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The authors thank Raymond Qui&#241;ones Alvarado (Yale University), Yitong Chen (Ragon Institute of Mass General, MIT, and Harvard), Angelique Hoelzemer and Katharina Schiering (Institute for Infection and Vaccine Development, University Medical Center Hamburg-Eppendorf), and Abner Louissaint, Jessica Duffy, Genna Mullen, and Gail Newton (Massachusetts General Hospital and Harvard Medical School) for scientific and technical advice and insightful discussions. The authors also thank Michael Waring, Kat Folz-Donahue, and Nathalie Bonheur of the Ragon Institute Flow Cytometry Core for expert technical support. W.F.G.-B. is supported by the Ragon Institute of Mass General, MIT, and Harvard, the Department of Pathology at Massachusetts General Hospital, and the National Institutes of Health Director&#8217;s New Innovator Award (DP2CA311214). J.O.T. is supported by the Kinghorn Foundation as a Fulbright Future Postgraduate Scholarship grantee.

<ol><li>Chen, S., Zhu, H., Jounaidi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+snapshots+of+natural+killer+cells+functions%2C+signaling%2C+molecular+mechanisms+and+clinical+utilization%5BTitle%5D)+AND+%22Signal+Transduct+Target+Ther%22%5BJournal%5D)">Comprehensive snapshots of natural killer cells functions, signaling, molecular mechanisms and clinical utilization.</a> Signal Transduct Target Ther. , (2024).</li>
<li>Liesche, C., Sauer, P., Prager, I., Urlaub, D., Claus, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-fluorescent+protein+reporters+allow+parallel+quantification+of+natural+killer+cell-mediated+granzyme+and+caspase+activities+in+single+target+cells%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Single-fluorescent protein reporters allow parallel quantification of natural killer cell-mediated granzyme and caspase activities in single target cells.</a> Front Immunol. , (2018).</li>
<li>Hartmann, J. A., Cardoso, M. R., Talarico, M. C. R., Kenney, D. J., Leone, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evasion+of+NKG2D-mediated+cytotoxic+immunity+by+sarbecoviruses%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Evasion of NKG2D-mediated cytotoxic immunity by sarbecoviruses.</a> Cell. , (2024).</li>
<li>Garcia-Beltran, W. F., Hölzemer, A., Martrus, G., Chung, A. W., Pacheco, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Open+conformers+of+HLA-F+are+high-affinity+ligands+of+the+activating+NK-cell+receptor+KIR3DS1%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Open conformers of HLA-F are high-affinity ligands of the activating NK-cell receptor KIR3DS1.</a> Nat Immunol. , (2016).</li>
<li>Rezvani, K., Rouce, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+application+of+natural+killer+cell+immunotherapy+for+the+treatment+of+cancer%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">The application of natural killer cell immunotherapy for the treatment of cancer.</a> Front Immunol. , (2015).</li>
<li>Coënon, L., Villalba, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+CD16a+biology+to+antibody-dependent+cell-mediated+cytotoxicity+improvement%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">From CD16a biology to antibody-dependent cell-mediated cytotoxicity improvement.</a> Front Immunol. , (2022).</li>
<li>Ruggeri, L., Mancusi, A., Capanni, M., Martelli, M. F., Velardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploitation+of+alloreactive+NK+cells+in+adoptive+immunotherapy+of+cancer%5BTitle%5D)+AND+%22Curr+Opin+Immunol%22%5BJournal%5D)">Exploitation of alloreactive NK cells in adoptive immunotherapy of cancer.</a> Curr Opin Immunol. , (2005).</li>
<li>Liu, E., Marin, D., Banerjee, P., Macapinlac, H. A., Thompson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+CAR-transduced+natural+killer+cells+in+CD19-positive+lymphoid+tumors%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors.</a> N Engl J Med. , (2020).</li>
<li>Li, Y., Hermanson, D. L., Moriarity, B. S., Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+iPSC-derived+natural+killer+cells+engineered+with+chimeric+antigen+receptors+enhance+anti-tumor+activity%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Human iPSC-derived natural killer cells engineered with chimeric antigen receptors enhance anti-tumor activity.</a> Cell Stem Cell. , (2018).</li>
<li>Marin, D., Li, Y., Basar, R., Rafei, H., Daher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safety%2C+efficacy+and+determinants+of+response+of+allogeneic+CD19-specific+CAR-NK+cells+in+CD19%2B+B+cell+tumors%3A+a+phase+1%2F2+trial%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19+ B cell tumors: a phase 1/2 trial.</a> Nat Med. , (2024).</li>
