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. Curr Protoc Immunol. 2020 Dec;131(1):e115 Authors: Sadiq BA, Mantel I, Blander JM Abstract 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 differentiatio...
Source: Current Protocols in Immunology - Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research