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抗溴化脱氧尿嘧啶核苷(BrdUrd)单克隆抗体荧光染色过程涉及到以下一些重要步骤:BrdUrd脉冲标记时间长短,核DNA部分变性处理所用盐酸的浓度与处理时间等。应以BrdUrd阳性细胞与阴性细胞之间平均荧光强度之比值,酸处理过程中细胞凝集的比例,细胞群体G_1峰DNA荧光强度及变异系数作为整个染色过程最佳条件的评价标准。在30分钟BrdUrd标记时间,2.4mol/L盐酸30分钟处理时间及抗BrdUrd单克隆抗体20℃1小时温育时间等条件下,可获得满意的染色结果,并且不需要核糖核酸酶的处理。本实验室将KF-1、KFr、HeLa、IK-90等几种培养细胞系用此方法染色均可获得满意的染色结果。
The BrdUrd monoclonal antibody fluorescent staining process involves the following important steps: the length of BrdUrd pulse labeling, the concentration of hydrochloric acid used in the partial denaturation of nuclear DNA, and the processing time. The ratio of the average fluorescence intensity between BrdUrd-positive cells and negative cells, the ratio of cell aggregation during acid treatment, the fluorescence intensity and coefficient of variation of cell population G1 peak DNA should be the evaluation criteria for the best conditions for the entire staining process. With a BrdUrd labeling time of 30 minutes, a 30 minute treatment time of 2.4 mol/L hydrochloric acid, and a 1-hour incubation time of the anti-BrdUrd monoclonal antibody at 20°C, satisfactory staining results can be obtained without the need for ribonuclease treatment. In this laboratory, several cultured cell lines such as KF-1, KFr, HeLa, and IK-90 can be stained by this method to obtain satisfactory staining results.