Salk Institute
Flow Cytometry Core Facility

LSR II Quick Reference & Troubleshooting Guide

Startup

  • Ensure that you signed up for instrument use using the on-line calendar (even if it is night or weekend).
  • Before switching on (or when starting your run):
    • Check sheath reservoir. A full tank provides about 3 hours' run time. Refill as necessary from large plastic canister containing 1x sheath solution. If machine is on, depressurize sheath tank before opening it. Make sure tank is tightly closed after filling.
    • Check waste reservoir. Empty if it seems full. Discard contents into sink, flush with water. Put approx. 1 liter bleach into empty waste tank before returning it to the machine.
  • Switch on instrument, if not already on.
  • Switch on computer and/or log into Windows (FCCF domain).
  • Purge bubbles from sheath filter if necessary.
  • Turn sample fine adjust knob to the center.

Run

  • Launch laser control software and activate the lasers you need to use. Note: you must always turn on the 488 nm laser.
  • Launch FACSDiva software and log into it. Wait for cytometer to connect. If cytometer will not connect, reset the cytometer computer and restart DiVa.
  • If you have run the same type of experiment before, open that experiment and duplicate it without data.
  • Otherwise, create a new experiment ("Blank Experiment with Sample Tube") or use a template from the USER tab.
  • Run compensation controls and calculate compensation.
  • Run samples.

Shutdown / Finished

  • From the laser control software, turn off all lasers.
  • Set fluidics switch to HIGH.
  • Potentially biohazardous samples: Run TriGene (1:50 or 1:100) for 5 minutes followed by three changes of DI water for 1 minute each.
  • Sytox: Run 100% bleach or 70% ethanol for 5 minutes followed by three changes of DI water for 1 minute each.
  • Make a new tube containing no more than 1 ml (1/2 inch/ 1 cm depth) filtered DI water and put onto SIP.
  • Run for 2 minutes.
  • Export your data and/or experiment to the FCCF data server during the clean-up procedure.
  • Set fluidics control to STBY
  • If you are the last user of the day, or there is a gap of more than 3 hours:
    • TURN OFF LSR unit and shut down computer.
    • Do not allow LSR to run overnight! Charge for instrument left on overnight is 50% of the time until it is turned off

Troubleshooting

  • 1. Neither cytometer nor computer will not turn on.
    Make sure that UPS under bench is turned on.
  • 2. Can't log in to computer.
    Make sure you are logging on to the FCCF domain.
    Make sure you put in the correct user name and password.
  • 3. Computer will not connect to cytometer.
    Restart Diva software.
  • 4. Computer STILL will not connect to cytometer.
    Restart Diva hardware: Open the "Cytometer Status" application (the red telephone on the Windows desktop) and press the Enter key a few times. If you get a prompt such as "-->", type "reboot" and press Enter. If this fails, reset the cytometer's internal computer.
  • 5. No PE signal.
    Ensure the 561 nm laser is turned on.
  • 6. No events.
    Laser(s) not turned on.
    Fluidics control not set to "RUN".
    Tube is cracked.
    Check level in sheath tank.
    Ensure that sheath tank lid is fitted properly.
    Stuck fluidics - PRIME.
    No cells in sample.
    Clumpy sample - filter it.
    Blocked SIP or flow cell - Remove sample, and press PRIME.
    Waste not venting - check waste line for flow.
    Air bubbles in sheath filter - purge filter.
  • 7. Sample runs for a few seconds then stops
    Clumpy sample - filter it.
    Blocked SIP or flow cell - PRIME.
    Waste not venting - check waste line.
    Air bubbles in sheath filter - purge filter.
  • 8. Very slow sample rate.
    Sample very dilute.
    Fluidics set to LOW.
    Fluidics fine control turned down.
  • 9. Sample still will not run
    Air bubbles in sheath filter - purge filter.
    SIP blocked - Set fluidics to "HIGH". Run 100% bleach for 30 sec. followed by at least 2 changes of DI water for 1 minute.

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