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)