Liegler T J, Hyun W, Yen T S, Stites D P
Laboratory for Cell Analysis, University of California, San Francisco, USA.
Clin Diagn Lab Immunol. 1995 May;2(3):369-76. doi: 10.1128/cdli.2.3.369-376.1995.
Regulation of peripheral lymphocyte number involves a poorly understood balance between cell renewal and loss. Disrupting this balance leads to a large number of disease states. Methods which allow qualitative and quantitative measurements of cell viability are increasingly valuable to studies directed at revealing the mechanisms underlying apoptotic and necrotic cell death. Here, we have characterized a method using single-laser flow cytometry that differentiates and quantifies the relative number of live, apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes. Following in vitro gamma irradiation and staining with acridine orange in combination with ethidium bromide, three distinct populations were seen by bivariate analysis of green versus red fluorescence. The identity of each distinct fluorescent population (whether live, apoptotic, or necrotic) was determined by sorting and examination of cellular morphology by electron microscopy. This flow cytometric method is directly compared with the techniques of trypan blue exclusion and DNA fragmentation to quantify cell death following exposure to various doses of in vitro gamma irradiation and postirradiation incubation times. We extend our findings to illustrate the utility of this method beyond analyzing radiation-induced apoptotic peripheral blood mononuclear cells (PBMC); similar fluorescent patterns are shown for radiation- and corticosteroid-treated murine thymocytes, activated human PBMC, and PBMC from human immunodeficiency virus-infected individuals. Our results demonstrate that dual-parameter flow cytometric analysis of acridine orange-ethidium bromide-stained lymphocytes is overall a superior method with increased sensitivity, greater accuracy, and decreased subjectivity in comparison with the other methods tested. By using standard laser and filter settings commonly available to flow cytometric laboratories, this method allows rapid measurement of a large number of cells from a heterogeneous sample.
外周淋巴细胞数量的调节涉及细胞更新与丢失之间一种尚不清楚的平衡。打破这种平衡会导致大量疾病状态。能够对细胞活力进行定性和定量测量的方法对于旨在揭示凋亡和坏死性细胞死亡潜在机制的研究越来越有价值。在此，我们描述了一种使用单激光流式细胞术的方法，该方法可区分并定量活的、凋亡的以及晚期凋亡和坏死的外周淋巴细胞的相对数量。在体外γ射线照射并用吖啶橙与溴化乙锭联合染色后，通过对绿色与红色荧光的双变量分析可观察到三个不同的群体。通过分选以及用电子显微镜检查细胞形态来确定每个不同荧光群体（无论是活的、凋亡的还是坏死的）的身份。将这种流式细胞术方法直接与台盼蓝排斥法和DNA片段化技术进行比较，以量化暴露于不同剂量的体外γ射线照射及照射后孵育时间后的细胞死亡情况。我们扩展了研究结果以说明该方法在分析辐射诱导的凋亡外周血单个核细胞（PBMC）之外的用途；辐射和皮质类固醇处理的小鼠胸腺细胞、活化的人PBMC以及来自人类免疫缺陷病毒感染个体的PBMC也显示出类似的荧光模式。我们的结果表明，与所测试的其他方法相比，对吖啶橙 - 溴化乙锭染色的淋巴细胞进行双参数流式细胞术分析总体上是一种更优的方法，具有更高的灵敏度、更大的准确性以及更低的主观性。通过使用流式细胞术实验室常用的标准激光和滤光片设置，该方法能够快速测量来自异质样本的大量细胞。
