SIGNING UP

When logging your use of the FACSCalibur, include your Principle Investigator's name, your first name and last initial and your telephone number.

DAILY START UP

  1. Turn on the cytometer.
  2. Turn on the computer.
  3. Fill the sheath tank and verify the following:
    • Tank is filled (~3L).
    • Cap is tight.
    • Tank is pressurized.
    • Tubing is not kinked.
  4. Empty the waste tank and verify the following:
    • Tank contains 400 mL bleach.
    • Tubing is not kinked.
  5. Purge bubbles from sheath filter.
  6. Allow the cytometer to warm up for 5 minutes.

DAILY CALIBRATION (FACSCOMP)

  1. Label one 12 x 75 mm tube A and another B.
  2. Add 1 mL of sheath to tube A. Add 3 mL to tube B.
  3. Add one free-falling, complete drop of the appropriate CaliBRITE beads to each tube:
    • Unlabeled and APC beads to tube A.
    • Unlabeled, FITC, PE, PerCP and APC beads to tube B.
  4. Empty the waste tank and verify the following:
    • Tank contains 400 mL bleach.
    • Tubing is not kinked.
  5. Cap the tubes and mix by gentle inversion.
  6. Select FACSComp from the Apple Menu.
  7. Fill in the Operator field. Click Accept.
  8. The set up information window appears. Unless a new lot of CaliBRITE beads is used for the first time, this information does not need to be changed. Click Run.
  9. Make sure the sample flow rate is set to HI and the cytometer is set to RUN. Install tube A.
  10. Click Start to begin PMT adjustment and time-delay calibration.
  11. Remove tube A and install tube B.
  12. Click Start to begin compensation adjustment and sensitivity tests.
  13. Remove tube B, install distilled water tube and set cytometer to STANDBY.
  14. Click Quit to quit FACSComp. The report will be printed automatically. Please put it in the appropriate section of the blue FACSCalibur folder.

SAVING DATA

  1. When designating your data file name, place your initials and the date (ABCyymmdd) in the custom prefix box. Assign a file count number and this will be appended to the date and will increment as you run each sample.
  2. The designation folder should be Flowdata. You may create a folder within Flowdata with your name.
  3. At the end of your run, transfer your data files to a Zip disk or your directory on Ireland.

DAILY SHUTDOWN

  1. Install a 12x75mm tube containing 3 mL of 10% bleach on the sample injection port (SIP).
  2. Leave the support arm to the side for 1 minute.
  3. Place the support arm under the tube, make sure the cytometer is in RUN, and let it run at HI flow rate for 5 minutes.
  4. Repeat steps 1 through 3 using distilled water.
  5. Leave a tube containing 1 mL of distilled water on the SIP with the support arm under the tube.
  6. Set the cytometer to STANDBY mode.
  7. Shut down the computer.
  8. Turn off the cytometer.

PROBLEMS

Record any problems in the Problem Log or let Angélique (2-6077) or Sherry (2-6067) know directly.

Department of Microbiology
School of Medicine
University of Washington
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