An In Vitro Artificial Activation Assay for Studying Fc Receptor Activation by Antibodies

Take a suspension of natural killer, or NK, cells.

Add anti-CD20 monoclonal antibodies and incubate.

In the absence of target cancer cells expressing the CD20 antigen, the Fc region of the antibodies bind to the FcγRIIIa receptors on the NK cell surface.

Post-incubation, add chilled media. Centrifuge and remove unbound antibodies.

Next, add anti-human κ light chain antibodies. Incubate.

These secondary antibodies bind to the κ light chain of the monoclonal antibodies.

The antibody crosslinking mimics target antigen binding, triggering an intracellular signaling cascade.

This causes artificial stimulation of NK cells in the absence of cancer cells.

Activated NK cells undergo cytoskeletal rearrangement and release cytotoxic granules.

Further, the cells produce chemokines and cytokines.

Following the desired duration, add chilled media to stop cell stimulation.

Centrifuge the tube. Collect the supernatant and cell pellet for downstream analysis of antibody-mediated effector NK cell functions.