Application of the BrdU/thymidine method to flow cytogenetics: Differential quenching/enhancement of Hoechst 33258 fluorescence of late-replicating chromosomes

Discrimination between many types of isolated mammalian chromosomes can be accomplished by dual-beam flow cytometry following DNA staining with Hoechst 33258 (HO) and Chromomycin A3 (CA3). In this report, we show that the bivariate discrimination of selected late-replicating Chinese hamster M3-1 chromosomes can be improved by appropriate treatment of the cells with 5-bromo-2′-deoxyuridine (BrdU) prior to chromosome isolation and staining. Two labeling schemes are reported. In one scheme the chromosomes are collected from cells labeled with BrdU only during late S phase. The Hoechst fluorescence of the 10, 11, M2, and Y chromosomes is substantially quenched by the incorporated BrdU, thus improving their discrimination. In the other scheme, chromosomes are collected from cells labeled with thymidine (dT) during late S phase following 20 h of growth in BrdU-containing medium. The Hoechst fluorescence of the 10, 11, M2, and Y chromosomes is quenched less than the other chromosomes, again improving their discrimination. Y chromosomes from chromosome suspensions of untreated controls, of cells labeled with BrdU during late S phase, and of cells labeled with dT during late S phase following 20 h growth in BrdU were separated by dual-parameter sorting. While the purity of the sorted Y chromosome was 15% in untreated controls, it was 70-75% using the BrdU/dT labeling protocols.