Chiu L, Cherwinski H, Ransom J, Dunne J F
Syntex Discovery Research, Palo Alto, CA 94304, USA.
J Immunol Methods. 1996 Feb 5;189(2):157-71. doi: 10.1016/0022-1759(95)00214-6.
The apoptotic response to various stimuli is an important part of immune regulation, and the ability to identify apoptotic lymphocytes within a complex population is a prerequisite to a more detailed understanding of its role in vivo, We described a flow cytometric technique which utilizes viable cells and enables simultaneous identification of apoptotic cells and analyses of immunophenotype, cell cycle progression, membrane integrity and light scatter properties. It is based upon analysis of two regions of the emission spectrum of the DNA-binding vital dye hoechst 33342. We established a precise correlation between the ratio of red to blue fluorescence emission and apoptosis based upon nuclear morphology and the presence of characteristic DNA degradation patterns. In human peripheral blood lymphocytes (PBL) and mouse thymocytes we incorporated light scatter properties, cell cycle stage, relevant cell surface immunophenotypic markers (CD25 or CD4) and CD8) and a marker of plasma membrane integrity (merocyanine 540) to enable multiparametric phenotyping of apoptotic cells. We show that staurosporine-induced apoptosis of ConA-stimulated PBL is not correlated with cell cycle stage but is selective for activated cells since the frequency of large, CD25+ cells is decreased by staurosporine. Dexamethasone and ionomycin differ in their ability to induce apoptosis selectively in murine thymocyte subsets. Dexamethasone kills a broad spectrum of the CD4/8 immunophenotypes with no selectively for cell cycle stage. Ionomycin selectively deplete CD4+8+ cells, especially those in the Go/G1 region of the cell cycle, and spared CD4-8+ cells. This technique is broadly advantageous for in vitro and ex vivo models of apoptosis in that it interrogates individual viable cells and correlates membrane and nuclear apoptotic changes with standard flow cytometric immunophenotyping.
对各种刺激的凋亡反应是免疫调节的重要组成部分，而在复杂细胞群体中识别凋亡淋巴细胞的能力是更详细了解其在体内作用的先决条件。我们描述了一种流式细胞术技术，该技术利用活细胞，能够同时识别凋亡细胞并分析免疫表型、细胞周期进程、膜完整性和光散射特性。它基于对DNA结合活性染料Hoechst 33342发射光谱两个区域的分析。我们根据核形态和特征性DNA降解模式的存在，建立了红色与蓝色荧光发射比率与凋亡之间的精确相关性。在人外周血淋巴细胞（PBL）和小鼠胸腺细胞中，我们纳入了光散射特性、细胞周期阶段、相关细胞表面免疫表型标志物（CD25或CD4和CD8）以及质膜完整性标志物（部花青540），以实现凋亡细胞的多参数表型分析。我们表明，星形孢菌素诱导的ConA刺激的PBL凋亡与细胞周期阶段无关，但对活化细胞具有选择性，因为星形孢菌素可降低大的CD25+细胞的频率。地塞米松和离子霉素在选择性诱导小鼠胸腺细胞亚群凋亡的能力上有所不同。地塞米松杀死广泛的CD4/8免疫表型，对细胞周期阶段无选择性。离子霉素选择性地耗尽CD4+8+细胞，尤其是细胞周期G0/G1期的细胞，而使CD4-8+细胞免受影响。该技术在体外和体内凋亡模型中具有广泛的优势，因为它可以检测单个活细胞，并将膜和核凋亡变化与标准流式细胞术免疫表型分析相关联。
