Terry Nicholas H A, White R Allen
Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.
Nat Protoc. 2006;1(2):859-69. doi: 10.1038/nprot.2006.113.
This protocol describes methods for calculating the proliferative parameters of cell populations. The basis of the technique is to label cells, either in vitro or in vivo, with halogenated thymidine analogs, such as bromodeoxyuridine (BrdU). Bivariate DNA-BrdU flow cytometry is used to analyze the BrdU-labeled and unlabeled cells. The enumeration of specific cohorts of cells that either have or have not divided in the interval between labeling and cell/tissue sampling permits the calculation of the potential doubling time (T(pot)) of the population, plus the durations of DNA synthesis (T(S)) and the G2+M phase (T(G2+M)) of the cell cycle. The method provides information that is not otherwise available, namely inhibition of DNA synthesis and the separate evaluation of cell-cycle effects in BrdU-labeled and unlabeled subpopulations. Ethanol-fixed samples take 1 d to prepare and stain, and reliable parameter estimates might be obtained from measurements made at a single time point after labeling.
本方案描述了计算细胞群体增殖参数的方法。该技术的基础是在体外或体内用卤代胸腺嘧啶类似物（如溴脱氧尿苷，BrdU）标记细胞。双变量DNA-BrdU流式细胞术用于分析BrdU标记和未标记的细胞。通过对在标记和细胞/组织取样间隔期间已分裂或未分裂的特定细胞群体进行计数，可以计算群体的潜在倍增时间（T(pot)），以及细胞周期中DNA合成期（T(S)）和G2+M期（T(G2+M)）的持续时间。该方法提供了其他方法无法获得的信息，即DNA合成的抑制以及对BrdU标记和未标记亚群中细胞周期效应的单独评估。乙醇固定的样本制备和染色需要1天时间，并且通过标记后单个时间点的测量可能获得可靠的参数估计值。
