A National Cancer Institute Comprehensive Cancer Center

 
 

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How to Use the FACScan

  1. Prepare the flow cytometer (check fluidics - sheath fluid level, bubbles, waste container level)
  2. Open a CellQuest software template to run the flow cytometer
  3. Acquire your unstained control cells
  4. Acquire your compensation control beads (or cells)
  5. Acquire your positive control cells (fully stained sample)
  6. Check daily Flow Core QC and ensure your PMT settings are similar to QC settings
  7. Acquire your sample tubes
  8. Clean the flow cytometer (RHSSCRAQ)
  9. Save your files to a memory stick
  10. Close CellQuest
  11. Remove your gloves, tubes and other belongings and clean up any trash
  12. Sign up for next appointment
  13. Analyse your Data on any of the work station computers.

If you need any explanation of any of these procedures, please ask Gary Ward

A detailed description of how to run a FACScan can be found on the excellent website for the FlowLab at the Instituto Gulbenkian de Ciencia.

When you have finished using the FACScan or Calibur, you must follow the 5 steps of the RJRHSSCRAQ Procedure. Otherwise you could run the sheath tank dry or fill the fluid lines with bubbles or clog the flow cell.

  1. Run dilute JetDry for 2 minutes
  2. Run distilled H2O for 2 minutes
  3. Switch to Standby and remove the sheath pressure (push Valve to the back). Install a sample tube with 2 ml (NOT MORE) of distilled water.
  4. Check Reservoirs fill or empty as required.  Please clean up spills.
  5. Quit CellQuest.
DRAIN AND FILL PROCEDURE TO REMOVE BUBBLES ON FACSCAN

To be used when a known sample will not flow properly. This usually indicates bubbles in the sample lines. Must be used if the cytometer has been "back flushed".

  1. Check to make sure that waste tank is not full. If it is, it needs to be emptied into the sink and the sheath tank needs to be filled with FACS buffer from the box.
  2. Look for bubbles in the Pall Filter (spherical plastic sheath filter inside lower case). This must be purged of air before a successful "drain and fill" may be accomplished. This filter is rubber mounted and may be tapped lightly to bring air bubbles to the top. The bubbles may then be drained into a beaker after the white plastic cap is removed from the rubber tubing. WARNING! Contents under pressure! Have beaker in place before removing cap.
  3. Remove tube from the sample probe.
  4. Place the fluidics knob (black knob lower right hand corner of cytometer) in the "BACK FLUSH" position for 20 seconds.
  5. Place the fluidics knob in the "DRAIN" position. Watch the waste line (1/4 inch Tygon tubing with orange connector inside lower case) to see when fluid has drained.
  6. After fluid has drained place fluidics knob in "fill" position. Watch the quartz cuvette (top right hand corner inside upper case) to make sure the meniscus is level and there are no air bubbles or obstructions in the fluid as it rises.
  7. If an uneven fluid level is present or if there is an air bubble or obstruction in the quartz cuvette repeat from 4 above.
  8. After quartz cuvette has evenly filled, watch waste line to see that a continuous flow of sheath is moving. An occasional air bubble here is OK.
  9. Place fluidics knob in "run" position to continue sample acquisition.