Flow cytometric analysis of bromodeoxyuridine-substituted cells stained with 33258 Hoechst.
This paper describes a flow-cytometric application of the quenching of fluorescence from 33258 Hoechst stained Chinese hamster ovary-line cells due to the incorporation of 5-bromo-deoxyuridine (BrdU) into the cellular deoxyribonucleic acid. Cells were grown for 24 hr in medium containing BrdU in concentrations ranging from 1 x 10(-8) to 1 x 10(-4) M. For each concentration we measured the average fluorescence as determined by flow cytometry, the  extent of BrdU substitution and the effect of the BrdU on cell growth. We determined that a BrdU concentration of 1 x 10(-5) M resulted in sufficient substitution to quench the fluorescence from 33258 Hoechst by a factor of 4, allowing discrimination between cycling and noncycling cells. The extent of BrdU substitution after growth for 24 hr in this concentration of BrdU was 64%. These data indicate the feasibility of detecting deoxyribonucleic acid synthesis in whole cells using the 33258 Hoechst-BrdU methodology.
D001562:Benzimidazoles; D006690:Bisbenzimidazole; D001973:Bromodeoxyuridine / Q000378:metabolism* / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis*; D005453:Fluorescence; D013050:Spectrometry, Fluorescence
D001562:Benzimidazoles; D004247:DNA; D001973:Bromodeoxyuridine; D006690:Bisbenzimidazole