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

Dispense 100 microliters of resuspended natural killer cells into 1.5-milliliter tubes or a 96-well U-bottom plate, and place the cells on ice. Prepare rituximab or other antibody of interest at a concentration of 100 micrograms per milliliter.

Add 1 microliter to each tube of cells for a final concentration of 1 microgram per milliliter, and incubate the cells on ice for 30 minutes. During incubation, prepare a solution of anti-human kappa light chain antibody in media at a concentration of 50 micrograms per milliliter. The amount of antibody solution needed will be 50 microliters per cell sample.

Warm the antibody solution to 37 degrees Celsius on a heat block or in a water bath. When incubation of the cells is complete, add 1 milliliter of ice-cold media to each tube and centrifuge the cells at 135 times g and 4 degrees Celsius for 5 minutes. Wash the cells again with 1 milliliter of ice-cold media. Aspirate the supernatant after the last wash and add 50 microliters of the anti-human kappa light chain antibody solution to activate the cells. Immediately place cell samples at 37 degrees Celsius on a heat block or in a water bath and incubate them until the desired time points for analysis or other procedures.

The best way to handle multiple samples is to use a floating rack in a 37-degree water bath, which can be removed and placed on ice to stop all reactions immediately.