Quantitative determination of phagocytosis by bone marrow-derived dendritic cells via imaging flow cytometry

Immunogenic cell death (ICD), induced by certain anticancer chemotherapeutics, leads to the emission of danger associated molecular patterns (DAMP) by cancer cells, which facilitates the attraction, activation and maturation of dendritic cells (DC) as well as the subsequent priming of effector T cells. In this context calreticulin (CALR) exposed at an early stage of ICD at the surface of the cancer cells serves as phagocytic signal and triggers the formation of immunological synapses between malignant cells and DC. Subsequent phagocytosis facilitates the transfer of tumor associated antigen and thus depicts a fundamental step in the generation of anticancer immunity. Here we provide an imaging flowcytometric protocol for the quantification of ICD-associated DC phagocytosis of cancer cells. As compared to the traditional flowcytometry-based analysis, the presented method offers additional means of differentiation between the transient formation of immunological synapses and the final DC-mediated phagocytosis of cancer cells.