Krutzik Peter O, Clutter Matthew R, Nolan Garry P
Department of Microbiology and Immunology, Baxter Laboratory of Genetic Pharmacology, Stanford University, Stanford, CA 94305, USA.
J Immunol. 2005 Aug 15;175(4):2357-65. doi: 10.4049/jimmunol.175.4.2357.
Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze intracellular signaling events in complex populations of cells because of its ability to simultaneously discriminate cell types based on surface marker expression and measure levels of intracellular phosphoproteins. This has provided novel insights into the cell- and pathway-specific nature of immune signaling. However, we and others have found that the fixation and permeabilization steps necessary for phosphoprotein analysis often negatively affect the resolution of cell types based on surface marker analysis and light scatter characteristics. Therefore, we performed a comprehensive profile of >35 different murine surface marker Abs to understand the effects of fixation and permeabilization on surface Ag staining. Fortuitously, approximately 80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform.
最近，磷酸特异性流式细胞术已成为分析复杂细胞群体中细胞内信号转导事件的强大工具，因为它能够基于表面标志物表达同时区分细胞类型，并测量细胞内磷酸化蛋白的水平。这为免疫信号的细胞特异性和通路特异性本质提供了新的见解。然而，我们和其他人发现，磷酸化蛋白分析所需的固定和通透步骤通常会对基于表面标志物分析和光散射特征的细胞类型分辨率产生负面影响。因此，我们对35种以上不同的小鼠表面标志物抗体进行了全面分析，以了解固定和通透对表面抗原染色的影响。幸运的是，大约80%的测试抗体能够分辨出感兴趣的细胞群体，尽管阳性和阴性群体之间的分离度降低，且效价与活细胞上使用的效价非常不同。另外20%的抗体要么显示群体之间完全失去分离，要么显示中间染色群体消失。我们能够通过在固定后、通透前进行染色来挽救其中几种抗原的染色，尽管荧光团选择有限。淋巴细胞的散射特征得到了很好的保留，但单核细胞和中性粒细胞群体的散射特征发生了显著变化。这些结果为有兴趣将磷酸特异性流式细胞术应用于复杂细胞群体的研究人员汇编了一份全面的资源，同时概述了成功将新的表面标志物抗体应用于该平台所需的步骤。
