An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes

Take fluorescently labeled microspheres conjugated to an antigen of interest. The immobilized antigens are bound to antigen-specific antibodies.

Add leukocytes, including non-phagocytic lymphocytes, phagocytic granulocytes, and monocytes. Incubate the mixture under physiological conditions.

The Fc region of the immobilized antibody binds to the Fc receptor on the phagocyte, triggering antibody-dependent cellular phagocytosis, or ADCP.

Centrifuge to pellet the cells and discard the supernatant containing non-internalized microspheres.

Add a viability stain that enters dead cells, fluorescently labeling them.

Centrifuge to pellet the cells and remove the supernatant.

Add fluorescently labeled antibodies that bind to leukocytes.

Centrifuge to discard the supernatant and fix the cells.

Using flow cytometry, first, eliminate the dead cells that are positive for viability stain.

Then, select the viable leukocytes and discriminate between granulocytes, monocytes, and lymphocytes based on their granularity and scattering properties.

Measure the fluorescence intensity of the microsphere-positive phagocytic cells to quantify their ADCP activity.