A Versatile In Vitro Quantitative Assay for Macrophage Efferocytosis in Diverse Research Applications

Macrophage efferocytosis is a previously unrecognized key pathogenic event, engulfing apoptotic targets, preventing inflammation and necrosis, and maintaining immune homeostasis. The phagocytic function can be disrupted by harmful factors and toxic substances. This protocol describes a versatile visualized in vitro method that can be used for the detection of general efferocytosis. This method is applicable to a wide range of research scenarios. As a representative application, it can be used to evaluate macrophage efferocytosis dysfunction in diseases linked to harmful exposures, including atherosclerosis, chronic inflammation, and malignant tumors. Among them, the detection of the effects of oxidized low-density lipoprotein (ox-LDL) and arsenite on macrophage efferocytosis capacity is an exemplary application of this protocol. Primary macrophages collected from mice were labeled with a cell-tracking dye and exposed to ox-LDL or arsenite, then co-cultured with apoptotic thymocytes or hepatocytes (labeled with another cell-tracking dye) for 2 h at a ratio of 5:1. Macrophage efferocytosis was visualized using a laser confocal microscope. The results indicate that arsenite impaired macrophage efferocytosis, leading to insufficient clearance of apoptotic thymocytes or hepatocytes. This method can be extended to subsequent studies, including those involving different types of phagocytes, apoptotic cell models, and research related to exposure to various factors. Key features • This protocol harvests primary mouse macrophages, treats them with different factors (ox-LDL, sodium arsenite), and assesses efferocytosis of apoptotic/necrotic thymocytes or hepatocytes. • Dye-swap red-green staining eliminates fluorescence interference and staining bias and validates dye effects on efferocytosis.