In Vitro Bone Marrow–Derived Dendritic Cells (BMDC) Generation for Antigen Presentation Assay

Dendritic cells (DC) are sentinel cells of the immune system that process and present antigens to activate T cells, thus serving to bridge the innate and adaptive immune systems. DCs are particularly efficient at cross-presentation whereby exogenously acquired antigens are processed and presented in context with MHCI molecules to activate CD8⁺ T cells. Assaying antigen presentation by DCs is a critical parameter in assessing immune functionality. However, the low abundance of bona fide DCs within the lymphoid compartments limits the utility of such assays. An alternative approach employing the culturing of bone marrow cells in the presence of factors needed for DC lineage commitment can result in the differentiation of bone marrow dendritic cells (BMDCs). This protocol details the process of in vitro generation of BMDCs and demonstrates their subsequent utility in antigen presentation assays. The protocol described can be adapted to various conditions and antigens. Key features • BMDCs can serve as surrogate antigen-presenting cells (APCs) for assessing in vitro and in vivo antigen presentation. • Co-culture of antigen-stimulated BMDCs with CFSE-labeled T cells can help quantify the responsiveness of both the antigen presenters and responders. • In vivo analysis of antigen presentation by BMDCs can be assessed using an adoptive transfer approach. • CFSE labeling can help track in vivo the fate of adoptively transferred BMDCs as well as T cells.