Flow Contract Site Laboratory, LLC, Bothell, Washington.
Curr Protoc Cytom. 2020 Mar;92(1):e69. doi: 10.1002/cpcy.69.
Understanding how immune cells respond to external stimuli such as pathogens or drugs is a key component of biomedical research. Critical to the immune response are the expression of cell-surface receptors and the secretion of cytokines, which are tightly regulated by gene expression and protein synthesis. Previously, cytokine mRNA expression levels have been measured from bulk analysis of heterogeneous or sorted cell populations, and the correlation between cytokine mRNA expression and protein levels using these techniques can be highly variable. Flow cytometry is used to monitor changes in cell-surface and intracellular proteins, but some proteins such as cytokines may be transient and difficult to measure. Thus, a flow cytometry method that can simultaneously measure cytokine mRNA and protein levels in single cells is a very powerful tool. We defined a flow cytometry method that combines the conventional measurement of T cell surface proteins (CD45, CD3, CD4, CD8) and intracellular cytokines (IL-2, INF-γ) with fluorescent in situ hybridization and branched DNA technology for amplification and detection of IL-2 and INF-γ mRNA transcripts in activated T cells. This method has been applied to frozen peripheral mononuclear blood cells (PBMCs) and frozen blood samples, making it applicable to clinical trial specimens that require shipment to the test site. In CD4 cells from activated PBMCs, the concordance between mRNA and protein levels was 41% for IL-2 and 21% for and INF-γ. In CD8 cells from activated PBMCs, the concordance was 15% for IL-2 and 32% for INF-γ. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen PBMCs Alternate Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen blood.
理解免疫细胞如何对外界刺激（如病原体或药物）作出反应是生物医学研究的一个关键组成部分。免疫反应的关键是细胞表面受体的表达和细胞因子的分泌，这受到基因表达和蛋白质合成的严格调控。此前，细胞因子 mRNA 表达水平是通过对异质或分选细胞群体的批量分析来测量的，而使用这些技术测量细胞因子 mRNA 表达与蛋白水平之间的相关性具有高度可变性。流式细胞术用于监测细胞表面和细胞内蛋白的变化，但一些蛋白（如细胞因子）可能是短暂的，难以测量。因此，一种能够同时测量单细胞中细胞因子 mRNA 和蛋白水平的流式细胞术方法是一种非常强大的工具。我们定义了一种流式细胞术方法，该方法将 T 细胞表面蛋白（CD45、CD3、CD4、CD8）和细胞内细胞因子（IL-2、INF-γ）的常规测量与荧光原位杂交和分支 DNA 技术相结合，用于扩增和检测活化 T 细胞中的 IL-2 和 INF-γ mRNA 转录本。该方法已应用于冷冻外周血单核细胞（PBMC）和冷冻血液样本，使其适用于需要运送到检测地点的临床试验标本。在活化 PBMC 中的 CD4 细胞中，IL-2 的 mRNA 和蛋白水平之间的一致性为 41%，而 INF-γ 的一致性为 21%。在活化 PBMC 中的 CD8 细胞中，IL-2 的一致性为 15%，而 INF-γ 的一致性为 32%。© 2020 年由 John Wiley & Sons, Inc. 基本方案：检测冷冻 PBMC 中的 IL-2 和 IFN-γ mRNA 和蛋白表达 备选方案：检测冷冻血液中的 IL-2 和 IFN-γ mRNA 和蛋白表达
