This method can be used to dissect the cellular molecular mechanism of FC gamma R3A driven events without the need for target cells that express the antigen. Set a refrigerated micro centrifuge to four degrees Celsius or place a micro centrifuge into a refrigerated space. 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 one microliter to each tube of cells for a final concentration of one 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 one milliliter of ice cold media to each tube and centrifuge the cells at 135 times G and four degrees Celsius for five minutes.
Wash the cells again with one 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. At the desired time point for western blood analysis or assessment of cytoskeletal rearrangement, stop activation by adding one milliliter of ice cold media and immediately spin samples in a refrigerated centrifuge at 135 times G for five minutes. Wash once more with one milliliter of ice cold media.
Lyse the cells by adding 20 microliters of RIPA buffer containing phosphatase and protease inhibitors mix. Place the tubes on ice for 30 minutes, then centrifuge tubes at 2, 100 times G for 15 minutes at four degrees Celsius. Transfer the lysate to a clean 1.5 milliliter tube and add the reagents required for protein separation.
To stop activation, remove the cell samples from the water bath and place them on ice. Immediately spin in a refrigerated centrifuge at 135 times G for five minutes. Transfer the supernatant to a clean vessel and freeze it for later assessment of chemokine or cytokine production.
Wash the pellet twice with one milliliter of ice cold PBS. Extract RNA and produce cDNA. Freeze the cDNA at negative 20 degrees Celsius until gene expression analysis.
Re-suspend the cell pellet with 50 microliters of 3.7%paraformaldehyde. After 10 minutes at room temperature, add one milliliter of PBS and spin at 135 times G for five minutes at room temperature. Repeat this washing once more.
Add 100 microliters of 0.1%Triton X 100 PBS to permeabilize cells. After five minutes, pellet the cells by centrifugation at 135 times G and room temperature for five minutes. Then add 100 microliters of phalloidin, and incubate for 20 minutes at room temperature.
To wash the cells, add one milliliter of PBS and spin the tubes at 135 times G for five minutes at room temperature. Re-suspend the cell pellet in the desired volume for flow cytometric analysis. While the cells are incubating on ice with rituximab or other antibody of interest, prepare 50 microliters of antibody mixture per sample.
After the final wash and aspiration of the cells, add 50 microliters of the antibody CD107A mixture. Then incubate at 37 degrees Celsius. At the desired time points, add one milliliter of PBS to the cells and spin at 135 times G for five minutes at room temperature.
Aspirate the supernatant and add 100 microliters of 4%paraformaldehyde. Incubate at room temperature for 10 minutes. Add one milliliter of PBS and spin at 135 times G for five minutes at room temperature to wash.
Re-suspend the cell pellet in the desired volume for flow cytometric analysis. Natural killer cells were isolated from PBMCs and stained with CD56 and CD3. The cells had a greater than 90%purity and viability of greater than 95%Cell lysates were separated on an SDS page gel.
After transfer onto a PVDF membrane, Western blots for phospho AKT, phospho PRAS 40 and phospho ERK 1 and 2 were performed. An accumulation of these molecules indicates that events were driven by the FC gamma R3A. Natural killer cells were stimulated with rituximab.
MRNA was collected, reverse transcribed, and subjected to qPCR analysis. Activated natural killer cells expressed MIP-1 alpha, MIP-1 beta, RANTES, interferon gamma and TNF alpha. After stimulation with rituximab for zero to 30 minutes, natural killer cells underwent phalloidin staining and flow cytometry.
Cytoskeletal rearrangement was observed in activated cells. Natural killer cells were assessed for degranulation by CD107A and flow cytometry. Rituximab increased the percentage of cells expressing CD107A.
Natural killer cells were stimulated for zero, 0.5, one, three and six hours with rituximab. Supernatant was collected and analyzed by a flow and bead based cytokine assessment method. As expected based on previously published studies, the chemokine's MIP-1 alpha, MIP-1 beta and RANTES and the cytokines interferon gamma and TNF alpha were detected in the supernatant.
A small subset of stimulated CD8 T cells express FC gamma R3A and this protocol may help in the identification of biomarkers for therapeutics that mediate their effect through FC gamma R3A driven events.
