FACS-Based Detection of Phosphorylated Histone H3 for the Quantitation of Mitotic Cells

The G2 checkpoint blocks cells from entering mitosis when DNA is damaged, and helps to protect the integrity of the genome. Tumor cells contain mutations that can inactivate checkpoints, and the inactivation of the G2 checkpoint can induce genomic instability and alter cellular responses to chemotherapeutic agents that damage DNA. The traditional method to assess whether the G2 checkpoint is normal is to microscopically count mitotic cells. A method using the fluorescence-activated cell scanner (FACS) is described, based on the presence of an intranuclear antigen present only in mitotic cells, detected using a specific, commercially available antibody. Cell staining and FACS analysis can be done in a single day, making this a rapid and reliable method to quantitate mitotic cells for various applications.