<li>Chava, S., Bugide, S., Gupta, R., Wajapeyee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+natural+killer+cell-mediated+cytotoxicity+and+migration+in+the+context+of+hepatic+tumor+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Measurement of natural killer cell-mediated cytotoxicity and migration in the context of hepatic tumor cells.</a> J Vis Exp. (156), e60714(2020).</li>
<li>Liu, E., Tong, Y., Dotti, G., Shaim, H., Savoldo, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cord+blood+NK+cells+engineered+to+express+IL-15+and+a+CD19-targeted+CAR+show+long-term+persistence+and+potent+antitumor+activity%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity.</a> Leukemia. , (2018).</li>
<li>Mensali, N., Dillard, P., Hebeisen, M., Lorenz, S., Theodossiou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NK+cells+specifically+TCR-dressed+to+kill+cancer+cells%5BTitle%5D)+AND+%22EBioMedicine%22%5BJournal%5D)">NK cells specifically TCR-dressed to kill cancer cells.</a> EBioMedicine. , (2019).</li>
<li>Nowak, J., Bentele, M., Kutle, I., Zimmermann, K., Lühmann, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CAR-NK+cells+targeting+HER1+%28EGFR%29+show+efficient+anti-tumor+activity+against+head+and+neck+squamous+cell+carcinoma+%28HNSCC%29%5BTitle%5D)+AND+%22Cancers+%28Basel%29%22%5BJournal%5D)">CAR-NK cells targeting HER1 (EGFR) show efficient anti-tumor activity against head and neck squamous cell carcinoma (HNSCC).</a> Cancers (Basel). , (2023).</li>
<li>Granger, J. E., Appledorn, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+measurement+of+apoptosis+and+immune+cell+killing+using+live-cell+imaging+and+analysis%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Kinetic measurement of apoptosis and immune cell killing using live-cell imaging and analysis.</a> Methods Mol Biol. , (2021).</li>
<li>Duffy, J., Rider, A., Loneman, D., Thakkar, D., Mullen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ALK%2B+LBCL+and+PBL+share+a+similar+plasmablastic+transcriptional+identity+but+show+divergent+signaling+dependencies+and+therapeutic+vulnerabilities%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">ALK+ LBCL and PBL share a similar plasmablastic transcriptional identity but show divergent signaling dependencies and therapeutic vulnerabilities.</a> Blood. , (2025).</li>
<li>Kandarian, F., Sunga, G. M., Arango-Saenz, D., Rossetti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+flow+cytometry-based+cytotoxicity+assay+for+the+assessment+of+human+NK+cell+activity%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">A flow cytometry-based cytotoxicity assay for the assessment of human NK cell activity.</a> J Vis Exp. (126), e56191(2017).</li>
<li>Klingemann, H., Boissel, L., Toneguzzo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Natural+killer+cells+for+immunotherapy+-+advantages+of+the+NK-92+cell+line+over+blood+NK+cells%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Natural killer cells for immunotherapy - advantages of the NK-92 cell line over blood NK cells.</a> Front Immunol. , (2016).</li>
<li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">CD107a as a functional marker for the identification of natural killer cell activity.</a> J Immunol Methods. , (2004).</li>
<li>Valiante, N. M., Uhrberg, M., Shilling, H. G., Lienert-Weidenbach, K., Arnett, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functionally+and+structurally+distinct+NK+cell+receptor+repertoires+in+the+peripheral+blood+of+two+human+donors%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors.</a> Immunity. , (1997).</li>
<li>Wang, Y., Zhang, Y., Hughes, T., Zhang, J., Caligiuri, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fratricide+of+NK+cells+in+daratumumab+therapy+for+multiple+myeloma+overcome+by+ex+vivo-expanded+autologous+NK+cells%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">Fratricide of NK cells in daratumumab therapy for multiple myeloma overcome by ex vivo-expanded autologous NK cells.</a> Clin Cancer Res. , (2018).</li>
<li>Cichocki, F., Bjordahl, R., Gaidarova, S., Mahmood, S., Abujarour, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((iPSC-derived+NK+cells+maintain+high+cytotoxicity+and+enhance+in+vivo+tumor+control+in+concert+with+T+cells+and+anti-PD-1+therapy%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti-PD-1 therapy.</a> Sci Transl Med. , (2020).</li>
</ol>

W.F.G.-B. received an honorarium from BioSciences.sas for a scientific presentation on spectral cytometry in 2025. The authors declare no other competing financial or non-financial interests.

