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Troubleshooting

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Analytical Cytometer Problems:

SYMPTOM: No Signal with Calibration Beads

Possible causes: Improper size or FSC settings, FSC threshold too high, poor pressure, sample tube cracked sample line clog, laser problems

  1. Make sure instrument settings for beads have been located and SET into cytometer
  2. Make sure the sheath tank is pressurized, pressure valve should be UP (Scan) or away from you (Calibur)
  3. Make sure there is no crack in the tube
  4. Perform prime or drain and fill again
  5. Run 20% bleach on high for 15 minutes, followed by fresh DI water

SYMPTOM: Noisy FSC, no visible peak for beads

Possible causes: Debris or gunk at the intercept point, FSC threshold too low, air bubbles

  1. Run 20% bleach for 15 minutes, followed by fresh DI water.
  2. Check the threshold setting under CYTOMETER menu and ensure the threshold is defined for FSC and is set at 52.
  3. Drain and Fill or Prime again

SYMPTOM: Bead FSC signal OK when signals for other parameters are way off

Possible causes: Laser power fault, compensation values still engaged

  1. Open the compensation box under cytometer menu and ensure all settings are at 0.0%
  2. Open the status box under the cytometer menu and write down the laser power and laser current values with your sample tube on and fluidics in RUN

SYMPTOM: Red diode won't calibrate for FL4 Stains (on Calibur)

Possible causes: Red diode fault, FSC signal too low, event count too low.

  1. Turn fluidics to medium
  2. view particles on E01 with increase amplitude
  3. Make new solution of 1 drop BD APC beads in 1 ml saline or FACSFLOW
  4. Use APC compensation control to calibrate- set FL4 voltage to last recorded setting.

SYMPTOM: Continuous Standby Fault or Cytometer "Not Ready"

Possible causes: Alligator clips on sheath tank have slipped off, improper pressure levels, No sheath fluid, Laser Power fault

  1. Reseat the clips around the two metal prongs of male connector.
  2. Ensure sheath tank is full and pressurized
  3. Make sure sensors are properly tightened
  4. Check status box under cytometer menu and record laser power and current values

Software Problems:

SYMPTOM: Counters won't count: Save the template and close it. Quit Cell Quest. Reopen your template which will also open Cell Quest again. Locate Acquisition Control under Windows and proceed with acquisition.

SYMPTOM: Sample ID box blacks out when you try to write in it: Click in the Patient ID box and then click in the Sample ID box

SYMPTOM: Sentinel EvE error: Repeat what you were trying to do.

SYMPTOM: Unexpected quits with type "x" error: Reopen Cell Quest template you were working on. These errors are file construction errors/ file memory allocation errors.

SYMPTOM: Anything involving GPI0: Unfortunately, you'll have to shut down both computer and Cytometer and wait for help the next day or issue a dispatch call. See below

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