Rubina Pal, Jayne Schaubhut, Darcey Clark, Lynette Brown, Jennifer J. Stewart
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Abstract
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
单细胞细胞因子mRNA和蛋白表达的流式细胞术分析
了解免疫细胞如何对病原体或药物等外部刺激作出反应是生物医学研究的关键组成部分。免疫应答的关键是细胞表面受体的表达和细胞因子的分泌,它们受到基因表达和蛋白质合成的严格调控。以前,细胞因子mRNA表达水平是通过异质或分类细胞群的大量分析来测量的,使用这些技术的细胞因子mRNA表达和蛋白质水平之间的相关性可能是高度可变的。流式细胞术用于监测细胞表面和细胞内蛋白质的变化,但一些蛋白质如细胞因子可能是短暂的,难以测量。因此,一种可以同时测量单个细胞中细胞因子mRNA和蛋白质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,将传统的T细胞表面蛋白(CD45, CD3, CD4, CD8)和细胞内细胞因子(IL-2, INF-γ)的测量与荧光原位杂交和支链DNA技术相结合,用于扩增和检测活化T细胞中IL-2和INF-γ mRNA转录物。该方法已应用于冷冻外周血单核细胞(PBMCs)和冷冻血液样本,适用于需要运送到试验点的临床试验样本。在活化PBMCs的CD4+细胞中,IL-2和INF-γ的mRNA和蛋白水平的一致性分别为41%和21%。在活化pbmc的CD8+细胞中,IL-2的一致性为15%,INF-γ的一致性为32%。©2020 by John Wiley &基本方案:检测冷冻pbmcs中IL-2和IFN-γ mRNA和蛋白表达。备选方案:检测冷冻血液中IL-2和IFN-γ mRNA和蛋白表达
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