Effects of denaturation with HCL on the immunological staining of bromodeoxyuridine incorporated into DNA

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Abstract
Immunochemical procedures for detection of BrdUrd incorporated into DNA require a denaturation step of DNA. Denaturation with HCl is widely used for flow cytometric analysis of the cell cycle and for histological preparations. This brief communication describes an attempt to standardize a denaturation procedure with HCl. Various denaturation conditions at 20°C were examined for human promyelocytic leukemia cells (HL‐60 cells) fixed in ethanol. After denaturation of DNA, the cells were stained by an indirect immunofluorescence method using a commercially available monoclonal anti‐BrdUrd antibody or by propidium iodide. The relative fluorescence intensities of stained BrdUrd and double‐stranded DNA were altered reciprocally by changing HCl concentration and/or denaturation time. Treatment with 4N HCl for 10‐20 min at 20°C allowed denaturation of more than 80% of DNA and the maximum BrdUrd‐linked immunofluorescence. Under this condition, the coefficient of variation of the DNA histograms remained relatively small.