Cell-cycle analysis using a monoclonal antibody to BrdUrd

The flow cytometric measurement of DNA distributions of cells has many applications in biomedical research. Phase fractions estimated (calculated) from such distributions are used to study the growth characteristics of various types of cells, particularly when the cells have been exposed to perturbing agents such as chemotherapeutic drugs. For more than 10 years many methods for resolving DNA distributions into the three cell subpopulations (G1, S and G2 + M) have been reported in the literature. A new method of analysis utilizing a monoclonal antibody to bromodeoxyuridine (BrdUrd) has been developed (Gratzner, 1982; Dolbeare et al., 1983) which makes it possible in most cases to accurately determine phase fractions without resorting to mathematical models. The procedure involves the incorporation of BrdUrd by growing (DNA synthesizing) S phase cells, labelling the BrdUrd with a fluorescent monoclonal antibody, and the bivariate measurement of the antibody and of total DNA content, the latter through propidium-iodide staining. The resulting bivariate distributions clearly and simply resolve the three subpopulations. This paper describes the method and illustrates its use in the analysis of various fractions of elutriated exponentially growing Chinese hamster ovary (CHO) cells.