Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

Background: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence int ensity of the membrane-permeable Hoechst dyes is reduced by the incorporati on of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU) cells from viable BrdU negative (BrdU-) cells. Methods: Cultures of proliferating cells mere supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two pro liferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells mere sorted into different fractions from a mixture of BrdU+ and BrdU- cel ls based on Hoechst fluorescence intensity and the ability to exclude the v ital dye, propidium iodide. Subsequently, samples from the original mixture , the sorted BrdU+ cell population, and the sorted BrdU- cell population we re immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. Results: Two mixtures consisting of approximately 55% and 69% BrdU+ cells w ere sorted into fractions consisting of greater than 93% BrdU+ cells and 92 % BrdU- cells. The separated cell populations were maintained in vitro afte r sorting to demonstrate their viability. Conclusions: Hoechst fluorescence intensity in combination with cell sortin g is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro aft er sorting to demonstrate their viability. Cytometry 41: 89-95, 2000. (C) 2 000 Wiley-Liss, Inc.