These protocols provide two complementary approaches for quantifying NK-cell cytotoxicity in a standardized 96-well plate format. Step 1 uses real-time live-cell imaging to continuously monitor target-cell loss and is well suited for assessing the kinetics of natural and CAR-mediated killing across multiple E:T ratios. Step 2 uses flow cytometry to quantify ADCC after a defined co-incubation period while simultaneously separating target and effector compartments for phenotypic analysis17. These methods, which are presented as complementary assay platforms that can be used to model distinct biological processes, are also readily interchangeable. Primary human NK cells can be used for live-cell-imaging-based ADCC assays and can also be engineered for CAR-mediated killing in flow cytometry assays. Furthermore, although wild-type NK-92 cells lack CD16 expression18, introduction of CD16 (e.g., via lentiviral transduction) readily enables NK-92 cells to mediate potent ADCC. As such, both assays can be tailored to individual scientific and translational needs.
There are several steps that are particularly important for reproducibility in both assays. Small changes in effector-cell number and phenotype can drastically alter target-cell survival. Accurate cell counting and thorough resuspension during plating are essential to minimize deviations in cell counts and incomplete mixing. Similarly, cell preparation must be kept consistent between experiments, including viability, recovery time, and prior cytokine exposure, to ensure uniform effector functionality. Plate geometry is another key determinant of assay performance. In the live-cell imaging workflow, flat-bottom coated plates support stable image acquisition and consistent field analysis, whereas in the flow cytometry workflow, round-bottom plates promote cell&#8211;cell contact and facilitate recovery of non-adherent co-cultures at the endpoint. In addition, the quality of fluorescent labeling must be confirmed before the assay, as poor target-cell discrimination by imaging or weak CFSE separation by flow cytometry will compromise quantification and interpretation.
These two workflows present distinct technical considerations. In the live-cell imaging assay, common causes of poor data quality include uneven cell plating, dense cell clustering or clumping, and suboptimal image-analysis thresholds, all of which are readily correctable. These issues may distort cell counts and result in inconsistent tracking over time. In the flow cytometry&#8211;based assay, suboptimal outcomes are more often caused by high background target-cell death, weak separation of target and effector populations, poor antibody staining, or inconsistent pipetting between replicate wells. Because these assays are designed for comparative analysis, inclusion of appropriate internal controls is essential for interpretation, including target-only controls for normalization and matched negative controls for antibody- or CAR-specific conditions.
Several shared and assay-specific limitations must be considered with these methods. The live-cell imaging assay provides temporal resolution but depends on accurate fluorescent labeling and image-analysis parameters, both of which may be influenced by cell clustering, spectral overlap, or changes in morphology during co-culture15. In some cases, particularly when assays involve primary effector cells, achieving adequate fluorescent labeling without altering killing dynamics is difficult or not feasible. The flow cytometry&#8211;based assay provides higher phenotypic resolution at a defined endpoint, but it does not independently distinguish early from late killing unless multiple matched time points are collected, which can be costly and time-consuming17. In addition, endpoint measurements may reflect not only target-cell death but also differences in target-cell proliferation, effector attrition, or recovery during sample handling. Mechanistic readouts such as CD107a are therefore best interpreted as complementary indicators of effector-cell activation rather than direct substitutes for target-cell elimination19. More broadly, neither platform fully reproduces the spatial architecture, stromal interactions, cytokine gradients, or trafficking constraints that shape NK-cell function in vivo.
Several biological factors must also be considered when interpreting NK-cell killing assays. Donor-to-donor variability is intrinsic to experiments with primary human NK cells, which differ in cytotoxic potential and subset composition due to donor age, sex, infectious disease history, and receptor polymorphisms20. Use of NK-92 cells greatly reduces variability, but these cells can still exhibit source- and passage-related changes18. Our protocol mitigates technical or experimental differences through standardized parameters and same-plate comparisons to more accurately assess these biological factors. Where feasible, we recommend using defined NK-92 passage ranges with fixed viability thresholds. For primary NK cells, assays should use NK cells from multiple donors for each condition, with statistical analyses accounting for donor-to-donor variability (e.g., paired or mixed-effects models), given that relative differences between conditions are generally preserved across donors. Furthermore, additional NK-cell markers (e.g., KIRs, NKG2A/C/D, and CD57) can be incorporated into antibody panels used for flow cytometry to compare subset frequencies across donors and assess subset-specific NK-cell activation (i.e., CD107a and CD69 expression), further facilitating interpretation of results. Finally, for all NK-cell killing assays, certain additional scenarios must be considered. Expression of targeted antigens on NK cells, either by endogenous expression or by acquisition of antigens from target cells via trogocytosis, can result in NK-cell fratricide and confound results. For example, daratumumab can target CD38 expressed on NK cells and induce fratricide21, limiting the magnitude of ADCC against the intended targets. Fortunately, live-cell imaging and flow cytometry assays allow for quantification of NK cells to assess fratricide.
Despite these limitations, the combined use of live-cell imaging and flow cytometry provides a practical framework for reproducible NK-cell functional testing across a range of basic and preclinical applications. The imaging workflow is advantageous when temporal behavior is important, such as distinguishing rapid from delayed cytotoxic responses or comparing killing kinetics across CAR designs22. The flow cytometry&#8211;based workflow is advantageous when functional readouts must be interpreted alongside effector- and target-cell phenotypes within the same sample. Together, these approaches enable quantitative assessment of natural killing, CAR-mediated cytotoxicity, and ADCC using widely accessible experimental platforms. By establishing a standardized and scalable framework, this system facilitates rigorous benchmarking and mechanistic interrogation across NK cell&#8211;based modalities, accelerating the development and clinical translation of next-generation immunotherapies.

