Protocol for assessing anti-Siglec-9 antibody-mediated tumor phagocytosis by macrophages using pH-sensitive dyes and flow cytometry

Here, we present a protocol to assess and quantify macrophage-mediated cancer cell phagocytosis using pH-sensitive dyes and flow cytometry. We describe steps for isolating and sorting CD14 + monocytes from leukocyte reduction system (LRS) cones, differentiating monocyte-derived macrophages, and preparing K562 leukemic cells for co-culture. We then detail procedures for quantifying phagocytic activity using flow cytometry and pH-sensitive dyes and visualizing internalized K562 cells using a fluorescence microscope. This technique enables the study of macrophage phagocytic function and supports application in cancer immunotherapy research. For complete details on the use and execution of this protocol, please refer to Muneekaew et al. 1