A Comprehensive Experimental Guide to Studying Cross-Presentation in Dendritic Cells In Vitro.
A Comprehensive Experimental Guide to Studying Cross-Presentation in Dendritic Cells In Vitro.
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DOI：
10.1002/cpim.115
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发表时间：
2020-12
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交叉呈现首次在 20 世纪 70 年代偶然被观察到。它的重要性很快就被认识到，并随后引起了免疫学家的高度关注。从那时起，我们对某些抗原呈递细胞内化、处理外源抗原并将其加载到 MHC-I 分子上以交叉引发 CD8+ T 细胞的能力的了解显着增加。树突状细胞 (DC) 是特殊的交叉呈递者，因此使其成为研究交叉呈递的重要工具，但循环和组织中 DC 的相对稀有性使得分离足够数量的细胞来在体外研究这一过程具有挑战性。在本文中，我们详细描述了两种从骨髓祖细胞培养 DC 的方法，以及一种扩大体内 DC 数量作为内源性真正交叉呈递 DC 来源的方法。我们还描述了使用初级 CD8+ T 细胞的激活作为读数来评估 DC 交叉呈递的方法。分离骨髓祖细胞 用 GM-CSF 体外分化树突状细胞 从产生 GM-CSF 的 J558L 细胞制备条件培养基 用 Flt3L 体外分化树突状细胞 从 B16-Flt3L 细胞制备含有 Flt3L 的培养基 在体内扩增 cDC1，用于离体实验 表征静息和活化的树突状细胞 树突状细胞刺激、抗原货物和固定 模型的制备抗原包被的微珠的制备 凋亡细胞的制备 重组细菌的制备 免疫细胞化学免疫荧光 (ICC/IF) 制备 Alcian 蓝包被的盖玻片 CD8+ T 细胞活化以评估交叉呈递 使用 CFSE 分离和标记 CD8+ T 细胞
Cross-presentation was first observed serendipitously in the 1970s. The importance of it was quickly realized and subsequently attracted great attention from immunologists. Since then, our knowledge of the ability of certain antigen presenting cells to internalize, process, and load exogenous antigens onto MHC-I molecules to cross-prime CD8+ T cells has increased significantly. Dendritic cells (DCs) are exceptional cross-presenters, thus making them a great tool to study cross-presentation but the relative rarity of DCs in circulation and in tissues makes it challenging to isolate sufficient numbers of cells to study this process in vitro. In this paper, we describe in detail two methods to culture DCs from bone-marrow progenitors and a method to expand the numbers of DCs present in vivo as a source of endogenous bona-fide cross-presenting DCs. We also describe methods to assess cross-presentation by DCs using the activation of primary CD8+ T cells as a readout. Isolation of bone marrow progenitor cells In vitro differentiation of dendritic cells with GM-CSF Preparation of conditioned medium from GM-CSF producing J558L cells In vitro differentiation of dendritic cells with Flt3L Preparation of Flt3L containing medium from B16-Flt3L cells Expansion of cDC1s in vivo for use in ex vivo experiments Characterizing resting and activated dendritic cells Dendritic cell stimulation, antigenic cargo, and fixation Preparation of model antigen coated microbeads Preparation of apoptotic cells Preparation of recombinant bacteria Immunocytochemistry immunofluorescence (ICC/IF) Preparation of Alcian blue-coated coverslips CD8+ T cell activation to assess cross-presentation Isolation and labeling of CD8+ T cells with CFSE