This article discusses the process of efferocytosis, where apoptotic cells display eat-me signals for clearance by phagocytes. The methodology for conducting an efferocytosis assay using dual-stained apoptotic cells and macrophages is detailed.
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.
After preparing human macrophages and apoptotic Jurkat cells, use a hemocytometer to count the apoptotic cells. Then, transfer a sufficient quantity of apoptotic cells to a 1.5-milliliter microcentrifuge tube.
Pellet the Jurkat cells via centrifugation at 500 times g for 5 minutes. Then, resuspend the cells in 500 microliters of PBS. While the Jurkat cells are in the centrifuge, aliquot 10 microliters of DMSO into a new microcentrifuge tube.
Next, dissolve a minimal amount of NHS-biotin in the DMSO. After this, transfer 500 microliters of the apoptotic cell and PBS suspension to the tube containing DMSO and NHS-biotin. Then, dilute an appropriate cell-tracking dye according to the manufacturer's instructions.
Incubate the cell suspension at room temperature for 20 minutes in the dark. Then, add an equal volume of RPMI 1640 with 10% FBS, and incubate the suspension at room temperature in the dark for an additional 5 minutes.
After this, pellet the cells via centrifugation at 500 times g for 5 minutes. Then, discard the supernatant and resuspend the stained cells in 100 microliters of RPMI 1640 with 10% FBS. Add 100 microliters of stained cell suspension drop-wise to each well of macrophages. Then, centrifuge the plate at 200 times g for 1 minute. Incubate the plate at 37 degrees Celsius with 5% CO2 in a tissue culture incubator.
After the appropriate incubation period, remove the cells from the incubator. Then, wash the cells twice with 1 milliliter of PBS. After this, add diluted FITC-conjugated streptavidin to each well and incubate the plate for 20 minutes in the dark.
Wash the cells three times with 1 milliliter of PBS, and gently shake the samples for 5 minutes after each rinse. Then, fix the cells with 4% paraformaldehyde in PBS for 20 minutes at room temperature. Rinse the cells once with PBS to remove any excess PFA.
Mount coverslips on an imaging slide, and transfer the slide to a fluorescence microscope. Capture z-stacks of a sufficient number of cells for quantification.
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