Natural killer (NK) cells are innate lymphocytes that play a central role in immune surveillance against virally infected and malignant cells, exhibiting rapid and potent cytotoxicity1. Unlike B and T cells, NK cells do not express antigen-specific receptors and instead rely on an array of activating and inhibitory receptors to detect loss of major histocompatibility complex class I expression or cellular stress. This enables them to mediate direct cytotoxicity by releasing cytotoxic granules containing perforin and granzymes or, in some cases, by activating death receptors1,2,3,4. In addition to natural cytotoxicity, NK cells express Fc&#947;RIII (CD16), a receptor that recognizes the Fc portion of immunoglobulin G (IgG) and mediates antibody-dependent cellular cytotoxicity (ADCC) against IgG-coated target cells5,6.
Because of their intrinsic cytotoxic mechanisms and favorable safety profile, NK cells have emerged as promising candidates for cellular immunotherapy. With the potential for allogeneic use, NK cells can function as an &#8220;off-the-shelf&#8221; therapy with minimal risk of graft-versus-host disease, enabling broader clinical applicability and reduced costs7. Recent advances have further expanded the therapeutic potential of NK cells through genetic engineering strategies, including the introduction of chimeric antigen receptors (CARs) that redirect NK cells to recognize specific tumor antigens. CAR-NK cells have demonstrated antitumor efficacy in in vivo&#160;xenograft models and clinical trials, with reduced toxicity compared to CAR-T cells8,9,10. Therefore, there is growing interest in developing engineered NK-cell platforms and antibody-based therapeutic strategies. In this context, accurate and quantitative measurement of NK-cell killing capacity is essential for understanding NK-cell biology, assessing the efficacy of CAR or other engineered NK-cell therapies, and evaluating monoclonal antibodies that rely on ADCC.
A variety of in vitro&#160;assays have been widely used across laboratories to evaluate NK-cell cytotoxicity. Common methods include radioactive chromium-51 release assays, lactate dehydrogenase release assays, and calcein&#8211;acetoxymethyl release assays11,12. While these traditional methods have been widely used to measure cytotoxicity, they rely on indirect readouts of cell death, provide endpoint measurements rather than kinetic data, and offer limited sensitivity and dynamic range. They also do not provide information about the phenotype or activation state of effector cells. More recently, live-cell imaging platforms and flow cytometry have been increasingly adopted because they enable direct quantification of target-cell survival, monitoring of killing over time, and simultaneous phenotypic analysis of effector and target cells13,14. However, even among these newer approaches, substantial variability exists across laboratories and published studies, including differences in target-cell preparation, effector-to-target (E:T) ratios, and co-incubation times, limiting reproducibility and cross-study comparisons, particularly in preclinical evaluations of NK cell&#8211;based immunotherapies.
This article presents a standardized workflow for quantitative assessment of NK-cell cytotoxicity using two independent, complementary platforms: real-time, live-cell imaging (IncuCyte SX5) and flow cytometry&#8211;based endpoint assays. Live-cell imaging enables continuous monitoring of target-cell survival during co-culture with effector cells, allowing kinetic evaluation of cytotoxicity across multiple E:T ratios and experimental conditions with minimal assay manipulation15. In contrast, flow cytometry provides a highly sensitive endpoint measurement of target-cell death, along with simultaneous phenotypic characterization of NK cells and target cells using a multimarker staining panel.
Additionally, this protocol incorporates standardized experimental parameters, including E:T ratios, incubation conditions, plate format, and analytical workflows, to improve reproducibility and facilitate comparison of cytotoxicity measurements across laboratories and studies. The workflow enables quantitative assessment of multiple NK effector functions, including natural cytotoxicity, CAR-mediated killing, and ADCC, and is compatible with primary human NK cells and NK cell lines. This protocol provides a practical framework for studies of NK-cell biology and preclinical evaluation of NK cell&#8211;based immunotherapy development.

