This article describes a method for activating natural killer (NK) cells using anti-CD20 monoclonal antibodies. The process involves incubating NK cells with antibodies to trigger an intracellular signaling cascade, leading to cell activation and cytotoxic responses.
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.
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.
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