Salk Institute
Flow Cytometry Core Facility

FACScan Quick Reference & Troubleshooting Guide

Startup

  • Ensure that you signed up for instrument use using the on-line calendar (even if it is night or weekend).
  • Before switching on (or when starting your run):
    • Open instrument lower door
    • Check sheath reservoir. A full tank provides about 3 hours' run time. Refill as necessary from large plastic canister near sink. Do not overfill!
    • Check waste reservoir. Empty if it looks over half full. Discard contents into sink, flush with water. Put approx. 100ml bleach into empty waste tank before returning it to the machine.
    • Empty waste whenever you fill the sheath. When replacing tanks, make sure that the tubing does not become kinked or trapped, otherwise the machine may not run properly, if at all. Ensure that the sheath reservoir cap is screwed tight.
    • Verify that the sheath reservoir tubing is not caught between the tank and the machine's frame.
    • Set vent valve to the "up" position (pressure on).
    • Close instrument lower door
  • Switch on instrument, if not already on.
  • Switch on computer. If the computer produces a message saying that it cannot connect to the cytometer, restart the computer.
  • Take off tube from SIP. Discard tube. Set fluid flow switch to HIGH.
  • Clear flow cell:
    • Open instrument upper door.
    • Ensure that there is no tube on the SIP.
    • Set fluidics control to DRAIN. Observe flow cell. Wait until liquid drains out of flow cell.
    • Set fluidics control to FILL. Observe flow cell. Wait until flow cell fills completely.
    • Repeat DRAIN/FILL cycle for a total of three times.
  • Set fluidics control to STANDBY
  • Make a new tube containing 1 ml (1/2 inch/ 1 cm depth) filtered DI water and put onto SIP.

Run

  • Launch desired acquisition document. (Apple menu→Acquisition setups).
  • Double-click CytConnect icon. This runs a macro that connects to the cytometer, opens all the windows you will need and loads a set of default instrument settings. The macro gets confused if you move the mouse or type anything while it is running; if you do this you may have to restart the computer, so be patient until the instrument settings have loaded.
  • Create and select a folder in which to store your data.
  • Create a filename or prefix. Set file count to 1.
  • If you have run the same sort of experiment before, use the Cytometer menu to load instrument settings from a previous data file. Don't forget to click SET!
  • If you are doing cell cycle, make sure that DDM is switched on in the detectors/amps control panel.
  • Ensure the Setup box is checked.

Shutdown / Finished

  • Set fluidics switch to HIGH.
  • Potentially biohazardous samples: Run TriGene (1:50 or 1:100) for 5 minutes followed by three changes of DI water for 1 minute each.
  • Sytox: Run 100% bleach or 70% ethanol for 5 minutes followed by three changes of DI water for 1 minute each.
  • Make a new tube containing no more than 1 ml (1/2 inch/ 1 cm depth) filtered DI water and put onto SIP.
  • Run for 2 minutes.
  • Transfer your data to the FCCF data server during the clean-up procedure.
  • Set fluidics control to STANDBY
  • If you are the last user of the day:
    • Set vent valve to the "down" position (pressure off and vent sheath tank).
    • Wait 5 minutes to allow laser to cool
    • TURN OFF FACSCAN unit and computer.
  • Do not allow FACScan to run overnight! Charge for instrument left on overnight is 50% of the time until it is turned off

Troubleshooting

1. Neither cytometer nor computer will not turn on.
Make sure that UPS under bench is turned on.

2. Computer will not connect to cytometer.
Restart computer.

3. Cytometer status is "NOT READY".
Allow to warm up for 5 minutes.
Check sheath is full and waste is empty.
If status window says that waste is full and you know it is empty, rap on waste tank to dislodge sensor float.
Check that tubing leading to tanks is not pinched or obstructed.

4. Cytometer status remains at "STANDBY".
Fluidics control not turned to "RUN"
Tube is cracked.
Check that pressure is ON (vent valve UP).
Check that sheath tank is actually pressurized - it should not be moveable.
Ensure that sheath tank cap is screwed down tightly.
Stuck fluidics - turn to STANDBY and back to RUN.

5. Status READY, but sample will not run.
No cells in sample.
Clumpy sample - filter it.
Blocked SIP or flow cell - Remove sample, then DRAIN/FILL 3 times.
Waste not venting - loosen waste tank cap. Report problem.

6. Sample runs for a few seconds then stops.
Clumpy sample - filter it.
Blocked SIP or flow cell - Remove sample, then DRAIN/FILL 3 times.
Waste not venting - loosen waste tank cap. Report problem.

7. Sample still will not run.
Air bubbles in sheath filter - purge filter.
SIP blocked - Set fluidics to "HIGH". Run 100% bleach for 30 sec. followed by at least 2 changes of DI water for 1 minute.

 


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