Quick Reference Guide - FACSCalibur
1. Turn on the BD FACSCalibur instrument.
2. Turn on the computer & login (instrument MUST be on before turning on the
3. If needed, fill the sheath reservoir to ¾ of its capacity.
4. If needed, empty the waste reservoir & add 200-400 mL of undiluted bleach
into the empty reservoir.
5. Check for proper system pressurization (place vent valve toggle switch in
6. Press PRIME to drain and fill the flow cell to remove air bubbles. The system
will cycle to STANDBY. REPEAT.
1. Open up CellQuestPro
2. Choose Acquire > Connect to Cytometer, Counters.
3. Open all instrument control windows: Choose Cytometer > Detectors/Amps,
Threshold, and Compensation.
4. If you are using FL4, select the Four Color checkbox in the Detectors/Amps
5. Open an existing template or set up an Experiment document to view data.
6. Put on Control Sample.
7. In the Detectors/Amps window, adjust the FSC and SSC detector levels to
position the population properly on graph.
8. In the Threshold window, adjust threshold to eliminate low-level signals.
9. In palette, use the gate tool to draw a region around the cell population of
10. Adjust all graphs to exhibit only target population.
11. In the Detectors/Amps window, adjust the fluorescence detector voltages.
12. Put on compensation controls and adjust compensation for all fluorochromes.
Defining Acquisition Information
1. Under acquire, in the Acquisition & Storage dialog box, enter all information
necessary for your run.
2. Make sure that all needed parameters are checked (under “parameters saved”)
and click OK.
3. If needed, edit the reagent list and panels.
4. From the Parameter Description window, select “Change,” and set-up your
file name and file storage location.
5. Choose a panel or label parameters (under Untitled Parameter Settings).
1. Type in the Sample ID.
2. Press the RUN fluid control button on the instrument.
3. Install the sample tube on the SIP (Sample Injection Port) and quickly center
the tube support arm under the tube.
4. Deselect the Setup checkbox only if you are ready to acquire your data.
5. Click Acquire.
6. Remove the sample when acquisition is complete.
7. Repeat Steps 3-6 with the rest of your sample tubes.
If you have problems running your samples, you should refer to the
Cleaning & System Shutdown
1. Install a tube containing roughly 10% bleach on the sample injection port.
Move the tube arm to the side and allow the vacuum to aspirate 2 mL of the
2. Center the tube support arm and allow the bleach to run on HI for 5 minutes.
3. Install a tube containing distilled water on the SIP. Move the tube support
arm to the side and allow the vacuum to aspirate 2 mL of DI water.
4. Center the tube support are and allow the DI water to run on HI for 5 minutes.
5. Press the STANDBY fluid control button.
NOTICE: Allow the laser to cool down by leaving the ‘Calibur in
STANDBY for 5 minutes before turning off the power. Leave the tube of
DI water on the SIP. The tube should contain no more than 1 mL of DI
6. Release pressure from sheath tank with vent valve toggle switch.
7. Exit the software.
8. Log off of the computer.
9. Turn off the FACSCalibur instrument.