Flow cytometric analysis of p53-induced apoptosis

The p53 protein is a key player in the cellular response to stress conditions. Activation of p53 induces growth inhibition in the form of cell growth arrest or apoptosis. The latter plays an important role in the tumor suppression function of p53. It is therefore of great interest to understand in detail the mechanisms by which p53 induces apoptosis. In this chapter, we describe a flow cytometric assay for the measurement of p53 apoptotic activity. This assay is applicable for exogenously expressed p53 by transfection as well as for endogenous p53. The p53 protein is detected by intra-cellular fluorescent staining of p53 or by tagging p53 with GFP. The extent of apoptosis in cells expressing p53 is determined by cell cycle distribution using flow cytometry. The protocol described here can be employed to study the regulation of p53-mediated apoptosis and can be broadly applied to other apoptotic proteins.