Quantitating MHC class II trafficking in primary dendritic cells using imaging flow cytometry

•Assessment of MHCII trafficking in primary human and mouse DCs•Correlation of activation status with receptor internalization•Correlating vesicle size, location, and contents with MHCII fate

Presentation of antigenic peptides in MHC class II (MHCII) on dendritic cells (DCs) is the first step in the activation of antigen-specific CD4+T cells. The expression of surface MHCII–peptide complexes is tightly regulated as the frequency of MHCII–peptide complexes can affect the magnitude, as well as the phenotype of the ensuing CD4+T cell response. The surface MHCII–peptide levels are determined by the balance between expression of newly generated complexes, complex internalization, and their subsequent re-emergence or degradation. However, the molecular mechanisms that underpin these processes are still poorly understood. Here we describe a multispectral imaging flow cytometry assay to visualize MHCII trafficking that can be used as a tool to dissect the molecular mechanisms that regulate MHCII homeostasis in primary mouse and human DCs.