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 non-efferocytosed fraction, differentiating the non-internalized and internalized portions of the apoptotic cell.

Under a fluorescence microscope, the non-internalized apoptotic cell fraction shows streptavidin labeling, whereas efferosomes appear as discrete dots free of streptavidin.

To quantify efferocytosis, determine the number of discrete efferosomes, and measure the intensity of the efferocytosed and non-efferocytosed apoptotic cell fractions.