Quantifying NK-Cell Cytotoxicity Potentiated by Chimeric Antigen Receptors and Therapeutic Antibodies Using Live-Cell Imaging and Flow Cytometry

Our laboratory studies the role of natural killer cells in health and disease, aiming to harness their potential to improve treatments for solid and blood cancers. Existing NK cell cytotoxicity assays lack standardization across platforms and setups. This protocol provides reproducible live-cell imaging and flow cytometry methods for accurate cytotoxicity assessment.

To begin, prepare 10 milliliters of 2x SYTOX Orange by diluting SYTOX Orange stock solution into NK-92 culture media in a 15-milliliter conical bottom centrifuge tube. Count the target cells using a Luna Counter after mixing with Trypan Blue or use any validated dye exclusion-based cell counting method. Transfer the required amount of target cells into a 15-milliliter conical bottom centrifuge tube.

Centrifuge the tube at 300G for five minutes at room temperature and discard the supernatant. Then re-suspend the target cells to a final concentration of 250, 000 cells per milliliter in 2X SYTOX Orange media. To prepare the effector cell, assess NK-92 cell viability and concentration, and proceed only if viability is greater than 80%as lower viability may affect assay performance.

Count and transfer the required number of NK-92 cells into individual 15 milliliter conical bottom centrifuge tubes. After centrifugation, re-suspend the cells to a final concentration of 500, 000 cells per milliliter. Prepare effector cell dilutions by adding 240 microliters of effector cells to wells of a 96 well round bottom plate for the two to one effector to target ratio, and add 120 microliters of media to the remaining wells.

Perform twofold serial dilutions from two to one to 0.25 to one effector to target ratios, mixing thoroughly by pipetting at least five times and changing tips between transfers. Next, to set up the co-culture, obtain the polylysine coated, flat bottom 96 well plate. Immediately before transfer, thoroughly re-suspend the target and effector cells without creating bubbles, then add 100 microliters of each cell suspension to the appropriate wells.

Then, add 200 microliters of PBS to the surrounding unused wells. Centrifuge the plate at 100G for two minutes at room temperature. Place the plate in the live cell imaging system.

Acquire images every one hour for 24 hours at 37 degrees Celsius with 5%carbon dioxide. Verify the image quality after the first scan and adjust the imaging settings if necessary. Adjust the spectral unmixing to correct for SYTOX Orange spillover into the green channel.

Verify that orange positive dead cells are not misidentified as green positive cells. To configure non-adherent cell-by-cell analysis, set the expected object diameter to 15 micrometers to match the approximate cell diameter. Set the threshold sensitivity and texture sensitivity to five, and the edge sensitivity to 10 to facilitate the separation of cell clusters.

Set the accepted object area filter range to 50 to 2, 000 square micrometers to exclude debris and large objects. Set the eccentricity maximum to 0.95 to exclude non-cellular objects. Obtain primary human NK cells cultured for 16 to 24 hours.

Transfer 500, 000 target cells into a 15 milliliter conical bottom centrifuge tube. Wash the cells once with 10 milliliters of PBS. Centrifuge the cells at 300G for five minutes at room temperature.

Then remove the supernatant. Prepare a 0.5 micromolar working solution of carboxyfluorescein succinimidyl ester or CFSC in PBS by diluting one microliter of five millimolar CFSE stock solution into 10 milliliters of PBS. Re-suspend the obtained pellet at approximately one million cells per milliliter in the CFSE solution.

Incubate the cells for 15 minutes at 37 degrees Celsius, wrapped in aluminum foil to protect from light. Then quench the reaction by adding five to 10 volumes of culture medium, and incubate for five minutes at 37 degrees Celsius. After centrifuging the cells, re-suspend the cell pellet at 250, 000 cells per milliliter so that 100 microliters contain 25, 000 cells.

Add 100 microliters per well of target cells to the designated wells of a round bottom 96 well plate using a multichannel pipette. Plate each condition in triplicate. Next, prepare 200 microliters of 4X antibody solution in NK cell culture medium for each test antibody and matched isotype control.

Transfer the antibody solutions to a deep well plate. Add 50 microliters of antibody solution to each well containing the target cells, and mix thoroughly by pipetting up and down at least five times. Add 50 microliters of NK cell culture medium per well to the no antibody control wells and mix thoroughly by pipetting.

Incubate the plate for 25 to 30 minutes under standard incubation conditions. Count and obtain 200, 000 NK cells and proceed only if viability is greater than or equal to 75%Re-suspend the cells to 250, 000 cells per milliliter in 800 microliters of primary human NK cell culture. Add 50 microliters per well of NK cells to the corresponding wells and mix thoroughly by pipetting up and down at least five times.

Next, pipette 50 microliters per well of culture medium into the control wells and mix thoroughly. Carefully inspect the round bottom 96 well plate to confirm that all wells contain 200 microliters with uniform meniscus levels and no bubbles. Centrifuge the plate for two minutes at 100G, then incubate it for 16 to 18 hours under standard incubation conditions.

Centrifuge the plate for five minutes at 300G and discard the supernatant. Wash all wells with 200 microliters per well of fluorescence activated cell sorting or FACS buffer. Prepare a surface antibody master mix in FACS buffer as presented here.

Re-suspend the cells in 50 microliters per well of antibody master mix and mix thoroughly by pipetting up and down at least five times. Incubate the plate for 15 minutes at four degrees Celsius, wrapped in aluminum foil to protect from light. Add 150 microliters per well of FACS buffer.

Following centrifugation, re-suspend the cells in 100 microliters per well of five micromolar DAPI in FACS buffer. Proceed immediately to flow cytometry acquisition without pre-acquisition incubation. Perform flow cytometry using the Cytek Aurora instrument and record fluorescence channels for CFSE, BV421, BV785, PE-Cy7, DAPI, and any additional fluorophores.

During co-culture, effector cells, live target cells and dead cells were visualized using real-time live cell microscopy. CAR2 and CAR3, NK-92 cells exhibited faster killing kinetics than CAR1-NK-92 cells, particularly at earlier time points at an equal effector to target ratio. Target cell killing increased with higher effector to target ratios, including control NK-92 cells, which demonstrated natural cytotoxicity.

CAR2 and CAR3 NK-92 cells demonstrated greater potency at lower effector to target ratios at later time points. While enhanced potency at intermediate effector to target ratios was observed at earlier time points. Three parameters, KR50, KT50 and IKI-50 were defined to quantify CAR-NK-92 cell potency, killing kinetics and overall cytotoxic performance across effector populations.

The addition of daratumumab alone did not reduce target cell survival, confirming that the antibody had no direct effect on target cell viability in the absence of effector cells. Co-culture with NK cells alone reduced target cell survival to approximately 70%indicating baseline natural cytotoxicity. The addition of daratumumab further reduced cell survival to approximately 37%consistent with enhanced antibody-mediated cytotoxicity.

In contrast, the isotype control condition showed approximately 62%survival comparable to the NK only condition. NK cell degranulation was lowest in NK cell target cell co-culture without antibody and highest in the daratumumab treated condition at approximately 31%while the isotype control showed intermediate degranulation at approximately 19%This protocol enables researchers to quantify NK cell natural cytotoxicity, evaluate tumor killing potency of CAR-NK designs, and compare ADCC efficacy of monoclonal antibodies. A key consideration while performing this protocol is maintaining accurate consistent cell counting, pipetting, and plating of NK end target cells to ensure reliable interpretable results.

Future studies can leverage these protocols to explore NK cell immune evasion, compare tumor susceptibility, and evaluate novel NK cell-based immunotherapies across cancers.