Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Curr Protoc. 2023 Jul;3(7):e838. doi: 10.1002/cpz1.838.
This article discusses methods to assess invariant natural killer T (iNKT) cell subsets isolated from the thymus, as well as the spleen, the liver, and the lung. iNKT cells can be subdivided in distinct, functional subsets based on the transcription factors they express and the cytokines they produce to regulate the immune response. Basic Protocol 1 focuses on characterizing murine iNKT subsets ex vivo by flow cytometry by evaluating the expression of lineage-specifying transcription factors such as PLZF and RORγt. The Alternate Protocol describes a detailed approach to define subsets based on expression of surface markers. This approach can be very useful for maintaining the subsets alive, without fixing them, in order to isolate them for downstream molecular assays such as DNA/RNA isolation, genome-wide analysis to assess gene expression (such as RNA-seq), assessment of chromatin accessibility (for instance, by ATAC-seq), and assessment of DNA methylation by whole-genome bisulfite sequencing. Basic Protocol 2 describes the functional characterization of iNKT cells, which are activated in vitro with PMA and ionomycin for a short period of time and subsequently stained and characterized for production of cytokines, such as IFNγ and IL-4, by flow cytometry. Basic Protocol 3 describes the process of activating iNKT cells in vivo using α-galactosyl-ceramide, a lipid that can be recognized specifically by iNKT cells, allowing assessment of their functionality in vivo. Cells are then isolated and directly stained for cytokine secretion. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identifying iNKT cell subsets based on transcription factor expression by flow cytometry Alternate Protocol: Identifying iNKT cell subsets based on surface marker expression by flow cytometry Basic Protocol 2: iNKT cell functional characterization based on in vitro activation and assessment of cytokine secretion Basic Protocol 3: iNKT cell in vivo activation and assessment of cytokine secretion by flow cytometry.
本文讨论了从胸腺以及脾、肝和肺中分离不变自然杀伤 T(iNKT)细胞亚群的方法。iNKT 细胞可以根据它们表达的转录因子和产生的细胞因子进行细分，以调节免疫反应。基本方案 1 侧重于通过流式细胞术评估谱系特异性转录因子（如 PLZF 和 RORγt）的表达，来描述体外鉴定小鼠 iNKT 亚群的方法。备选方案描述了一种基于表面标志物表达来定义亚群的详细方法。这种方法对于在不固定它们的情况下使亚群保持存活非常有用，以便将它们分离出来进行下游分子分析，例如 DNA/RNA 分离、全基因组分析以评估基因表达（例如 RNA-seq）、染色质可及性评估（例如，通过 ATAC-seq）以及通过全基因组亚硫酸氢盐测序评估 DNA 甲基化。基本方案 2 描述了 iNKT 细胞的功能特征，iNKT 细胞在体外用 PMA 和离子霉素短时间激活，随后通过流式细胞术对细胞因子（如 IFNγ 和 IL-4）的产生进行染色和特征分析。基本方案 3 描述了使用α-半乳糖神经酰胺（一种可以被 iNKT 细胞特异性识别的脂质）在体内激活 iNKT 细胞的过程，允许评估它们在体内的功能。然后分离细胞并直接染色以检测细胞因子分泌。© 2023 Wiley Periodicals LLC. 基本方案 1：通过流式细胞术根据转录因子表达鉴定 iNKT 细胞亚群 备选方案：通过流式细胞术根据表面标志物表达鉴定 iNKT 细胞亚群 基本方案 2：基于体外激活和细胞因子分泌评估的 iNKT 细胞功能特征 基本方案 3：通过流式细胞术进行 iNKT 细胞体内激活和细胞因子分泌评估。
