Perez Omar D, Krutzik Peter O, Nolan Garry P
The Baxter Laboratory for Genetic Pharmacology, Department of Microbiologyand Immunology, Stanford University School of Medicine, Stanford, CA, USA.
Methods Mol Biol. 2004;263:67-94. doi: 10.1385/1-59259-773-4:067.
Flow cytometry offers the capability to assess the heterogeneity of cellular subsets that exist in complex populations, such as peripheral blood, based on immunophenotypes. We describe methodologies to measure phospho-epitopes in single cells as determinants of intracellular kinase activity. Multiparametric staining, using both surface and intracellular stains, allows for the study of discrete biochemical events in readily discernible lymphocyte subsets. As such, the usage of multiparameter flow cytometry to obtain proteomic information provides several major advantages: (1) the ability to perform multiparametric experiments to identify distinct signaling profiles in defined lymphocyte populations, (2) simultaneous correlation of multiple active kinases involved in signaling cascades, (3) profiling of active kinase states to identify signaling signatures of interest rapidly, and (4) biochemical access to rare cell subsets such as those from clinically derived samples or populations that comprise too few in numbers for conventional biochemical analysis.
流式细胞术能够基于免疫表型评估复杂群体（如外周血）中存在的细胞亚群的异质性。我们描述了测量单细胞中磷酸化表位作为细胞内激酶活性决定因素的方法。使用表面和细胞内染色的多参数染色允许研究易于识别的淋巴细胞亚群中的离散生化事件。因此，使用多参数流式细胞术获得蛋白质组学信息具有几个主要优点：（1）能够进行多参数实验以识别特定淋巴细胞群体中的不同信号谱，（2）参与信号级联的多个活性激酶的同时相关性，（3）活性激酶状态的分析以快速识别感兴趣的信号特征，以及（4）对罕见细胞亚群的生化分析，例如来自临床样本或数量太少而无法进行常规生化分析的群体。
