Hoy C A, Seamer L C, Schimke R T
Department of Biological Sciences, Stanford University, California 94305.
Cytometry. 1989 Nov;10(6):718-25. doi: 10.1002/cyto.990100608.
Significant inter- and intraexperimental variations of the relative antibromodeoxyuridine fluorescence were found during measurement of DNA synthesis rates using flow cytometric analysis of 5-bromodeoxyuridine (BrdUrd)-labeled cells with an anti-BrdUrd antibody. Fluctuations in other endpoints associated with levels of denaturation (integrity of DNA and cell size) were also observed to vary widely among samples that were otherwise thought to have been treated identically. Therefore, the denaturation step has been carefully re-examined, and several critical factors were identified that influence the denaturation and subsequent binding of the anti-BrdUrd to the labeled DNA. These factors include cell density, volume of water, and pH of the sample during heating. Appropriate adjustments are now included in the protocol, resulting in more consistent anti-Brd-Urd measurements in the face of routine (and sometimes necessary) experimental variations.
在使用抗溴脱氧尿苷抗体对5-溴脱氧尿苷（BrdUrd）标记的细胞进行流式细胞术分析来测量DNA合成速率的过程中，发现实验间和实验内相对抗溴脱氧尿苷荧光存在显著差异。与变性水平（DNA完整性和细胞大小）相关的其他终点指标的波动在原本认为处理方式相同的样本中也被观察到有很大差异。因此，对变性步骤进行了仔细的重新审视，并确定了几个影响变性以及随后抗BrdUrd与标记DNA结合的关键因素。这些因素包括加热过程中的细胞密度、水量和样本的pH值。现在方案中包含了适当的调整，从而在面对常规（有时也是必要的）实验变化时能获得更一致的抗Brd-Urd测量结果。
