Gasiorowski K, Brokos B, Kulma A, Ogorzałek A, Skórkowska K
Wrocław Medical University, Department of Basic Medical Sciences, Poland.
Cell Mol Biol Lett. 2001;6(2):141-59.
The sensitivity of the available methods of apoptosis detection in lymphocyte cultures was tested. Cells were preincubated with genotoxic agents: hydrogen peroxide (0.2 mM; 20 min.) and benzo[a]pyrene (40 microM; 90min.), and then cultured for 36h in the presence of a lectin (PHA-M; 1% v/v) and one of the following potentially antimutagenic agents: alkylresorcinols, anthocyanins, todralazine and fluphenazine. It was established that staining with a mixture of fluorochromes (ethidium bromide and acridine orange) provided the highest amount of detected apoptotic cells, and the best repeatability of the results in subsequent experiments. Calculation of the Spearman's rank correlation coefficients proved that there was a high correlation between the results obtained by the ethidium bromide/acridine orange method and those obtained by identifying genomic DNA fragmentation by means of FIGE-electrophoresis. Therefore, these two methods were chosen for further studies of the tested antimutagens' impact on apoptosis in genotoxically-damaged lymphocytes.
对淋巴细胞培养物中现有凋亡检测方法的敏感性进行了测试。细胞先用遗传毒性剂进行预孵育：过氧化氢（0.2 mM；20分钟）和苯并[a]芘（40 microM；90分钟），然后在存在凝集素（PHA-M；1% v/v）和以下潜在抗诱变剂之一的情况下培养36小时：烷基间苯二酚、花青素、托屈嗪和氟奋乃静。结果表明，用荧光染料混合物（溴化乙锭和吖啶橙）染色可检测到的凋亡细胞数量最多，且在后续实验中结果的重复性最佳。Spearman等级相关系数的计算证明，溴化乙锭/吖啶橙法获得的结果与通过FIGE电泳鉴定基因组DNA片段化获得的结果之间存在高度相关性。因此，选择这两种方法进一步研究受试抗诱变剂对遗传毒性损伤淋巴细胞凋亡的影响。
