acquired events to twice the above recommendations e Save your instument settings in the InstrSettings window under the Cytometer menu bar Post run Procedure 1 Allow several drops of fluid to fall from the probe to flush the probe and flow cell 2 Run 1 hypochlorite for 3 minutes on HI flow rate last user of the day 10 minutes Keep cap on tubes contaiing 1 hypochlorite as the bleach will oxidize and be useless RUN MilliQ water for 3 minutes on HI flow rate last user of the day 5 minutes Press the STANDBY button Depressurise the sheath tank and refill with millQ water Empty the waste tank and refil with a small amount of 1096 bleach Quit CELLQuest Transfer files to your own computer and ensure that the files have been transferred by checking on your computer Old files are routinely deleted from the Apple Mac computers and no responsibility is taken by AMREP Flow for lost data if you have not removed it in a timely manner 9 Unless you can have contacted and confirmed that the next user will arrive within
5. 10 and 10 on the FL1 histogram 11 Adjust the FL2 FL1 compensation while viewing the FL1 vs FL2 dot plot Often the positive cells will have a wider scatter than the negative cells Adjust so that the median of each population is the same There is a risk of overcompensation if you try to get the population within the same quadrant 12 Place your single stained PE control sample on the probe Check that the negative population still sits between 10 and 10 on the FL2 histogram Adjust the FL1 FL2 compensation while viewing the FL1 vs FL2 dot plot Adjust the FL3 FL2 compensation while viewing the FL2 vs FL3 dot plot 13 Place your single stain control sample for the FL3 detector eg TRI COLOR or PERCP on the probe Check the negative population still sites between 10 and 10 on the FL3 histogram 14 Adjust the FL2 FL3 compensation while viewing the FL2 vs FL3 dot plot and the FL4 FL3 compensation while viewing the FL3 vs FL4 dot plot as before 15 If doing four colour work place your single stained APC control sample on the probe Check the negative population still sits between 10 and 10 on the FLA histograms 16 Adjust the FL3 FL4 compensation while viewing the FL3 vs FLA dot plot as before NOTE If you have to change the voltage of any detector during compensation you must check the compensation on ALL single stained tubes again Aquiring amp Saving data Uncheck the Setup box in the Acquisition Control window of
6. NOTES ON FACSCalibur USE AND TROUBLESHOOTING PROCEDURES Department of Immunology Monash University 14 12 07 Paul U Cameron Updated 22 08 2014 Geza Paukovics paukovic burnet edu au Jeanne Le Masurier jeanne burnet edu au Phil Donaldson phil burnet edu au Contents Responsibilities of all flow cytometer users ccecececeesceceececeeeeeceeneeceeceeceeeeceeeeeneeeenaeeees Data Storage POC yc naceenie hel ote Le aati ie ete tae ee Preparaaom OF Samples eiiis do een Padi d taux ded ees psi ccs tul A Preparation of Contro Samples eoa etas o rr qe de Maec diaue en geese a ade Starting up the PACSCalib r 55 tette ete REI SPD REA UT IG E SERVA U Ta aera Pre r m Procedure oa opera a tua ba cu a ee lee quud Jeu L R nnine San plese A a diia ee eU C ede FACS analysis of Human and Infectious Samples esee Setting detectors amps and compensation windows eese Poster n Procedute sins seas ote hot net oe aaa sated sou dee was ioo ee tete Baltes Sect aaa edes Responsibilities of all flow cytometer users These guidelines suggest ways in which all users can help to ensure that the FACSCalibur instruments remain operational and in excellent condition This is every user s responsibility so please pay attention to these guidelines for the sake of yourself and all other FACSCalibur users Users deliberately or carelessly ignoring the su
7. at the sink e Run MilliQ water at HI flow rate for 5 minutes e Ifthe cytometer is still blocked seek help from Geza or Michael Repeat offenders who continually block the instrument run the risk of having their access reviewed Contact Numbers If you have any problems or queries please contact the following people FACS Lab 9903 0601 Geza Paukovics 9282 2246 paukovic Q burnet edu au Jeanne Le Masurier 8506 2363 jeanne G burnet edu au Phil Donaldson 8506 2332 phil burnet edu au
8. ggestions in these guidelines and thereby creating problems with the flow cytometers may have their licence revoked The responsibilities of all FACSCalibur users are 1 Careful use and care of the FACSCaliburs 2 Proper sample preparation 3 Make bookings on the online system before using the FACSCaliburs 4 To alert Geza or Michael to problems or potential problems with the FACSCaliburs If after hours please send an email detailing the problem 5 To let the next user know if you have finished early or to changes your booking time If you cannot confirm the next user will be there within an hour SWITCH THE CALIBUR OFF 6 For the last booked user of the day including weekends to turn off theFACSCalibur and computer If you are booked to use the FACSCalibur and you change your mind but don t remove your booking from the computer system you must still ensure that the FACSCalibur is turned off including on weekends EXTENED PERIODS OF THE INSTRUMENT BEING LEFT ON WHILST NOT USED WILL NOT BE TOLORATED OFFENDERS RISK HAVING THEIR ACCESS REVIEWED Data Storage Policy At the conclusion of your session transfer files to your own computer via USB or Novell Account and ensure that the files have been transferred by checking on your computer Old files are routinely deleted from the Apple Mac computers and no responsibility is taken by AMREP Flow for lost data if you have not removed it in a timely manner Please use the smaller USB memo
