Abstract

Cell proliferation assays are used for a large variety of applications in the life sciences. This unit describes a flow-cytometry-based method that uses BrdU labeling in conjunction with multiple fluorescently labeled cell surface markers, allowing extensive phenotypic characterization of dividing cells in addition to assessment of their frequency. BrdU labeling has the advantage of constituting a nonradioactive technique that, when combined with additional fluorescent-based reagents, avoids time-consuming and often costly cell isolation procedures. Moreover, because BrdU is stably integrated into the DNA, it can be measured in a cell for many months. The method presented in this unit is based on paraformaldehyde fixation and reversible saponin-based cell membrane permeabilization, which maintains cell integrity as well as fluorescent labeling with a large number of different fluorochromes, allowing 10- to 12-color flow cytometric analysis of proliferating cells.