Lsrii troubleshooting tips
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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

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LSRII Troubleshooting Tips

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Lsrii troubleshooting tips

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


Lsrii troubleshooting tips1

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?


Lsrii troubleshooting tips2

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.


Lsrii troubleshooting tips3

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.


Lsrii troubleshooting tips4

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!!!


Lsrii troubleshooting tips5

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)


Lsrii troubleshooting tips6

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


Lsrii troubleshooting tips7

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.


Lsrii spare parts to have on hand

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


Lsrii reagents to have on hand

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)


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