1. LSRII Troubleshooting Tips
Before starting each acquisition, check the following items:
Waste tank is empty, but contains ~1 liter of bleach
FACSFlow Sheath Fluid tank is full
FACSFlow Sheath Fluid is connected correctly to instrument/reservoir
No bubbles in the Sheath filter
Tap the filter against a hard surface to move bubbles to the drain valve. A screwdriver is often useful to dislodge & move or “direct” bubbles to the drain line.
Drain the Sheath filter
Repeat process until all bubbles are removed from the Sheath filter
Check that there are no bubbles in Sheath line from Sheath filter into the side of the instrument
Drain the tubing line if necessary

2. LSRII Troubleshooting Tips
No events/Very few events
Restart the LSRII, FACSFlow Supply System, & Computer
Often there is a disconnect between the LSRII & the computer. A simple restart will sometimes fix this problem.
Has the correct tube been selected in FACSDiva?
Check that the correct tube has a Pointer next to it in FACSDiva.
Ensure that the Acquire Button has been selected on the Acquisition Control Panel.
Is tube pressurized?
Is LSRII in RUN mode?
Check that the GREEN RUN light is illuminated.
Is tube seated properly on SIP (Sample Injection Port)?
Wiggle tube to make sure tube is seated properly on support arm.
Ensure that tube is pushed up tightly against the Bal Seal.
Check that the Bal Seal is not worn. Change Bal seal if necessary.
Are you using the appropriate tube for the LSRII?
Is the tube cracked?

3. LSRII Troubleshooting Tips
No events/Very few events (continued)
Is the SIP (Sample Injection Port) clogged?
Perform Daily Clean (in RUN HIGH mode , ~15 minutes required)
1 minute with 0.1M NaOH with support arm to side followed by
5 minutes of 0.1M with support arm under the tube
1 minute with FACSRinse with support arm to side followed by
5 minutes of FACSRinse with support arm under the tube
1 minute with dI H2O with support arm to side followed by 5 minutes
of dI H2O with support arm under the tube
Perform Daily Calibration. If improvement is seen, repeat until Daily Calibration
passes. If no improvement is seen, continue to next step.
Perform Aggressive Debubbling Procedure
Alternate between pressing the PRIME button and the RUN button
every 3 seconds.
This forces the Flow Cell to empty & refill very quickly with
FACSFlow Sheath Fluid.

4. LSRII Troubleshooting Tips
No events/Very few events (Continued)
Carefully insert a stylet into the Sample Probe
This will often dislodge a piece of debris or clump of cells from
the Sample Probe.
Perform Daily Calibration. If improvement is seen, repeat until Daily Calibration passes. If no improvement is seen, continue to next step.
Perform a Monthly Clean (in RUN HIGH mode, ~1.5 hours required)
Bypass/disconnect the Sheath filter
45 minutes with 0.1M NaOH in Sheath tank and with 0.1M NaOH
in tube on SIP
45 minutes with dI H2O in Sheath tank and with dI H2O in tube on SIP
5 minutes with FACSFlow Sheath Fluid in Sheath tank & with dI H2O
Reconnect the Sheath filter & ensure that ALL bubbles are out of the Sheath filter before continuing

5. LSRII Troubleshooting Tips
No events/Very few events (Continued)
Flush Flow Cell with syringe filled with 0.1M NaOH
Fill 10-20mL syringe with 0.1M NaOH & disconnect sheath line
from top of Flow Cell.
Ensure that an empty vessel is under SIP & tube is off SIP with support arm to side.
Connect syringe tubing to top of Flow Cell & force 0.1M NaOH into Flow Cell.
Repeat 3 times.
Reconnect sheath line.
Perform Daily Calibration. If improvement is seen, repeat until Daily Calibration passes. If no improvement is seen, continue to next step.
Check that the Blue Laser is not blocked with the cover.
Check that the HTS toggle switch is on TUBE & not on PLATE!
Call BD Service!!!

6. LSRII Troubleshooting Tips
High CV or Median is Outside Acceptable Range when Daily Calibration is performed
If Beads were not made fresh, prepare new Beads & re-run
Daily Calibration Assay
Check for air bubbles in the Sheath Filter & Sheath line
Check for air bubbles in Flow Cell
Perform Aggressive Debubbling (see Slide 4 for details)
Perform a Daily Clean (see Slide 4 for details)
Perform a Monthly Clean (See Slide 4 for details)

7. LSRII Troubleshooting Tips
Sample being aspirated very quickly from tube
Is the DCM (Droplet Containment Module) audible when a tube is on the SIP & the LSRII in RUN mode?
Yes - Move the support arm fully under the tube & ensure that tube is truly pressurized.
No - The DCM is Failing. Call BD service!!!
Tube is backfilling rapidly or SIP is dripping with support arm to side
Check that the SIP is not clogged (see Slide 4 for details)
Change the O-Ring
Call BD Service!!! - Possible clog in DCM tubing that must be repaired by a BDB Service Technician
For some samples that are known to clump (BAL, mono/mac, etc), filter fixed/stained samples using 12x75 filter top tubes prior to putting tube on SIP

8. LSRII Troubleshooting Tips
For some samples that are known to clump (BAL, mono/mac, etc), filter fixed/stained samples using 12x75 filter top tubes prior to putting tube on SIP.

9. LSRII Spare Parts to have on Hand
Sheath filter (BDB Catalog # )
Bal seals (BDB Catalog # )
Stylets (BDB Catalog # )
O-Ring (BDB Catalog # )
Tubing (ask your service engineer for extra tubing)
Screwdriver

10. LSRII Reagents to have on Hand
FACSFlow Sheath Fluid (BDB Catalog # )
FACSRinse (BDB Catalog # )
D-PBS (Invitrogen/Gibco Catalog # )
8 Peak Rainbow Beads (Spherotech Catalog # RCP-30-5A)
1X Rainbow Beads (Spherotech Catalog # RFP-30-5A)
Ultra Rainbow Beads (Spherotech Catalog # URFP-30-2)
anti-Mouse Compensation Beads (BDB Catalog # )
Use Negative Beads from kit for Daily Calibration
Sodium hydroxide (Sigma-Aldrich Catalog # S8045)
1M NaOH pellets
dI H2O (any source is OK)