Flow cytometric analysis of factors which influence the BrdUrd‐Hoechst quenching effect in cultivated human fibroblasts and lymphocytes

Human fibroblasts and lymphocytes were grown in bromodeoxyuridine (BrdUrd) containing medium, harvested after 27–71 hours and stained with the fluorochrome Hoechst 33258. Parameters that influence the BrdUrd quenching effect of Hoechst fluorescence at a given pH and ionic strength of the staining solution were determined. The parameters were determined in order to obtain high resolution histograms with differentiation of each cell cycle compartment for noncycling cells and for cycling cells in the first and second cycle. The BrdUrd concentration was found to be the most significant determinant, but cell density, cell type, dye concentration, type of Hoechst stain and incubation temperature during staining also contribute to the modulation of fluorescence of BrdUrd substituted nuclei. Optimization of these factors is required in order to achieve complete and well resolved cell cycle histograms by the BrdUrd-Hoechst flow cytometric technique.