树突状细胞体外交叉呈递研究的综合实验指南

Q2 Immunology and Microbiology Current Protocols in Immunology Pub Date : 2020-12-14 DOI:10.1002/cpim.115

Barzan A. Sadiq, Ian Mantel, J. Magarian Blander

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引用次数: 3

摘要

交叉呈现是在20世纪70年代偶然发现的。它的重要性很快被认识到，并随后引起了免疫学家的极大关注。从那时起，我们对某些抗原呈递细胞内化、加工和将外源抗原装载到MHC-I分子上以交叉引物CD8+ T细胞的能力的了解显著增加。树突状细胞(dc)是一种特殊的交叉呈递细胞，因此使它们成为研究交叉呈递的一个很好的工具，但在循环和组织中树突状细胞的相对稀缺性使得分离足够数量的细胞来体外研究这一过程具有挑战性。在本文中，我们详细描述了从骨髓祖细胞培养dc的两种方法和一种扩大体内dc数量的方法，作为内源性真正交叉呈递dc的来源。我们还描述了使用原代CD8+ T细胞激活作为读数来评估DCs交叉呈现的方法。©2020 Wiley期刊公司。基本方案1:骨髓祖细胞的分离基本方案2:用GM-CSF体外分化树突状细胞支持方案1:从产生J558L细胞的GM-CSF中制备条件培养基基本方案3:用Flt3L体外分化树突状细胞支持方案2:从B16-Flt3L细胞中制备含有Flt3L的培养基基本方案4:在体内扩增cDC1s用于离体实验基本方案5:表征静止和活化的树突状细胞基本方案6:树突状细胞刺激，抗原cargo和固定支持方案3:制备模型抗原包被微珠支持方案4:制备凋亡细胞支持方案5:制备重组细菌基本方案7:免疫细胞化学免疫荧光(ICC/IF)支持方案6:制备阿利新蓝包被覆盖基本方案8:CD8+ T细胞活化评估交叉呈现支持方案7:CD8+ T细胞与CFSE的分离和标记

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A Comprehensive Experimental Guide to Studying Cross-Presentation in Dendritic Cells In Vitro

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. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of bone marrow progenitor cells

Basic Protocol 2: In vitro differentiation of dendritic cells with GM-CSF

Support Protocol 1: Preparation of conditioned medium from GM-CSF producing J558L cells

Basic Protocol 3: In vitro differentiation of dendritic cells with Flt3L

Support Protocol 2: Preparation of Flt3L containing medium from B16-Flt3L cells

Basic Protocol 4: Expansion of cDC1s in vivo for use in ex vivo experiments

Basic Protocol 5: Characterizing resting and activated dendritic cells

Basic Protocol 6: Dendritic cell stimulation, antigenic cargo, and fixation

Support Protocol 3: Preparation of model antigen coated microbeads

Support Protocol 4: Preparation of apoptotic cells

Support Protocol 5: Preparation of recombinant bacteria

Basic Protocol 7: Immunocytochemistry immunofluorescence (ICC/IF)