<table><tbody><tr><td>Advanced RPMI-1640 medium</td><td>Thermo Fisher Scientific</td><td>12633020</td><td>Basal medium for NK-92 and primary human NK-cell culture media (see protocol for complete formulation)</td></tr><tr><td>Anti-human CD107a antibody, PE-Cy7 conjugated (clone H4A3)</td><td>BioLegend</td><td>328618</td><td>Degranulation marker for flow cytometry</td></tr><tr><td>Anti-human CD16 antibody, BV785 conjugated (clone 3G8)</td><td>BioLegend</td><td>302046</td><td>Flow cytometry</td></tr><tr><td>Anti-human CD56 antibody, BV421 conjugated (clone HCD56)</td><td>BioLegend</td><td>318328</td><td>Flow cytometry</td></tr><tr><td>Benchtop centrifuge with swinging bucket rotor compatible with 15-mL tubes and 96-well plates (Eppendorf Centrifuge 5810R)</td><td>Eppendorf</td><td>5811000015</td><td>Used for cell pelleting and plate centrifugation (100&amp;ndash;300 &amp;times; g)</td></tr><tr><td>Carboxyfluorescein succinimidyl ester (CFSE)</td><td>Thermo Fisher Scientific</td><td>C34554</td><td>Cell proliferation dye for target-cell labeling</td></tr><tr><td>Cytek Aurora spectral flow cytometer (or equivalent)</td><td>Cytek Biosciences</td><td>N7-00003</td><td>Used for flow cytometry acquisition and analysis</td></tr><tr><td>DAPI (4&amp;prime;,6-diamidino-2-phenylindole)</td><td>BioLegend</td><td>422801</td><td>Nuclear stain for live/dead discrimination</td></tr><tr><td>Deep-well 96-well plate</td><td>USA Scientific</td><td>1896-2110</td><td>Used for antibody preparation</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>BP231-100</td><td>Cryopreservation reagent</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Avantor</td><td>76419-584</td><td>Basal media supplementation; used in FACS buffer (2% FBS)</td></tr><tr><td>General Purpose Water Bath, 10 L (37 &amp;deg;C)</td><td>PolyScience</td><td>WBE10</td><td>For cell thawing</td></tr><tr><td>GlutaMAX (L-alanyl-L-glutamine supplement)</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>Stable glutamine source</td></tr><tr><td>Human AB serum</td><td>Sigma-Aldrich</td><td>H4522-100ML</td><td>Supplement for primary human NK-cell culture media</td></tr><tr><td>IncuCyte SX5 live-cell imaging system</td><td>Sartorius</td><td>4816</td><td>Real-time live-cell imaging and Cell-by-Cell analysis</td></tr><tr><td>LUNA-FL dual fluorescence cell counter (or equivalent)</td><td>Logos Biosystems</td><td>L20001</td><td>Automated cell counting</td></tr><tr><td>Parafilm M (4 in &amp;times; 250 ft roll)</td><td>Genesee</td><td>16-101</td><td>Plate sealing and storage</td></tr><tr><td>Phosphate-buffered saline (PBS), sterile (pH 7.2&amp;ndash;7.4)</td><td>Corning</td><td>21-020-CV</td><td>Used for washes and FACS buffer preparation</td></tr><tr><td>Poly-D-lysine (0.1 mg/mL)</td><td>Thermo Fisher Scientific</td><td>A3890401</td><td>Plate coating reagent</td></tr><tr><td>Primocin</td><td>InvivoGen</td><td>ant-pm-2</td><td>Antibiotic</td></tr><tr><td>Recombinant human interleukin-2 (IL-2)</td><td>R&amp;amp;D Systems</td><td>202-IL-500</td><td>Cytokine</td></tr><tr><td>RosetteSep Human NK-cell enrichment kit</td><td>STEMCELL Technologies</td><td>15065</td><td>Human NK-cell enrichment cocktail</td></tr><tr><td>SYTOX Orange dead cell stain</td><td>Thermo Fisher Scientific</td><td>S34861</td><td>Viability dye for live-cell imaging</td></tr><tr><td>Trypan Blue 0.4%</td><td>Gibco</td><td>15250-061</td><td>Viability dye for cell counting</td></tr><tr><td>TrypLE Express Enzyme (1X), no phenol red</td><td>Thermo Fisher Scientific</td><td>12604039</td><td>For adherent cell detachment</td></tr><tr><td>UltraPure EDTA (0.5 M, pH 8.0)</td><td>Thermo Fisher Scientific</td><td>15575020</td><td>Used for FACS buffer preparation</td></tr><tr><td>15-mL conical-bottom centrifuge tubes</td><td>Corning</td><td>352097</td><td>Sterile</td></tr><tr><td>24-well tissue culture plate (tissue culture&amp;ndash;treated)</td><td>Corning</td><td>3524</td><td>Used for NK-cell recovery</td></tr><tr><td>96-well clear round-bottom plate (tissue culture&amp;ndash;treated)</td><td>Corning</td><td>3799</td><td>Used for co-culture and flow cytometry assays</td></tr><tr><td>96-well flat clear-bottom black polystyrene plate (tissue culture&amp;ndash;treated)</td><td>Corning</td><td>3603</td><td>Compatible with live-cell imaging systems</td></tr></tbody></table>

