An improved immunocytochemical procedure for high‐sensitivity detection of incorporated bromodeoxyuridine

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Abstract
This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: (1) chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X‐100 to facilitate DNA denaturation and to minimize nonspecific staining; (2) cellular DNA is denatured by heating to 100°C in distilled water; (3) BrdUrd in single‐stranded DNA (ssDNA) is stained using an immunochemical procedure; (4) autofluorescence is reduced using sodium borohydride (NaBH4); and (5) DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.