Selective elimination of proliferating cells in human diploid cell cultures by treatment with BrdU, 33258 Hoechst and visible light.

We have attempted to obtain pure populations of postmitotic cells from cultures of human diploid fibroblasts by the selective elimination of replicating cells. The selective system consists of treating cultures with 5-bromodeoxyuridine (5-BrdU) followed by exposure to the fluorescent dye 33258 Hoechst and irradiation with visible light. The percentage of survivors after the combined treatment was observed to be inversely proportional to the pretreatment [3H] thymidine labeling index of the culture. The post-treatment labeling indices, determined on the basis of the number of cells plated prior to the initiation of treatment, was higher in late passage cultures. Even after prolonged exposure to 5-BrdU (10 days), a small fraction of cells in early passage human fibroblast-like cultures was capable of incorporating [3H] thymidine. In contrast, actively replicating cells could be completely eliminated from HeLa cultures in this selective system. Studies with [3H] BrdU indicated that the cells incorporating this analog are preferentially killed. These observations suggest that human diploid cell cultures contain a subpopulation of very slowly cycling or transiently noncycling cells.