Flow cytometric ratio analysis of the Hoechst 33342 emission spectrum: Multiparametric characterization of apoptotic lymphocytes
Flow cytometric ratio analysis of the Hoechst 33342 emission spectrum: Multiparametric characterization of apoptotic lymphocytes
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DOI：
10.1016/0022-1759(95)00214-6
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发表时间：
1996-02-05
影响因子：
2.2
通讯作者：
Dunne, JF
中科院分区：
文献类型：
--
作者：
Chiu, L;Cherwinski, H;Dunne, JF
对各种刺激的凋亡反应是免疫调节的重要组成部分，在复杂的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，细胞周期进展，膜完整性和光散射特性。它是基于对dna结合活性染料Hoechst 33342的发射光谱的两个区域的分析。我们根据核形态和特征DNA降解模式的存在建立了红蓝荧光发射比例与细胞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，我们结合了光散射特性、细胞周期阶段、相关细胞表面免疫表型标记（CD25或CD4和CD8）和质膜完整性标记（merocyanine 540），以实现凋亡细胞的多参数表型。我们发现，staurosporine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，因为频率大，staurosporine使CD25（+）细胞减少。地塞米松和离子霉素诱导小鼠胸腺细胞亚群选择性凋亡的能力不同。地塞米松杀死广泛的CD4/8免疫表型，对细胞周期阶段没有选择性。离子霉素选择性地消耗CD4(+)8（+）细胞，特别是细胞周期G(0)/G(1)区域的细胞，而保留CD4(-)8（+）细胞。该技术在体外和离体细胞凋亡模型中具有广泛的优势，因为它询问单个活细胞，并将膜和核凋亡变化与标准流式细胞术免疫表型相关联。
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 describe 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 selectivity for cell cycle stage. Ionomycin selectively depletes CD4(+)8(+) cells, especially those in the G(0)/G(1) 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.