A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification.
A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification.
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一种温和的固定和透化方法，用于组合细胞表面和细胞内染色，提高 DNA 定量的精度。
DOI：
10.1002/cyto.990120312
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Giorgi,JV
中科院分区：
文献类型：
--
作者：
Schmid,I;Uittenbogaart,CH;Giorgi,JV
开发了一种温和固定哺乳动物细胞并使其膜透化的方法。该方法可用于细胞内抗原的染色或与细胞表面染色同时定量DNA含量。将细胞在4°C下用0.25%缓冲的多聚甲醛处理1小时，然后在37°C下用PBS中的0.2%吐温20洗涤剂处理15分钟。该程序允许细胞内蛋白质的极好染色，DNA分布的G 0 G1-峰的变异系数（CV）非常低，并保持细胞表面抗原的完整性。细胞簇的低角与90°角光散射曲线得以保持，从而能够区分不同的细胞群，包括人外周血淋巴细胞和单核细胞，以用于门控和分析目的。该方法已成功用于多种其他细胞类型，包括人胸腺细胞、小鼠胸腺细胞和脾细胞以及几种白血病细胞系。在破伤风类毒素培养的外周血单核细胞（PBMC）上，双色表面抗原染色结合7-氨基放线菌素D（7-AAD）DNA染色，可确定优先刺激的细胞亚群。在CCRF-CEM上进行内部抗原染色以表达CD 3 β，在NALM-6上进行内部抗原染色以表达μ。我们开发的技术给出了明亮的内部抗原的特异性染色在这里提出的例子。它特别适用于内部抗原染色与DNA染色和/或表面免疫荧光的相关性。
A method was developed for gentle fixation of mammalian cells and permeabilization of their membranes. The method is useful for staining of intracellular antigens or quantification of DNA content simultaneously with cell surface staining. Cells are treated for 1 h at 4°C with 0.25% buffered paraformaldehyde then for 15 min at 37°C with 0.2% Tween 20 detergent in PBS. The procedure permits excellent staining of intracellular proteins, very low coefficients of variation (CV) on the G0G1‐peak of DNA distributions, and preservation of the integrity of cell surface antigens. The low vs. 90° angle light scatter profile of cell clusters is maintained thereby allowing discrimination of different cell populations including human peripheral blood lymphocytes and monocytes for gating and analytic purposes. The method was successfully used on a variety of other cell types, including human thymocytes, murine thymocytes and spleen cells, and several leukemic cell lines. Dual‐color surface antigen staining combined with DNA staining with 7‐amino‐actinomycin D (7‐AAD) on peripheral blood mononuclear cells (PBMC) cultured with tetanus toxoid allowed the determination of the cell subset that was preferentially stimulated. Staining for internal antigens was done on CCRF‐CEM for expression of CD3ϵ and on NALM‐6 for expression of μ. The technique we developed gave bright and specific staining of internal antigens in the examples presented here. It is particularly suited for correlations of internal antigen staining with DNA staining and/or surface immunofluorescence.