Lamoreaux Laurie, Roederer Mario, Koup Richard
Immunology Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, 40 Convent Drive, Bethesda, Maryland 20892, USA.
Nat Protoc. 2006;1(3):1507-16. doi: 10.1038/nprot.2006.268.
We describe here a method for optimizing the use of polychromatic flow cytometry (with up to 17 fluorochromes simultaneously) in surface and intracellular staining of human T lymphocytes. We will highlight and discuss how to procedurally optimize key steps in the experimental process before an intracellular cytokine staining assay protocol is finalized. These include but are not limited to the titration of monoclonal antibodies, use of a dead-cell discriminator and 'dump' channel, selection of a cytokine secretion inhibitor, selection of fixation and permeabilization reagents, and inclusion of compensation controls. Building on this basic protocol, we then establish a polychromatic assay designed to detect five separate functions of T lymphocytes (production of three cytokines and one chemokine, and degranulation) while simultaneously identifying multiple surface markers on the responding cells.
我们在此描述一种优化多色流式细胞术（可同时使用多达17种荧光染料）用于人T淋巴细胞表面和细胞内染色的方法。我们将重点介绍并讨论在确定细胞内细胞因子染色检测方案之前，如何在实验过程中对关键步骤进行程序优化。这些步骤包括但不限于单克隆抗体的滴定、死细胞鉴别器和“排除”通道的使用、细胞因子分泌抑制剂的选择、固定和通透试剂的选择以及补偿对照的设置。基于此基本方案，我们随后建立了一种多色检测方法，旨在检测T淋巴细胞的五种不同功能（三种细胞因子和一种趋化因子的产生以及脱颗粒），同时识别反应细胞上的多种表面标志物。
