Beisker W, Dolbeare F, Gray J W
Cytometry. 1987 Mar;8(2):235-9. doi: 10.1002/cyto.990080218.
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.
本报告描述了一种改进的免疫化学方法，用于对悬浮细胞中掺入的溴脱氧尿苷（BrdUrd）和总DNA含量进行染色。该方法包括五个步骤：用0.1 M HCl和0.7% Triton X-100处理以提取染色质蛋白，促进DNA变性并最大限度减少非特异性染色；细胞DNA在蒸馏水中加热至100℃进行变性；使用免疫化学方法对单链DNA（ssDNA）中的BrdUrd进行染色；用硼氢化钠（NaBH4）降低自发荧光；并用荧光染料碘化丙啶对DNA进行染色。通过该方法，使用流式细胞术可检测CHO细胞在短至几秒的时间内掺入的BrdUrd。此外，免疫荧光染色程序的化学计量比很高。
