Bromodeoxyuridine distribution patterns in L 929 fibroblasts and influence of tumor necrosis factor

Incorporation of bromodeoxyuridine during the S phase of the cell cycle provides an appropriate method to study the replication of DNA in proliferating cells. We analyzed the distribution patterns of incorporated bromodeoxyuridine in L 929 cells and studied the influence of tumor necrosis factor-alpha in vitro by indirect immunofluorescence. Using conventional epifluorescent and confocal laser scanning microscopy, we defined three types of bromodeoxyuridine distribution patterns in synchronized L 929 cells which showed typical differences in their percentages during the time course of the S phase. Type I with small spots and unstained areas was typical for the early S phase. Type II was characterized by a texture of homogeneously distributed fluorescence and increased during middle and late S phase. Type III with a small number of larger clusters revealed its highest proportion in the late S phase. Incubation with tumor necrosis factor reduced the number of bromodeoxyuridine-positive cells. We found that the cytokine, when present in late S phase, provoked an increase in the percentage of type III distribution pattern. The results show that bromodeoxyuridine distribution pattern analysis can be used to detect influences on cell cycle kinetics induced by therapeutics.