The DOI matches its Crossref record; 99 of the 100 references it cites were found in a registry or an index.
Comparative analysis of assays to measure CAR T cell-mediated cytotoxicity
Open accessAbstract
The antitumor efficacy of genetically engineered 'living drugs', including chimeric antigen receptor and T-cell receptor T cells, is influenced by their activation, proliferation, inhibition, and exhaustion. A sensitive and reproducible cytotoxicity assay that collectively reflects these functions is an essential requirement for translation of these cellular therapeutic agents. Here, we compare various in vitro cytotoxicity assays (including chromium release, bioluminescence, impedance, and flow cytometry) with respect to their experimental setup, appropriate uses, advantages, and disadvantages, and measures to overcome their limitations. We also highlight the US Food and Drug Administration (FDA) directives for a potency assay for release of clinical cell therapy products. In addition, we discuss advanced assays of repeated antigen exposure and simultaneous testing of combinations of immune effector cells, immunomodulatory antibodies, and targets with variable antigen expression. This review article should help to equip investigators with the necessary knowledge to select appropriate cytotoxicity assays to test the efficacy of immunotherapeutic agents alone or in combination.
Overview
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Verification
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“A U.S. company that seeks to manufacture CAR T-cells in the U.S. and E.U. must comply with both sets of regulations and work with strict oversight from both the FDA and the European Medicines Agency (EMA) to meet respective manufacturing standards (133,135,136).”
From Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy (2021) · cites this paper as [133]
Experimental — accuracy not yet measuredThe check wasn’t confident either way, so it gives no verdict — never disputed.
In the citing paper
“A similar clinical trial phase structure exists in the European Union, although some GMP regulations are stricter in Europe (132,133).”
From Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy (2021) · cites this paper as [133]
Experimental — accuracy not yet measured
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Citations
Cites
Genome engineering using the CRISPR-Cas9 system
Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013) · Nature Protocols · doi:10.1038/nprot.2013.143 · Cited by 11,133
Immune checkpoint inhibitors: recent progress and potential biomarkers
Darvin, P., Toor, S. M., Sasidharan Nair, V., & Elkord, E. (2018) · Experimental & Molecular Medicine · doi:10.1038/s12276-018-0191-1 · Cited by 2,035
Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection
Eyquem, J., Mansilla-Soto, J., Giavridis, T., van der Stegen, S. J. C., Hamieh, M., Cunanan, K. M., Odak, A., Gönen, M., & Sadelain, M. (2017) · Nature · doi:10.1038/nature21405 · Cited by 1,831
Analysis of apoptosis by propidium iodide staining and flow cytometry
Riccardi, C., & Nicoletti, I. (2006) · Nature Protocols · doi:10.1038/nprot.2006.238 · Cited by 1,685
Human chimeric antigen receptor macrophages for cancer immunotherapy
