Metabolic labeling and targeting of dendritic cells

Dendritic cells (DCs) play a central role in orchestrating immune responses, and methods that enable precise, modular, and non-invasive manipulation of DCs are essential for both mechanistic studies and translational applications. Here, we describe a protocol for the metabolic glycan labeling and bioorthogonal targeting of bone marrow-derived dendritic cells (BMDCs). This method leverages endogenous glycan biosynthetic pathways to incorporate azido-functionalized monosaccharides into DC surface glycans, followed by strain-promoted azide-alkyne cycloaddition (SPAAC) with diverse cargos conjugated with dibenzocyclooctyne (DBCO). The protocol details BMDC isolation and differentiation, optimization of azido-sugar labeling conditions, and subsequent in vitro and in vivo DBCO-cargo conjugation. Using flow cytometry and imaging-based validation, this approach enables efficient and reproducible surface functionalization of BMDCs while preserving cell viability and phenotype. The modular nature of the click chemistry step allows flexible attachment of fluorophores, proteins, or other functional molecules. Overall, this protocol provides a versatile platform for studying DC biology, enabling targeted modulation, imaging, and functional interrogation of DCs.