FACSCalibur Use
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
- Turn on the cytometer.
- Turn on the computer.
- Fill the sheath tank and verify the following:
- Tank is filled (~3L).
- Cap is tight.
- Tank is pressurized.
- Tubing is not kinked.
- Empty the waste tank and verify the following:
- Tank contains 400 mL bleach.
- Tubing is not kinked.
- Purge bubbles from sheath filter.
- Allow the cytometer to warm up for 5 minutes.
DAILY CALIBRATION (FACSCOMP)
- Label one 12 x 75 mm tube A and another B.
- Add 1 mL of sheath to tube A. Add 3 mL to tube B.
- 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.
- Empty the waste tank and verify the following:
- Tank contains 400 mL bleach.
- Tubing is not kinked.
- Cap the tubes and mix by gentle inversion.
- Select FACSComp from the Apple Menu.
- Fill in the Operator field. Click Accept.
- 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.
- Make sure the sample flow rate is set to HI and the cytometer is set to RUN. Install tube A.
- Click Start to begin PMT adjustment and time-delay calibration.
- Remove tube A and install tube B.
- Click Start to begin compensation adjustment and sensitivity tests.
- Remove tube B, install distilled water tube and set cytometer to STANDBY.
- 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
- 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.
- The designation folder should be Flowdata. You may create a folder within Flowdata with your name.
- At the end of your run, transfer your data files to a Zip disk or your directory on Ireland.
DAILY SHUTDOWN
- Install a 12x75mm tube containing 3 mL of 10% bleach on the sample injection port (SIP).
- Leave the support arm to the side for 1 minute.
- 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.
- Repeat steps 1 through 3 using distilled water.
- Leave a tube containing 1 mL of distilled water on the SIP with the support arm under the tube.
- Set the cytometer to STANDBY mode.
- Shut down the computer.
- Turn off the cytometer.
PROBLEMS
Record any problems in the Problem Log or let Angélique (2-6077) or Sherry (2-6067) know directly.