Klichinsky, M., Ruella, M., Shestova, O., Lu, X. M., Best, A., Zeeman, M., Schmierer, M., Gabrusiewicz, K., Anderson, N. R., Petty, N. E., et al. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0462-y · Cited by 1,495
CRISPR-engineered T cells in patients with refractory cancer
Stadtmauer, E. A., Fraietta, J. A., Davis, M. M., Cohen, A. D., Weber, K. L., Lancaster, E., Mangan, P. A., Kulikovskaya, I., Gupta, M., Chen, F., et al. (2020) · Science · doi:10.1126/science.aba7365 · Cited by 1,478
CD107a as a functional marker for the identification of natural killer cell activity
Alter, G., Malenfant, J., & Altfeld, M. A. (2004) · Journal of Immunological Methods · doi:10.1016/j.jim.2004.08.008 · Cited by 1,416
BRUNNER, K. T., Mauël, J., Cerottini, J. C., & Chapuis, B. (1968) · PubMed Central · Cited by 1,367
Decker, T., & Lohmann‐Matthes, M. (1988) · Journal of Immunological Methods · doi:10.1016/0022-1759(88)90310-9 · Cited by 1,366
Mass Cytometry: Single Cells, Many Features
Spitzer, M. H., & Nolan, G. P. (2016) · Cell · doi:10.1016/j.cell.2016.04.019 · Cited by 1,199
Cited by
Sorted by citations
Items 101–138 of 138
Advanced Cell Therapy product release containing CAR-T cells
de Godoy, J. A. P., Kerbauy, L. N., Paiva, R. M. A., Kondo, A. T., Okamoto, O. K., & Kutner, J. M. (2022) · JOURNAL OF BONE MARROW TRANSPLANTATION AND CELLULAR THERAPY · doi:10.46765/2675-374x.2022v3n1p156
Immunotherapy using CAR T: What we have learned from trials and where we are heading
Zhu, A., Bellis, R. Y., Saini, J. K., Fong, C., & Adusumilli, P. S. (2023) · Elsevier eBooks · doi:10.1016/b978-0-12-822620-9.00012-4
Self-citationData from Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy
Abou‐El‐Enein, M., Elsallab, M., Feldman, S. A., Fesnak, A. D., Heslop, H. E., Marks, P. W., Till, B. G., Bauer, G., & Savoldo, B. (2023) · doi:10.1158/2643-3230.c.6550449
Yin, D., Sun, Q., Li, Y., Shen, W. H., Shang, W., Xu, Y., Yang, J., Chen, J., Gao, W., Wu, Q., et al. (2023) · Research Square · doi:10.21203/rs.3.rs-3259314/v1
Optimizing ex vivo CAR-T cell-mediated cytotoxicity assay through multimodality imagingPreprint
Foulke, J. G., Chen, L., Chang, H., McManus, C. E., Tian, F., & Gu, Z. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.05.03.592348
Interfacial-engineered living drugs with “ON/OFF” switching for oral delivery
Feng, P., Bai, X., Ma, X., Kong, H., & Yang, R. (2024) · Nanoscale · doi:10.1039/d4nr01927j
Dogan, M., Kozhaya, L., Placek, L., Karhan, E., Yiğit, M., Mohammed, C., Zhao, Q., & Unutmaz, D. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.07.09.602762
The method for assessing the specificity of developing CAR therapies
Prikhodko, I. V., & Guria, G. (2024) · Biophysical Reports · doi:10.1016/j.bpr.2024.100172
Adeniran, A., Handy, S., & Baki, A. (2024) · Springer eBooks · doi:10.1007/978-3-031-62007-2_9
Considerations for nonclinical studies conducted to support the development of cellular therapies
Bolt, M. W., & Narayanan, P. K. (2024) · Elsevier eBooks · doi:10.1016/b978-0-443-13344-2.00011-0
Single-Cell Analysis of NK Cell Cytotoxicity in Cancer Therapy Using Microfluidic DropletsPreprint
Ozcan, R., Vahedi, F., Namakian, S., Ashkar, A. A., & Didar, T. F. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.05.04.651923
Analytical Methods Shaping Cell Therapy Characterization
