Krutzik Peter O, Irish Jonathan M, Nolan Garry P, Perez Omar D
Department of Molecular Pharmacology, School of Medicine, Stanford University, CA 94305, USA.
Clin Immunol. 2004 Mar;110(3):206-21. doi: 10.1016/j.clim.2003.11.009.
Analysis of protein phosphorylation with flow cytometric techniques has emerged as a powerful tool in the field of immunological signaling, allowing cellular subsets in complex populations to be analyzed accurately and rapidly. In this review, we examine the development of phospho-epitope, or phospho-specific, flow cytometry and the premises upon which the technique is based. Phospho-specific flow cytometry is compared to traditional biochemical methods, and its advantages, such as single cell analysis, multiparameter data acquisition, rapid protocols, and the ability to analyze rare cell subsets, are detailed. We also discuss the many technical considerations that must be addressed when developing new antibodies or analyzing new epitopes including antigen accessibility, stability of the phospho-epitope, fluorophore selection, surface phenotype integrity, and antibody suitability for staining epitopes inside fixed and permeabilized cells. The methods that have been used to date are described in light of these technical considerations. The importance of developing bioinformatic platforms in parallel with these techniques is emphasized due to the large, multiparameter datasets that are rapidly accumulated and which require more efficient data viewing and complex clustering methods than currently available for flow cytometric data. Finally, we discuss the potential clinical applications of phospho-specific flow cytometry in analyzing immune cell development and antigen-specific immune responses, as well as pharmacodynamic profiling of disease states or drug efficacy and specificity against particular signaling proteins.
利用流式细胞术技术分析蛋白质磷酸化已成为免疫信号领域的一种强大工具，可准确、快速地分析复杂群体中的细胞亚群。在本综述中，我们探讨了磷酸化表位或磷酸化特异性流式细胞术的发展及其技术基础。将磷酸化特异性流式细胞术与传统生化方法进行了比较，并详细阐述了其优势，如单细胞分析、多参数数据采集、快速检测方案以及分析稀有细胞亚群的能力。我们还讨论了开发新抗体或分析新表位时必须考虑的诸多技术因素，包括抗原可及性、磷酸化表位的稳定性、荧光团选择、表面表型完整性以及抗体对固定和通透细胞内表位染色的适用性。根据这些技术因素描述了迄今为止所使用的方法。由于快速积累的大量多参数数据集需要比目前用于流式细胞术数据更高效的数据查看和复杂的聚类方法，因此强调了与这些技术并行开发生物信息平台的重要性。最后，我们讨论了磷酸化特异性流式细胞术在分析免疫细胞发育和抗原特异性免疫反应以及疾病状态的药效学分析或药物疗效和对特定信号蛋白的特异性方面的潜在临床应用。
