LSRII Troubleshooting Tips

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.
• Perform Daily Calibration. If improvement is
  seen, repeat until Daily Calibration
• passes. If no improvement is seen, continue to
  next step.

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
• in tube on SIP
• Reconnect the Sheath filter ensure that ALL
  bubbles are out of the Sheath filter before
  continuing
• 5 minutes with FACSFlow Sheath Fluid in Sheath
  tank with dI H2O
• in tube on SIP
• Perform Daily Calibration. If improvement is
  seen, repeat until Daily Calibration passes. If
  no improvement is seen, continue to next step.

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 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 344678)
• Bal seals (BDB Catalog 343509)
• Stylets (BDB Catalog 343648)
• O-Ring (BDB Catalog 343615)
• Tubing (ask your service engineer for extra
  tubing)
• Screwdriver

10
LSRII Reagents to have on Hand

• FACSFlow Sheath Fluid (BDB Catalog 342003)
• FACSRinse (BDB Catalog 340346)
• D-PBS (Invitrogen/Gibco Catalog 14190-144)
• 8 Peak Rainbow Beads (Spherotech Catalog
  RCP-30-5A)
• 1X Rainbow Beads (Spherotech Catalog RFP-30-5A)
• FACSDiva 6.0 CST Beads (BDB Catalog 641319)
• FACSDiva 7.0 CST Beads (BDB Catalog 655051)
• anti-Mouse Compensation Beads (BDB Catalog
  552843)
• Use Negative Beads from kit for Daily Calibration
• Sodium hydroxide (Sigma-Aldrich Catalog S8045)
• 1M NaOH pellets
• dI H2O (any source is OK)