Amritha, L., Sudeshna, S., Rebecca, M., Rahamthulla, S., Egemen, T., & Amy, G. (2025) · doi:10.1002/9781394236145.ch14
Modern Laboratory Test-Systems as Platforms for Validation of Clinically Promising T-Cell Receptors
Mungalov, R. V., Vand, N. S., Chudakov, D. M., & Bryushkova, E. A. (2025) · Russian Journal of Bioorganic Chemistry · doi:10.1134/s1068162025601910
Flow-cytometry-based assay to study CD8+ T cell cytotoxicity against tumor cells
Sarapulov, A. V., Rodriguez, M.-A., & Zappasodi, R. (2025) · Methods in cell biology · doi:10.1016/bs.mcb.2025.10.016
Additional file 2 of Mechanism of action, potency and efficacy: considerations for cell therapies
Simon, C. G., Bozenhardt, E. H., Celluzzi, C. M., Dobnik, D., Grant, M., Lakshmipathy, U., Nebel, T., Peltier, L., Ratcliffe, A., Sherley, J. L., et al. (2024) · Figshare · doi:10.6084/m9.figshare.25744470
Leland, P., Kumar, D., Nimmagadda, S., Bauer, S. R., Puri, R. K., & Joshi, B. H. (2024) · Figshare · doi:10.6084/m9.figshare.26595290
Leland, P., Kumar, D., Nimmagadda, S., Bauer, S. R., Puri, R. K., & Joshi, B. H. (2024) · Figshare · doi:10.6084/m9.figshare.26595281
Additional file 1 of Mechanism of action, potency and efficacy: considerations for cell therapies
Simon, C. G., Bozenhardt, E. H., Celluzzi, C. M., Dobnik, D., Grant, M., Lakshmipathy, U., Nebel, T., Peltier, L., Ratcliffe, A., Sherley, J. L., et al. (2024) · Figshare · doi:10.6084/m9.figshare.25744317
Leland, P., Kumar, D., Nimmagadda, S., Bauer, S. R., Puri, R. K., & Joshi, B. H. (2024) · Figshare · doi:10.6084/m9.figshare.26595296
Tschaidse, T., Trefny, M. P., Carlini, E., Andreu-Sanz, D., Michaelides, S., Nguyen, T., & Kobold, S. (2025) · Journal of Visualized Experiments · doi:10.3791/69623
From Bench to Bedside: Innovations and Challenges in CAR-T Cell Therapy Manufacturing and Regulation
Talukdar, J., Baishya, D., Karmakar, S., Ghosh, A., Seth, T., Kumar, U., & Ghatak, A. (2026) · Springer eBooks · doi:10.1007/978-981-95-3628-3_8
Rajnpreht, I., Vimeux, L., & Donnadieu, E. (2026) · Methods in cell biology · doi:10.1016/bs.mcb.2026.01.017
Analysis of murine CAR-T cell–mediated cytotoxicity in solid tumor models
Martín-Otal, C., Gómez‐Morón, Á., Martin-Cofreces, N. B., & Lozano, T. (2026) · Methods in cell biology · doi:10.1016/bs.mcb.2026.01.018
Flow cytometry evaluation of cytotoxicity exerted by effector immune cells against tumor cells
Ripollés-Yuba, C., Giraldos, D., Galano-Frutos, E., Reina-Ortiz, C., & Anel, A. (2026) · Methods in cell biology · doi:10.1016/bs.mcb.2026.02.008
Plying potency assays for immunotherapy of solid tumors
Burns, J. S., Gjeta, R., Lesný, P., & Vives, J. (2026) · Frontiers in Immunology · doi:10.3389/fimmu.2026.1770222
Lehner, E., Riedl, S. M., Haberl, F., Skvorc-Winkler, N., Müller, A. M. S., & Zajc, C. U. (2026) · Journal of Visualized Experiments · doi:10.3791/70162
Advances in CAR-T cell function monitoring: From preclinical evaluation to clinical application
Tang, Y., Dong, S., Zhang, Q., Qi, Y., & Chen, Z. (2026) · TrAC Trends in Analytical Chemistry · doi:10.1016/j.trac.2026.118862
Dennebos, R., Corrales, M. G., Lysandrou, M., Smit, N. A. M., Qi, Y., van Meerten, T., Kosterink, J. G. W., Huls, G. A., Lourens, H.-J., Gareb, B., et al. (2026) · International Journal of Pharmaceutics X · doi:10.1016/j.ijpx.2026.100559