quantifying nk-cell cytotoxicity potentiated by chimeric antigen receptors and therapeutic antibodies using live-cell imaging and flow cytometry

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

Natural killer (NK) cells are innate lymphocytes that play a critical role in protective immunity against diverse intracellular pathogens and cancers. Their primary function is to kill target cells that are infected, malignantly transformed, or coated by antibodies via antibody-dependent cellular cytotoxicity (ADCC). NK cells can also be genetically engineered to express chimeric antigen receptors (CARs) that enable targeted recognition of specific antigens. Quantitative measurement of NK-cell cytotoxicity is essential for assessing baseline functionality and for preclinical evaluation of monoclonal antibodies and CAR-engineering strategies. However, in vitro&#160;functional assays remain highly variable across laboratories due to differences in cell preparation, target cells, effector-to-target ratios, co-incubation times, and readout methods, limiting reproducibility and cross-study comparisons. This article presents a standardized protocol for quantitative assessment of NK-cell cytotoxicity using flow cytometry and real-time, live-cell imaging. Primary human NK cells and CAR-expressing NK-92 cells were evaluated for their ability to kill cancer cells and antibody-coated target cells in a 96-well plate format to measure natural cytotoxicity, CAR-mediated killing, and ADCC. Target-cell survival was measured either continuously using live-cell imaging or at a defined time point by flow cytometry, which also enabled phenotypic characterization of NK cells and target cells. These protocols provide a robust framework using routine tissue culture, imaging, and flow cytometry methods to enable reproducible&#160;quantification of NK-cell effector functions for studies of innate immunity and NK cell&#8211;based immunotherapies.

