Sample Injection Tube (SIT) de-clogging guide
1. Check sheath tank for pressure.
2. Ensure tube is not cracked.
3. Attempt PRIME and check for bubbles.
a. Strong bubble stream
i. Clog may be gone, try sample again.
b. Weak bubble stream
i. Repeat PRIME 2x.
ii. Run FACS Clean for 5 min, then DI H2O for 5 min.
iii. Repeat PRIME, try sample again.
c. No bubbles
i. Use stylus wire to manually dislodge clog.
ii. Gently feed wire through aperture until there is 1 inch protruding from the bottom.
iii. Move wire up and down to dislodge clog.
iv. Remove wire and PRIME.
Cytometer Troubleshooting Guide
Observations
No events in acquisition display
and RUN button is green.
No events in acquisition display
and RUN button is orange.
Possible Causes
Recommended Solutions
Air in the sheath filter.
Purge the air from the sheath
filter.
Waste tank is full.
Empty the waste tank.
Sample injection tube (SIT) is
clogged.
See SIT de-clogging guide.
Connection issue with the
cytometer
Turn off cytometer and
computer and restart both.
Gating issue
Make sure plot is not gated on a
non-existent population.
Sheath container is not
pressurized.
Ensure that the sheath container
lid and all connectors are
securely seated.
Inspect the large O-ring and
replace if necessary.
Ensure that the sample tube is
not cracked, if so, replace it.
Sample tube is not pressurized.
Ensure that the correct tube is
being used. Corning 12x75mm
5mL polystyrene tubes.
Check the Bal seal (O-ring that
seals the tube onto the SIT). If it
is worn, and does not make a
tight seal, please ask core staff
for assistance.
Cytometer Troubleshooting Guide
Observations
Absent or weak fluorescent
signal
Possible Causes
Incorrect optical filter is
installed.
Laser malfunction
High event rate
Low event rate
Recommended Solutions
Make sure the appropriate
optical filter is installed for the
fluorochrome in question.
Check with core staff to run CST
to verify instrument
performance.
Sample is too concentrated.
Dilute the sample or use a lower
flow rate.
Threshold level is set too low.
Increase the threshold level to
ignore noise and debris.
Air bubble in the sheath filter
Bleed the bubbles from the
sheath filter.
Air bubble in flow cell
Perform a PRIME with a tube of
water. (de-bubble on CantoII).
Sample injection tube (SIT) is
clogged.
See SIT de-clogging guide.
Sample is too dilute.
Concentrate the sample. If the
flow rate setting is not critical to
the application, set the flow rate
to MED or HI.
Sample is not adequately mixed.
Vortex the sample to re-suspend
the cells.
Cytometer Troubleshooting Guide
Observations
Erratic event rate
Distorted scatter parameters
Possible Causes
Recommended Solutions
Sample tube is cracked.
Replace the sample tube
Bal seal is worn.
Replace the Bal seal. Please ask
core staff for assistance.
Sample injection tube (SIT) is
clogged.
PRIME with FACS Clean and H20.
Sheath filter is dirty.
Replace the sheath filter. Please
ask core staff for assistance.
Cytometer settings are
improperly adjusted.
Ensure that FSC and SSC are set
to linear scale and voltages are
optimized.
Air bubble in the sheath filter
Bleed the bubbles from the
sheath filter.
Air bubble in flow cell
Perform a PRIME with a tube of
water. (de-bubble on CantoII).
Flow cell is dirty.
Run a tube of FACS Clean for 5
minutes, followed by a tube of
dH20 for 5 minutes
Cytometer Troubleshooting Guide
Observations
Possible Causes
Excessive amount of debris in
display.
Threshold level is too low.
Sheath filter is dirty.
High CV
Recommended Solutions
Increase the threshold level or
lower the forward scatter
detector.
Replace the sheath filter. Ask
core staff for assistance.
Excessive debris in sample.
Filter sample with a 40um nylon
mesh.
Air bubble in sheath filter or flow
cell
Purge the air from the sheath
filter.
Sample flow rate is set too high.
Lower the sample flow rate.
Excessive debris in sample.
Filter sample with a 40um nylon
mesh.
Cytometer Troubleshooting Guide
Observations
Possible Causes
Recommended Solutions
Software Errors
Acquisition rate is too high.
Master DAQ Overflow
Canto: <15,000 evt/sec
Lower flow rate or dilute sample.
Increase FSC threshold to ignore
noise and debris.
Bad connection
Turn off both cytometer and
computer. Wait 30 sec. Turn on
cytometer. Turn on computer.
Allow windows to boot up
completely (including anti-virus)
before opening FACS Diva
software.
PMT Voltage Mismatch
Voltages are not consistent for
fluorescent parameters in
compensation control tubes.
Go through each compensation
control and find which voltage is
not consistent. Reacquire tube
with corrected voltage and
overwrite. Recalculate
compensation.
Software Freeze
Usually due to excessive amount
of data stored in the Browser.
Access ‘Task Manager’ by
pressing Control+Alt+Delete.
Force quit FACS Diva software.
Computer Crash
Usually due to excessive amount
of data stored on the hard drive.
If computer is totally
unresponsive, hold the power
button for 5 seconds. Wait 30
seconds. Turn computer back on.
Cannot Connect to Cytometer