Analysis of Cellular DNA Content by Flow Cytometry

Abstract Cellular DNA content can be measured by flow or laser‐scanning cytometry with the aim of (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating the frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing the DNA‐ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and proteolytic treatment to permeabilize cells. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells for subsequent culturing based on DNA‐content differences, is also described. Also presented are methods for staining of cell nuclei isolated from paraffin‐embedded tissues, and deconvolution of DNA‐content‐frequency histograms to estimate the percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content.