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Instrument Setup with BD FACSComp
™
Software
and BD CaliBRITE
™
Beads
1. Perform instrument start up.
2. Perform flow check.
3. Prepare tubes of BD CaliBRITE™ beads and open BD FACSComp™
software.
4. Launch BD FACSComp software.
5. Run BD FACSComp software in Lyse/No Wash mode.
6. Proceed to Instrument Setup with the Cytometer Setup Beads.
Note: For detailed information on using BD FACSComp with
BD CaliBRITE beads to set up the flow cytometer, refer to the
BD FACSComp Software User’s Guide and the BD CaliBRITE
Beads Package Insert. Version 4.2 contains a BD CBA preference
setting to automatically save a BD CBA calibration file at
the successful completion of any Lyse/No Wash assay. The BD
CBA calibration file provides the optimization for FSC, SSC,
and threshold settings as described in Instrument Setup with
the Cytometer Setup Beads, Steps 3 – 5. Optimization of the
fluorescence parameter settings is still required (ie, PMT and
compensation settings, see Instrument Setup with the Cytometer
Setup Beads, Step 6).
Instrument Setup with the Cytometer Setup Beads
1. Launch BD CellQuest™ software and open the BD™ CBA Instrument
Setup template.
Note: The BD CBA Instrument Setup template can be found on the
BD CBA Software or BD FACStation™ CD for Macintosh computers in
the BD CBA folder. Following installation on Macintosh computers
using BD CBA Software Version 1.0, the template can be found in
the BD Applications/BD CBA folder/Sample Files/Mouse Isotyping
Files/Instrument Setup folder. For BD CBA Software Version 1.1 or
higher, the template can be found in the BD Applications/BD CBA
folder. The template is not installed from the CD on PC-compatible
computers. This file and instrument set-up templates for dual-laser and
other flow cytometers may also be downloaded via the internet from:
bdbiosciences.com/pharmingen/CBA/downloads.shtml
2. Set the instrument to Acquisition mode.
Note: The BD CBA Software will evaluate data in five parameters
(FSC, SSC, FL1, FL2, and FL3). Turn off additional detectors.
3. Set SSC (side light scatter) and FSC (forward light scatter) to Log mode.
4. Decrease the SSC PMT voltage by 100 from what FACSComp set.
5. Set the Threshold to SSC at 650.
6. In setup mode, run Cytometer Setup Beads tube A. Follow the setup
instructions on pgs. 15 – 16.
Note: Pause and restart acquisition frequently during the instrument
setup procedure in order to reset detected values after settings
adjustments.
