Flow cytometry analysis using the Becton Dickinson FACS Calibur.

This unit details the operation of a FACS Calibur flow cytometer for cell analysis. The operation of the Becton Dickinson FACS Calibur and CELLQuest software version 3.0 is described, but the unit is general enough to be helpful for users of all flow cytometers. The FACS Calibur replaces both the FACSCan and FACSort; the information presented here is also applicable to older BD instruments. In this unit, particular emphasis is placed on data acquisition rather than data analysis. Single-color analysis using fluorescein isothiocyanate (FITC)-conjugated antibodies is described along with procedures to check instrument performance and sensitivity, single-color (FITC) analysis with simultaneous live/dead discrimination using propidium iodide (PI), simultaneous two-color analysis using FITC- and phycoerythrin (PE)-conjugated antibodies, two-color FITC/PE analysis with simultaneous live/dead discrimination using PI, simultaneous three-color immunofluorescence with FITC, PE, and the red fluorescent dyes, and finally, simultaneous four-color immunofluorescence with FITC, PE, red fluorescence dyes, and APC.