Work

Protocol for measuring exogenous antigen presentation triggered by membranes of bone marrow-derived dendritic cell purified phagosomes

Year: 2025

Type: article

Abstract: Dendritic cells are potent antigen-presenting cells that initiate T cell responses by presenting exogenous antigens associated with major histocompatibility complex (MHC) class I and MHC class II molecules. Here, we present a protocol to colorimetrically evaluate CD8+ and CD4+ T cell activation by using dendritic cell purified magnetic phagosomes previously coated with ovalbumin (OVA). We describe the procedure for recovering phagosomes through mechanical dendritic cell lysis. We also detail steps for optimal separation, fixation, and phagosomal membrane rupture to expose OVA-loaded MHC molecules to antigen-specific T cells. For complete details on the use and execution of this protocol, please refer to Cebrian et al. 1 • Instructions for bone marrow-derived dendritic cell production • Steps for magnetic bead preparation, phagocytosis, and phagosome purification • Guidance on LacZ-inducible T cell hybrid activation • Procedures for troubleshooting poor T cell activation signal Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Dendritic cells are potent antigen-presenting cells that initiate T cell responses by presenting exogenous antigens associated with major histocompatibility complex (MHC) class I and MHC class II molecules. Here, we present a protocol to colorimetrically evaluate CD8+ and CD4+ T cell activation by using dendritic cell purified magnetic phagosomes previously coated with ovalbumin (OVA). We describe the procedure for recovering phagosomes through mechanical dendritic cell lysis. We also detail steps for optimal separation, fixation, and phagosomal membrane rupture to expose OVA-loaded MHC molecules to antigen-specific T cells.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104225

Authors: Facundo Garrido, Franco Nieto, Luis S. Mayorga, Ignacio Cebrian

Institutions: Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Cuyo, Instituto de Histología y Embriología de Mendoza

Language: English

Topic: Immunotherapy and Immune Responses

Subfield: Immunology

Field: Immunology and Microbiology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,550