Cell Cycle Analysis by Flow Cytometry

Abstract Flow cytometry, the methodology that provides a way to measure physical and chemical attributes of individual cells rapidly and with high accuracy, is widely used in cell cycle analysis. The most common assays are: univariate analysis of cellular deoxyribonucleic acid (DNA) content discloses frequencies of cells in G 0/1 , S and G 2 /M phases; bivariate analysis of DNA versus ribonucleic acid (RNA) content reveals frequencies of G 0 , G 1 , S and G 2 M cells, of DNA content versus histone H3 phosphorylated on Ser10 defines frequencies of G 0/1 , S, G 2 and M cells. Detection of cells that incorporated DNA precursor, such as 5‐bromo‐2′‐deoxyuridine (BrdU) or 5‐ethynyl‐2′‐deoxyuridine (EdU) identifies cells that replicated DNA. Immunocytochemical detection of cyclin D1, cyclin E, Cyclin A and cyclin B1 concurrent with DNA content reveals the proteins that are essential for driving the cell through the cell cycle. The stathmokinetic approach makes it possible to assess the kinetics of progression through the cell cycle. Key Concepts: Cellular DNA content identifies cell position in the cell cycle. Fractional cellular DNA content (‘sub‐G 1 ’ cells) is a marker of apoptotic cells. Low cellular RNA content is a biomarker of G 0 (quiescent) cells. Incorporation of BrdU or EdU identifies DNA replication cells. Phosphorylation of histone H3 on Ser10 is a marker of mitotic cells. Detection of cyclin proteins is a marker of cell progression machinery. Stathmokinetic approach based on cell arrest in metaphase makes it possible to assess the entry of cells to mitosis (‘mitotic rate’, ‘cell birth rate’) and reveals kinetics of cell progression through the cycle.