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Troubleshooting

Software problems:

When you cannot fix the problem:

1. If your samples can remain unfixed on ice at 4C overnight, store them for running the next day.
2. If not, you can try fixing your samples in 0.5% paraformaldehyde (as long as there is no PI in your sample).

Analytical cytometer problems:

DO NOT ATTEMPT TO REMOVE ANY PARTS FROM THE CYTOMETERS.
YOU WILL BE HELD RESPONSIBLE FOR BENT OR BROKEN PIECES OR ANY DAMAGE INCURRED.

TIP: To resolve a minor clog or other minor problems (such as irregular FSC/SSC profile not due to parameter settings):
Prime the machine three times and see if that fixes it. If not, run bleach for 15 minutes followed by 15 minutes of DI water.

Observation Possible Causes Recommended solutions
Droplet containment vacuum not functioning Worn O-ring in retainer Replace the O-ring. Contact FMCF staff.
Outer sleeve is not seated in the retainer
  1. Loosen the retainer
  2. Push the outer sleeve up into the retainer until seated.
  3. Tighten the retainer
Outer sleeve is not on the sample injection tube Replace the outer sleeve.
  1. Loosen the retainer.
  2. Slide the outer sleeve over the sample injection tube until it is seated.
  3. Tighten the retainer.
Waste line is pinched, preventing proper aspiration Check the waste line.
Waste tank is full Empty the waste tank.
Sample tube not fitting on SIP Sample tube other than BD Falcon tubes used Use BD Falcon 12 x 75-mm sample tubes.
Tubes must be polystyrene NOT polypropylene
Worn Bal seal Replace the Bal seal. Contact FMCF staff
Rapid sample aspiration Support arm is to the side. Place the support arm under the sample tube.
Droplet containment module is failing Call your service representative. Contact FMCF staff.
No events in acquisition display and RUN button is green. Threshold is not set to the correct parameter (usually FSC). Set the threshold to the correct parameter for your application
Threshold level is too high Lower the threshold level.
PMT voltage for threshold parameter is set too low Set the PMT voltage higher for the threshold parameter.
Gating issue Ensure gating strategy is appropriate.
Air in the sheath filter. Purge the filter.
Sheath and air line not connected to the sheath tank. Ensure sheath and air line are connected to the sheath tank
O-ring is not in position in the sheath tank. Reposition O-ring in sheath tank.
Cracked tube/ Bal seal (tube to SIP connection) problem Replace tube.
No sample in the tube. Add sample to the tube or install a new sample tube.
Sample is not mixed properly. Mix the sample to suspend cells.
Waste tank is full Empty the waste tank.
PMT voltages set too low or too high for display parameter Reset the PMT voltages.
Too few events are displayed Increase the number of events to display.
Sample injection tube is clogged Remove the sample tube to allow backflushing and prime the instrument several times. Clean instrument. Contact FMCF staff if issue is not resolved.
Laser is not warmed up. Wait the recommended amount of time for the laser to warm up.
  • 30 min for the 488-nm (blue)
  • 30 min for the 355-nm (UV)
  • 15 min for the 405-nm (violet)
  • 20 min for the 633-nm (red)
Laser delay is set incorrectly Contact FMCF staff
Laser is not functioning Verify the malfunction by changing the threshold to an alternative laser while running the appropriate sample. If unsuccessful, contact FMCF staff
No events in acquisition display and RUN button is orange RUN is not activated Press the RUN button.
Sample tube is not installed or is not properly seated Install the sample tube correctly on the SIP.
Sample tube is cracked Replace the sample tube.
Sheath container is not pressurized
  • Ensure that the sheath container lid and all connectors are securely seated.
  • Inspect the O-ring and replace it if necessary. Contact FMCF staff.
Bal seal is worn Replace the Bal seal. Contact FMCF staff
Air leak at sheath container Ensure that the sheath container lid and all connectors are securely seated.
Sheath container is empty Fill the sheath container.
Air in sheath filter. Purge the filter.
No fluorescent signal Incorrect fluorochrome assignment Make sure the cytometer configuration in the software matches the optical filters in the cytometer.
Wrong filter is installed Make sure the appropriate filter is installed for each fluorochrome.
Laser is not functioning Verify the laser malfunction by changing the threshold to an alternative laser while running the appropriate sample. If unsuccessful, contact FMCF staff.
High event rate Air bubble in the sheath filter or flow cell. Remove the air bubble from filter.
Sample is too concentrated Dilute the sample.
Sample flow rate is set on HI Set the sample flow rate to MED or LO.
Threshold level is too low ncrease the threshold level.
PMT voltage for the threshold parameter set too high Set the PMT voltage lower for the threshold parameter.
Low event rate Threshold level is too high Lower the threshold level.
PMT voltage for the threshold parameter is set too low Set the PMT voltage higher for the threshold parameter.
Sample is not adequately mixed Mix the sample to suspend the cells.
Sample is too diluted Concentrate the sample. If the flow rate setting is not critical to the application, set the flow rate switch to MED or HI.
Sample injection tube is clogged Remove the sample tube to allow backflushing. If the event rate is still erratic, clean the sample injection tube.
Erratic event rate Sample tube is cracked Replace the sample tube.
Bal seal is worn Replace the Bal seal. Contact FMCF staff
Sample injection tube is clogged Remove the sample tube to allow backflushing and prime the instrument several times. Contact FMCF staff if the issue is not resolved.
Contaminated sample Prepare the specimen again. Ensure that the tube is clean.
Sheath filter is dirty Replace the filter. Contact FMCF staff.
Distorted scatter parameters Cytometer settings are improperly adjusted. Optimize the scatter parameters.
Air bubble in sheath filter or flow cell Purge the air from the filter.
Flow cell is dirty Perform the system flush procedure. Contact FMCF staff.
Air leak at sheath container Ensure that the sheath container lid is tight and all connectors are secure.
Hypertonic buffers or fixative Replace the buffers and fixative.
Excessive amount of debris in display Threshold level is too low Increase the threshold level.
Sheath filter is dirty Replace the filter. Contact FMCF staff.
Flow cell is dirty Prime the instrument several times. Contact FMCF staff. 
Dead cells or debris in sample Examine the sample under a microscope.
Sample is contaminated Re-stain the sample, ensure tube is clean.
Stock sheath fluid is contaminated Contact FMCF staff. 
High CV Air bubble in sheath filter or flow cell Purge the filter.
Sample flow rate is set too high Set the sample flow rate lower.
Air leak at sheath container Ensure that the sheath container lid is tight and all connectors are secure.
Flow cell is dirty Prime the instrument several times. Contact FMCF staff. 
Poor sample preparation Repeat sample preparation.
Sample not diluted in same fluid as sheath fluid Dilute the sample in the same fluid as you are using for sheath.
Poor QC results Air bubble or debris in flow cell Prime the fluidics system.
Old or contaminated QC particles Make new QC samples and perform the quality control procedure again.
Sample not diluted in same fluid as sheath fluid Dilute the sample in the same fluid as you are using for sheath.
Laser not warmed up Wait the recommended amount of time for the laser to warm up.
  • 30 min for the 488-nm (blue)
  • 30 min for the 355-nm (UV)
  • 15 min for the 405-nm (violet)
  • 20 min for the 633-nm (red)
Laser not functioning Contact FMCF staff
Optical alignment problem Contact FMCF staff