Okafor, S. S., Montgomery, S. K., Park, J., Liu, T., Safrega, M., Yu, J. S., O'Hare, C. P., Schab, A. M., Goestenkors, A. P., Espinoza, C. J. V., et al. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.05.07.719019
CAR-Cytotox: A Standardized Framework to Quantify Cytotoxic Potency in CAR T Cell ProductsPreprint
Sr, L., Janžič, L., Auersperger, I., Reberšek, K., Furlan, T., Melkić, E., Zver, S., Sever, M., Ihan, A., & Kopitar, A. N. (2026) · Research Square · doi:10.21203/rs.3.rs-9287376/v1
Impedance-Based Analysis of Functional Immunoassays Using Cultured Cells
Michaelis, S., Grimm, A.-K., Erl, J., Schmauss, J., Friedrich, S., Naber, T., Anczykowski, B., & Wegener, J. (2026) · Springer eBooks · doi:10.1007/978-3-662-71782-0_25
Flow Cytometry in Cell and Gene Therapy:Principles, Practices, and Perspectives
Aguilera-Sandoval, C. R. (2026) · Cytotherapy · doi:10.1016/j.jcyt.2026.102982
Bock, T., Willis, K. A., Krivosic, N., Smith‐Bell, S. W., Kuksin, D., Franco Nitta, C., Pierce, M., Chan, L. L., Fiorenza, S., & Turtle, C. J. (2026) · Cytotherapy · doi:10.1016/j.jcyt.2026.102997
Wang, Y., Chen, H., Ko, B.-S., Xu, N., Dwarshuis, N. J., Wood, J., DeRose, P., Putta, S., Qian, Y., Quinn, J., et al. (2026) · Frontiers in Immunology · doi:10.3389/fimmu.2026.1946801
Rathore, A. S., Guillarme, D., Beck, A., Peruri, V., & Dubey, S. (2026) · TrAC Trends in Analytical Chemistry · doi:10.1016/j.trac.2026.119179
Lazzaro, S., Leroux-Roels, G. G., Janetzki, S., Salaun, B., Cook, L. E., Franke, K., Poschke, I. C., Bunse, L., Welters, M. J. P., Fehlings, M., et al. (2026) · Nature Protocols · doi:10.1038/s41596-026-01453-8 · Cited by 1
Yang, Z., Li, S., Wang, L., Li, Y., Xu, H., Li, Y., Liu, L., Yuan, Z., Zhang, Y., Zhang, L., et al. (2026) · Cell stem cell · doi:10.1016/j.stem.2026.09.006
Price, S. A., del Rio, A., Papadopoulos, G., Clingan, J. M., Yang, Y., Brackenbury, L. S., Sancerni, S. C., Lopez, D. C., de Munnik, S., Delsing, L., et al. (2026) · Cytotherapy · doi:10.1016/j.jcyt.2026.103053
Metadata
- Authors
- Kiesgen, S., Messinger, J. C., Chintala, N. K., Tano, Z., & Adusumilli, P. S.
- Affiliations
- Memorial Sloan Kettering Cancer Center (US)
- Corresponding author
- Adusumilli, Prasad S.
- Venue
- Nature Protocols
- Published
- 2021-02-15
- Volume
- 16
- Issue
- 3
- Pages
- 1331-1342
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC8064272
- OpenAlex
- W3132904428
- Cited by
- 138
- References
- 100
- Citations by year
- 2026: 24 · 2025: 37 · 2024: 32 · 2023: 22 · 2022: 18 · 2021: 5
- FWCI
- 2.33
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Cancer Institute (R01CA236615, R01CA235667, P30CA008748, T32CA009501)
- Topics
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Keywords
- chimeric antigen receptor T cells, T cell cytotoxicity, chromium release assay, bioluminescence assay, flow cytometry, potency assay, T cell exhaustion, T cell proliferation, combination immunotherapy, immunomodulatory antibodies, T cell receptor T cells
- MeSH terms
- Receptors, Chimeric Antigen, Animals, Cytotoxicity Tests, Immunologic, Flow Cytometry, Humans, Receptors, Antigen, T-Cell, T-Lymphocytes, Immunotherapy, Adoptive, Xenograft Model Antitumor Assays
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
