Use of a flow cytometric assay to quantitate apoptosis in human lymphocytes

Apoptosis is an active process of cellular self-destruction which plays an important physiologic role in the maintenance of homeostasis. This form of cell death has classically been assessed by the appearance of a 'ladder-like' banding pattern upon gel electrophoresis of disrupted cells. The electrophoretic method is qualitative but offers no means of quantitation. We have optimized a flow cytometric method which allows quantitation of the apoptotic subset of cells. This assay uses propidium iodide and therefore can be performed on any flow cytometer with a blue-green excitation line. We initially verified the results of this method by using human thymocytes treated with dexamethasone, a known inducer of apoptosis. Gels were run simultaneously with flow cytometric analysis and assessed for the presence or the absence of apoptotic cells. Results obtained with gel electrophoresis correlated well with the flow cytometric method. In further studies, we applied this assay to human PBMC and obtained similar results. In conclusion, this assay is a significant improvement over currently available methodology for the determination of apoptosis. We have found it to be quantitative, reproducible, and applicable to different human cell types. The role of apoptosis in human disease remains to be elucidated and this method offers a convenient means to study this process.