Distinction between 5-bromodeoxyuridine labeled and unlabeled mitotic cells by flow cytometry

Exponentially growing Friend leukemia cells are exposed to 5-bromodeoxyuridine (BrdUrd) for the time period equivalent to one generation. After fixation and incubation with RNase, DNA in situ is partially denatured by acid and the cells are stained with acridine orange (AO). Staining with AO under these conditions reveals the extent of denatured versus double stranded DNA and enables one to distinguish mitotic from interphase cells. It is observed that all BrdUrd treated cells, regardless of cell cycle phase, have decreased both green and red fluorescence. This finding suggests that BrdUrd interacts with AO not only in the complexes of the dye with double stranded DNA, but also with the single stranded biopolymer. The BrdUrd-attributed suppression of cell fluorescence is large enough to separate totally BrdUrd labeled from unlabeled mitotic populations. The combination of these two flow cytometric techniques, one which allows discrimination between interphase and metaphase cells and the other which allows further subdivision of metaphase cells into those which have incorporated BrdUrd and those which have not, provides an alternative to the autoradiographic procedure necessary for obtaining the fraction of labeled mitoses.