Analysis of Cell Cycle by Flow Cytometry

Described are four widely used procedures to analyze the cell cycle by flow cytometry. The first two are based on univariate analysis of cellular DNA content following cell staining with either propidium iodide (PI) or 4′,6′-diamidino-2-phenylindole (DAPI) and deconvolution of the cellular DNA content frequency histograms. This approach reveals distribution of cells in three major phases of the cycle (G₁ vs S vs G₂ /M) and makes it possible to detect apoptotic cells with fractional DNA content. The third approach is based on the bivariate analysis of DNA content and proliferation-associated proteins. The expression of cyclin D, cyclin E, cyclin A, or cyclin B1 vs DNA content is presented as an example. This approach allows one to distinguish, for example, G₀ from G₁ cells, identify mitotic cells, or relate expression of other intracellular proteins to the cell cycle position. The fourth procedure relies on the detection of 5′-bromo-2′-deoxyuridine (BrdU) incorporation to label the DNA-replicating cells.