Srivastava V, Miller S, Busbee D
Department of Anatomy and Public Health, College of Veterinary Medicine, Texas A & M University, College Station 77843.
Cytometry. 1993;14(2):144-53. doi: 10.1002/cyto.990140206.
An improved differential fluorescence analysis technique which employs scanning laser cytometry for the detection of two DNA binding fluorochromes was used to quantitate both total DNA and cellular incorporation of the base analogue, bromodeoxyuridine (BrdUrd), into UV- or methyl methane sulphonate (MMS)-treated DNA. In this procedure DNA containing BrdUrd was partially denatured and immunolabeled by binding anti-BrdUrd monoclonal IgG to incorporated BrdUrd. Anti-BrdUrd was detected with a secondary fluorochrome-labeled polyclonal anti-IgG. Cells were counterstained with propidium iodide to allow the determination of total DNA. Fluorescence was determined using the Meridian ACAS 570 Scanning Laser Cytometer. The computer-analyzed images were generated from monodisperse populations of UV- or MMS-treated cells, allowing the evaluation of DNA synthesis associated with excision repair and of total DNA content in single cells. These data were compared with 3H-thymidine incorporation occurring as a function of excision repair. The analysis of DNA synthesis with this technique is consistent, relatively simple, and rapid with excellent sensitivity and provides a viable method for determining cycling vs. non-cycling cells, total cellular DNA, and excision repair-associated DNA synthesis of individual cells within heterogeneous cell populations.
一种改进的差分荧光分析技术利用扫描激光细胞术检测两种DNA结合荧光染料，用于定量经紫外线或甲基磺酸甲酯（MMS）处理的DNA中的总DNA以及碱基类似物溴脱氧尿苷（BrdUrd）的细胞掺入情况。在此过程中，含有BrdUrd的DNA被部分变性，并通过将抗BrdUrd单克隆IgG与掺入的BrdUrd结合进行免疫标记。用二级荧光染料标记的多克隆抗IgG检测抗BrdUrd。细胞用碘化丙啶复染以确定总DNA。使用Meridian ACAS 570扫描激光细胞仪测定荧光。计算机分析的图像由紫外线或MMS处理细胞的单分散群体生成，从而能够评估与切除修复相关的DNA合成以及单个细胞中的总DNA含量。将这些数据与作为切除修复函数的3H-胸腺嘧啶掺入情况进行比较。用该技术分析DNA合成具有一致性、相对简单、快速且灵敏度高的特点，并为确定异质细胞群体中单个细胞的循环与非循环细胞、总细胞DNA以及与切除修复相关的DNA合成提供了一种可行的方法。
