Mitotic index determination by flow cytometry.

Abstract

Chromosome condensation during mitosis is associated with phosphorylation of histone H3 at serine 10 (pS10H3). Detection of pS10H3 phosphorylation using phospho-specific antibodies in combination with DNA content flow cytometry provides a rapid and accurate means of determining the percentage of mitotic cells in a population. Using the spindle poison nocodazole to trap mitotic cells it is possible to determine the rate at which cells accumulate in mitosis with time. By comparing the rates of mitotic accumulation before and after DNA damage it is possible to gauge the efficiency with which the G2 checkpoint blocks entry to mitosis. Because DNA content is measured simultaneously with pS10H3 fluorescence this method can also be used to identify cells which enter mitosis with incompletely replicated DNA as a result of S-M checkpoint failure during DNA synthesis inhibition.