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.
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D006690:Bisbenzimidazole / Q000494:pharmacology*; D001973:Bromodeoxyuridine / Q000494:pharmacology*; D002460:Cell Line; D002875:Chromosomes / Q000187:drug effects / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D004261:DNA Replication / Q000187:drug effects*; D005434:Flow Cytometry / Q000379:methods; D005467:Floxuridine / Q000494:pharmacology; D007700:Kinetics; D013936:Thymidine / Q000378:metabolism*
D001562:Benzimidazoles; D005467:Floxuridine; D001973:Bromodeoxyuridine; D006690:Bisbenzimidazole; D013936:Thymidine