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American Family Children's Hospital
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FACSCalibur Protocol

Starting up the FACSCalibur

  1. Check levels of reservoirs. Left side (sheath tank) should be full and right side (waste tank) should be empty. If not, fill sheath tank up to line with sheath fluid and empty waste tank into sink. Add approximately 50 ml of bleach to waste tank and put it back in the cytometer. Make sure the caps are tight on both reservoirs and that the tubing is not kinked.
  2. Turn on the FACSCalibur first. Give it about 5 seconds to get going and then turn on the computer. Turn on the printer.
  3. Make sure vent toggle switch is in the forward position (towards you). After about 10 seconds, try to wiggle the sheath tank: if it's tight and you can't move it at all, then it's pressurized, if it's easy to move, then it's not.
  4. Prime the fluidics. Take the tube off the sipper and leave the sample arm swung to the side. Press the PRIME button and wait until the STANDBY button lights up. Swing the sample arm back to the middle, without a tube on the sipper. The sipper should drool. Prime 4 more times. If there are any air bubbles visible in the tubing coming from the sheath tank (white connector), prime until they are gone.
  5. Put the tube of water back on the sipper and press the RUN button until all bubbles are out of the tube (orange connector) that leads to the waste tank.
  6. When the RUN button is green (instead of yellow), the cytometer is ready to run calibration beads or samples. Press the STANDBY button to put the cytometer into Standby mode.

Shutting down the FACSCalibur

  1. Put a tube with approximately 2ml (half full) 10% bleach on the sipper. Leave the sample arm swung to the side and let the sipper suck up the bleach for about 1 min.
  2. Swing the sample arm back to the middle and run on HIGH for 5 minutes.
  3. Take the tube of bleach off the sipper. Put a tube with approximately 2ml (half full) dH2O on the sipper. Leave the sample arm swung to the side and let the sipper suck up the dH2O for about 1 min.
  4. Swing the sample arm back to the middle and run on HIGH for 5 minutes.
  5. Press the STANDBY button. Make sure there is not more than 1 ml of water in the tube before putting on STANDBY (otherwise, water may back up into the cytometer and short out a circuit board).
  6. Leave machine on in STANDBY for 10 min to let the laser cool down.
  7. Turn power off on machine.
  8. Shut down computer.
  9. Release the pressure in the sheath tank by pushing the vent toggle switch to the Vent position (away from you). After the tank finishes hissing, flip the vent toggle switch back to the Pressurize position (towards you).