Fixed Cell Efferocytosis Assay: A Method to Study Efferocytic Uptake of Apoptotic Cells by Macrophages Using Fluorescence Microscopy

Cells undergoing apoptosis - programmed cell death - display eat-me signals on their cell surface, facilitating degradation by phagocytes - specialized immune cells. This process of clearance of apoptotic cells is called efferocytosis.

To perform the efferocytosis assay, first, take a suspension of dual-stained apoptotic cells. The surfaces of these cells are labeled with biotin, and the cytoplasm is labeled with a cell-tracking dye, tagging apoptotic cell-derived materials.

Add the cell suspension into a culture of adhered macrophage cells, which function as efferocytes. Centrifuge, forcing contact between macrophages and apoptotic cells, and incubate.

Efferocytic receptors on macrophages bind to phosphatidylserine - an eat-me signal - on the apoptotic cell membrane, facilitating its engulfment. Small fragments of the apoptotic cell are taken up inside a membrane-bound vesicle, called an efferosome.

This results in an efferocytosed fraction - part of the apoptotic cell inside the macrophage - and a non-efferocytosed fraction - the non-internalized part of the apoptotic cell.

Add fluorescently-labeled streptavidin, which binds to the exposed biotin molecules on the surface of the n