Efferocytosis assay to quantify the engulfment and acidification of apoptotic cells by macrophages using flow cytometry

Efficient macrophage efferocytosis maintains homeostasis and resolves inflammation. Here, we provide a protocol to assess the engulfment and acidification of apoptotic cells (ACs) by macrophages. We describe steps for preparing bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs), fluorescent labeling of ACs using both a pH-sensitive dye, pHrodo-Red succinimidyl ester, and a pH-insensitive dye, Hoechst, and subsequent incubation with macrophages for efferocytosis. We then detail procedures for flow cytometry-based quantification of engulfment and acidification. For complete details on the use and execution of this protocol, please refer to Shi and Wu et al.1 1