Efferocytosis assay to quantify the engulfment and acidification of apoptotic cells by macrophages using flow cytometry
Xun Wu, Ziyi Wang, Tyler P Shern, Hanrui Zhang
Abstract
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 • Assess macrophage efferocytosis in vitro using flow cytometry • Co-label apoptotic cells (ACs) with both a pH-insensitive dye and a pH-sensitive dye • The pH-insensitive dye identifies macrophages that have engulfed ACs • The pH-sensitive dye fluoresces only after the ACs have been acidified post-engulfment Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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 macrophage (BMDM) 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.