9. one of the carboys containing milliQ water over to the instrument 1 Check that the tubing is not kinked and that the tank lids are all screwed on tight 2 Pressurise the sheath tank by ensuring the cover plate is on correctly and the pressure with is in the correct position 3 Check the filter lines for air bubbles and purge as necessary seek assistance from Geza or Michael if needed 4 Allow the cytometer to warm up for 5 minutes If using FL4 detector check the Four Color box at the bottom of the Detectors Amps window This will turn on the red diode laser Wait 15 20 min for this laser to warm up before proceeding Pre run Procedure Open the tube support arm and take off the MilliQ water tube Press the PRIME fluid control button Wait for the STANDBY button to light up Repeat Press the RUN fluid control button Install MilliQ water on the probe again NB The RUN button should turn green if machine is operating OK If the button is still orange there is a pressure problem Check that the O ring is supporting the tube that all is correct in the fludics drawer and that your tube has no cracks 5 RUN MilliQ water at HI flow rate for 2 3 minutes pho Na CellQuest 1 Open CellQuest from the Apple menu 2 Choose Connect to Cytometer from the Aquire menu 3 Openan existing document template or make a new one by creating dot plots or histograms using the plot tools in the tool palette 4 When creating a new dot plo
10. ry sticks to do this external hard drives have been known not to be compatible with the operating system the Caliburs run on Preparation of Samples Proper sample preparation will lessen the chance of blockages during your FACS run Blockages are caused by cell clumps and by DNA released by dead cells Remember that the internal diameter of the probe is small so please ensure your samples have been filtered and if necessary ad EDTA to a concentration of 5mM Preparation of Control Samples In addition to your test samples you will need to prepare a number of control samples on each occasion for compensating the FACSCalibur These include 1 cells alone ie no stain including no dead cell markers such as PI or 7AAD 2 Single stained samples for FL1 FL2 FL3 and FL4 as necessary Use an antibody that will stain a large proportion of cells but not all eg CD4 or CD8 on splenocytes or PBMC The use of these control samples for compensation is described further on Starting up the FACSCalibur Turn on the FACSCalibur first wait 30 seconds then start up the computer Check Fluidics Tanks 1 Check that the waste tank is empty it should have a small volume of 10 hypochlorite in the bottom of the tank 2 Fill the sheath tank milliQ water to 3 4 capacity To fill the tank release the pressure remove the probe BUT LEAVE THE TANK IN THE FLUDICS DRAWER Use one of the cylinders or beakers located near the sink to transport milliQ water from
11. t Format dot plot histogram window in CellQuest allows you to format your plot 5 Use the Aquisition Control and Parameter Description to define the acquisition and the data file location and description 6 Choose Dectectors Amps and Compensation Control from the Cytometer menu to set up your instrument settings or choose Instrument Settings from the Cytometer menu to open former settings Use the Aquire menu in CellQuest to access the counter and the acquisition dialogs Running Samples 1 2 3 4 Make sure your samples have been prepared properly ie no clumps DNA Press the RUN fluid control button before putting on your sample Be careful not to knock the probe while putting sample tubes on and off as this may cause the cell stream and laser to become misaligned Start with the flow rate on LO and move to MED or HI if there are few cells The flow rate should be no more than 1500 2000 events sec check on counter found under Aquire menu Pause a few seconds with the tube support arm open between samples to allow the probe to be flushed out Hluid levels in tubes should not be greater than 1 2 ml to prevent fluid getting into the air intake at the top of the probe NB When setting up Acquisition and Storage parameters see page 5 please o setthe Resolution to 256 rather than 1024 This aquires data in only 256 channels instead of 1024 channels which is adequate for most uses Furthermore the files saved will
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