A simplified method for the coordinate examination of apoptosis and surface phenotype of murine lymphocytes
A simplified method for the coordinate examination of apoptosis and surface phenotype of murine lymphocytes
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DOI：
10.1016/0022-1759(95)00216-2
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发表时间：
1995-12-27
影响因子：
2.2
通讯作者：
Moore, JS
中科院分区：
文献类型：
--
作者：
Douglas, RS;Tarshis, AD;Moore, JS
小鼠淋巴细胞在体外很容易发生自发和糖皮质激素诱导的凋亡。以往的研究表明，在细胞凋亡过程中，包括淋巴细胞在内的许多细胞类型都会被DNA酶切割，从而在碘化丙啶染色和流式细胞仪分析时，显示出一个亚G(0)DNA峰。在混合种群中，通常希望从表型上识别经历细胞凋亡的不同种群或亚群，因此需要对表面表型和DNA含量进行多参数分析。虽然多聚甲醛固定程序在表面评估中很常见，但还没有广泛应用于量化细胞凋亡的方法。为了测量混合淋巴细胞群体中的凋亡，我们评估了温和的洗涤剂渗透和多聚甲醛固定程序与碘化丙啶(PI)DNA染色相结合的方法，适用于细胞周期研究的现有方法。用这种方法和我们定义的严格的门控技术，我们检测到糖皮质激素处理的小鼠淋巴细胞株的亚G期细胞周期区域中的凋亡和碎片部分。使用这个细胞系，WEHI 231.7，作为一个淋巴细胞模型，我们开发了一种逻辑门控策略，将碎片排除在分析之外。我们进一步证明，用多聚甲醛固定和PI染色检测新鲜分离的小鼠淋巴细胞的凋亡在数量上与乙醇固定的PI染色或DNA链断裂缺口平移标记(TUNEL)法相当。最后，使用新鲜的小鼠脾细胞，我们证明了多聚甲醛固定保留了表面蛋白染色，允许对混合淋巴细胞群体中的免疫表型和细胞周期状态进行多参数分析。因此，这种方法提供了一种廉价和技术简单的替代方法来评估细胞凋亡和表面表型。
Murine lymphocytes readily undergo spontaneous and glucocortocoid-induced apoptosis in vitro. It has been previously demonstrated that during apoptosis, many cell types including lymphocytes, enzymatically cleave their DNA, thus demonstrating a sub-G(0) DNA peak when stained with propidium iodide and analyzed by flow cytometry. In a mixed population, it is often desirable to phenotypically identify distinct populations or subsets undergoing apoptosis, thus requiring multiparameter analysis of surface phenotype and DNA content. Paraformaldehyde fixation procedures, although common for surface evaluation, have not been extensively used in methods quantifying apoptosis. To measure apoptosis in a mixed lymphocyte population, we evaluated a gentle detergent permeabilization and paraformaldehyde fixation procedure combined with propidium iodide (PI) DNA staining, adapted from existing methods for cell cycle studies. With this method and rigorous gating techniques which we defined, we detected both apoptotic and debris fractions within the sub-G, cell cycle region of a glucocortocoid-treated murine lymphocyte cell line. Using this cell line, WEHI 231.7, as a lymphocyte model, we developed a logical gating strategy to exclude debris from analysis. We further demonstrated that apoptosis in freshly isolated murine lymphocytes detected with paraformaldehyde fixation and PI staining was quantitatively comparable to PI staining with ethanol fixation, or nick translation labeling of DNA strand breaks (TUNEL). Finally, using fresh murine spleen cells, we demonstrated that paraformaldehyde fixation preserves surface protein staining, allowing multiparameter analysis of immunophenotype and apoptotic or cell cycle status in a mixed lymphocyte population. Thus, this method offers an inexpensive and technically simple alternative for assessing apoptosis and surface phenotype.