To quantify NK-cell cytotoxicity in real time, NK-92 effector cells engineered to express anti-BCMA CARs were co-cultured with BCMA+ Ramos target cells in a black, clear flat-bottom 96-well plate and monitored using live-cell imaging (Figure 1A and Supplemental Figure 1A). Effector cells were added at E:T ratios of 0:1 (target only), 0.25:1, 0.5:1, 1:1, and 2:1, with each condition performed in triplicate. Three different CAR NK-92 constructs were evaluated: CAR1 with no co-stimulatory domain, CAR2 with a 4-1BB (CD137) co-stimulatory domain, and CAR3 with a DAP10 co-stimulatory domain, along with control NK-92 cells expressing a ZsGreen-only vector. All effector cells were generated via lentiviral transduction, as previously described4 using ZsGreen-P2A-CAR transgene cassettes. Ramos target cells were similarly transduced with an emiRFP670-encoding lentiviral vector. Target-cell growth and survival were tracked every hour over a 24-h period using the live-cell imaging instrument, measuring green fluorescence for ZsGreen+ NK-92 cells, orange fluorescence for SYTOX Orange+ dead cells, and NIR fluorescence for emiRFP670+ target cells (Figure 1A and Supplemental Figure 1B and 1C).
Microscopic images were acquired at defined time intervals to visualize effector cells, live target cells, and dead cells during co-culture (Figure 1B). Quantification of target-cell growth and survival demonstrated that CAR2 and CAR3 NK-92 cells exhibited faster killing kinetics than CAR1 NK-92 cells, particularly at earlier time points at an E:T ratio of 1:1. Target-cell killing increased with higher E:T ratios, including in control NK-92 cells, which demonstrated natural cytotoxicity (Figure 1C). These measurements highlight the ability of live-cell imaging to capture dynamic changes in cytotoxic activity. Target-cell survival was also analyzed as a function of E:T ratio at defined time points to compare CAR NK-92 potency. CAR2 and CAR3 NK-92 cells showed greater potency at lower E:T ratios at later time points (e.g., E:T = 0.25:1 at 24 h) and at intermediate E:T ratios at earlier time points (e.g., E:T = 1:1 at 4 h) (Figure 1D). No significant differences in killing were observed between CAR2 and CAR3 NK-92 effector cells across analyses.
A heatmap integrating E:T ratio, co-incubation time, and target-cell survival provides a comprehensive visualization of natural and CAR-mediated cytotoxicity across all effector populations and enables quantitative assessment. Three parameters were defined to quantify potency and kinetics: KR50, the killer-to-target ratio that achieves 50% killing (at 12 h); KT50, the co-incubation time required to achieve 50% killing (at an E:T ratio of 1:1); and IKI50, the integrated killer index representing the area of the heatmap below 50% survival, defined by the 50% survival isocline (Figure 1E). These metrics enable comparison of CAR NK-92 cell potency (KR50), killing kinetics (KT50), and integrated cytotoxic performance (IKI50) (Figure 1F). This analysis demonstrates how the assay can quantitatively compare engineered NK-cell platforms across multiple parameters within a single experiment.
Flow cytometry with cell counting was used to measure ADCC by co-culturing fluorescently labeled target cells with primary human NK cells in the presence or absence of therapeutic antibodies and quantifying the residual live target-cell population after 16–18 h of incubation (Figure 2A). Flow cytometers such as Cytek Aurora, BD Symphony, or BD Fortessa with a high-throughput sampler can be used. In the representative setup (Supplemental Figure 2), CD38+ patient-derived lymphoma target cells were plated in a round-bottom 96-well plate, and daratumumab (or isotype antibody) was added to appropriate wells. NK cells were then co-cultured at an E:T ratio of 0.5:1, a ratio optimized for these target cells to minimize natural killing while allowing detection of ADCC. After incubation, flow cytometry was performed to resolve cell populations by forward and side scatter, exclude non-viable events using DAPI, and separate CFSE+ target cells from CD56+CD16+ NK cells for parallel analysis (Figure 2B). This design enables simultaneous cytotoxicity measurement and phenotypic characterization.
<img alt="NK cell assay setup; ADCC assay results; flow cytometry diagram; cell survival and degranulation." class="xfigimg" src="/files/ftp_upload/71377/71377fig2v2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Flow cytometry–based antibody-dependent cellular cytotoxicity (ADCC) assay enables simultaneous quantification of target-cell survival and NK-cell activation. (A) Schematic overview of the ADCC assay. CFSE-labeled CD38+ lymphoma target cells were plated at 2.5 × 104 cells per well and cultured under five conditions: target cells alone; target cells with daratumumab (25 µg/mL); target cells with NK cells (E:T = 0.5:1); target cells with NK cells and daratumumab; and target cells with NK cells and isotype control antibody (25 µg/mL). (B) Representative flow cytometry gating strategy. Events were first gated by forward scatter and side scatter, followed by exclusion of non-viable cells using DAPI. Target cells were identified as CFSE+ events, and NK cells were identified as CD56+ and CD16+ events. NK-cell degranulation was assessed by CD107a expression. (C) Quantification of target-cell survival, expressed as a percentage relative to the target-only control condition (set to 100%). (D) Quantification of NK-cell degranulation, shown as the percentage of CD107a+ cells within the NK-cell population. Bars represent mean ± standard deviation, and overlaid points represent individual replicate wells (n = 3). Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc multiple-comparison test. Statistical significance is indicated as follows: *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. <a href="https://www.jove.com/files/ftp_upload/71377/71377fig2largev1.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The primary endpoint is target-cell survival, calculated within the live CFSE+ gate and normalized to the target-only condition, which is set to 100% survival. This normalization controls for spontaneous target-cell loss and enables direct comparison across conditions. In representative data (Figure 2C), the addition of daratumumab alone did not reduce target-cell survival, confirming that the antibody has 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 survival to approximately 37%, consistent with enhanced antibody-mediated killing, whereas the isotype control condition showed survival of approximately 62%, comparable to the NK-only condition. Successful experiments show clear separation between antibody-treated and control conditions with high reproducibility across replicates.
Secondary phenotyping of NK cells provides complementary confirmation of the link between target-cell killing and effector activation. NK-cell degranulation, measured as the percentage of CD107a+ NK cells, was lowest in NK cell–target cell co-culture without antibody and highest in the daratumumab-treated condition (approximately 31%), with the isotype control showing intermediate degranulation (approximately 19%), supporting antibody-mediated activation (Figure 2D). These results demonstrate that the assay can distinguish target-cell killing from effector activation. Although degranulation and survival trends are generally aligned, they do not fully overlap, indicating that they represent related but distinct biological processes.
Suboptimal experiments typically show reduced separation between conditions, variability in cell counts across wells, high target-cell death in control wells, weak CFSE staining, or elevated background degranulation. These outcomes may result from poor target-cell viability, inconsistent NK-cell recovery, inadequate mixing, or variability in staining and acquisition. Because the assay independently identifies target and effector compartments, it can be extended to include additional phenotyping of target cells (e.g., antigen density or ligand expression) and broader NK-cell characterization. Inclusion of an “NK only” condition can further quantify baseline degranulation and NK-cell viability. This multiparameter design is a key advantage, enabling functional cytotoxicity to be interpreted alongside mechanistic immune phenotypes.
Supplemental Figure 1. Representative plate layout and analysis workflow for live-cell imaging cytotoxicity assay. (A) Representative 96-well plate layout for the CAR NK-cell killing assay. Four effector cell conditions are arranged in quadrants, each tested across E:T ratios of 0:1 (target-only control), 0.25:1, 0.5:1, 1:1, and 2:1, with each condition performed in triplicate (total of 60 assay wells). (B) Representative configuration of non-adherent Cell-by-Cell analysis in IncuCyte 2021C software, illustrating spectral unmixing, segmentation, and masking parameters used to identify individual effector and target cells while excluding debris and aggregates. (C) Representative Cell-by-Cell classification gating strategy using NIR versus SYTOX Orange fluorescence to define the live target-cell population. Target-only wells and co-culture wells at an example time point (t = 4 h) are used to confirm separation between live and dead cell populations. <a href="https://www.jove.com/files/ftp_upload/71377/Supplemental%20Figure%201.pdf" target="_blank">Please click here to download this file.</a>
Supplemental Figure 2. Representative plate layout for flow cytometry–based ADCC assay. Representative 96-well plate layout illustrating five experimental conditions plated in triplicate across rows B–D and columns 2–6 (total assay wells = 15): (1) target-only control, (2) target cells with daratumumab, (3) target cells with NK cells, (4) target cells with NK cells and daratumumab, and (5) target cells with NK cells and isotype control antibody. Target cells were plated at 2.5 × 104 cells per well, and NK cells were added at 1.25 × 104 cells per well to achieve an E:T ratio of 0.5:1. Antibodies were used at a final concentration of 25 µg/mL. Surrounding wells were filled with PBS to minimize evaporation. The target-only condition was used to normalize target-cell survival, and the isotype control accounted for antibody formulation effects independent of antigen specificity. Optimization of E:T ratio may be required depending on the susceptibility of the target cell line. <a href="https://www.jove.com/files/ftp_upload/71377/Supplemental%20Figure%202.pdf" target="_blank">Please click here to download this file.</a>

This protocol provides a standardized in vitro. workflow for consistent measurement of natural, chimeric antigen receptor&#8211;mediated, and antibody-dependent cytotoxicity of human natural killer cells using live-cell imaging and flow cytometry.

Natural killer (NK) cells are innate lymphocytes that play a critical role in protective immunity against diverse intracellular pathogens and cancers. ...

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.

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Research

JoVE Journal

Immunology and Infection

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