title: An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine.
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: 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; cellular DNA is denatured by heating to 100 degrees C in distilled water; BrdUrd in single-stranded DNA (ssDNA) is stained using an immunochemical procedure; autofluorescence is reduced using sodium borohydride (NaBH4); and 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.
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001894:Borohydrides; D001973:Bromodeoxyuridine / Q000032:analysis*; D002021:Buffers; D002453:Cell Cycle; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000032:analysis*; D006358:Hot Temperature; D009691:Nucleic Acid Denaturation
chemical_list: D000911:Antibodies, Monoclonal; D001894:Borohydrides; D002021:Buffers; D004247:DNA; D001973:Bromodeoxyuridine