{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,14]],"date-time":"2026-03-14T00:55:50Z","timestamp":1773449750618,"version":"3.50.1"},"reference-count":16,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2021,10,23]],"date-time":"2021-10-23T00:00:00Z","timestamp":1634947200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J. Imaging"],"abstract":"<jats:p>The cytotoxic activity of T cells and Natural Killer cells is usually measured with the chromium release assay (CRA), which involves the use of 51Chromium (51Cr), a radioactive substance dangerous to the operator and expensive to handle and dismiss. The accuracy of the measurements depends on how well the target cells incorporate 51Cr during labelling which, in turn, depends on cellular division. Due to bystander metabolism, the target cells spontaneously release 51Cr, producing a high background noise. Alternative radioactive-free methods have been developed. Here, we compare a bioluminescence (BLI)-based and a carboxyfluorescein succinimidyl ester (CFSE)-based cytotoxicity assay to the standard radioactive CRA. In the first assay, the target cells stably express the enzyme luciferase, and vitality is measured by photon emission upon the addition of the substrate d-luciferin. In the second one, the target cells are labelled with CFSE, and the signal is detected by Flow Cytometry. We used these two protocols to measure cytotoxicity induced by treatment with NK cells. The cytotoxicity of NK cells was determined by adding increasing doses of human NK cells. The results obtained with the BLI method were consistent with those obtained with the CRA- or CFSE-based assays 4 hours after adding the NK cells. Most importantly, with the BLI assay, the kinetic of NK cells\u2019 killing was thoroughly traced with multiple time point measurements, in contrast with the single time point measurement the other two methods allow, which unveiled additional information on NK cell killing pathways.<\/jats:p>","DOI":"10.3390\/jimaging7110222","type":"journal-article","created":{"date-parts":[[2021,10,24]],"date-time":"2021-10-24T22:06:16Z","timestamp":1635113176000},"page":"222","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity"],"prefix":"10.3390","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5962-8642","authenticated-orcid":false,"given":"Claudia","family":"Coronnello","sequence":"first","affiliation":[{"name":"Fondazione Ri.MED, 90133 Palermo, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7546-7209","authenticated-orcid":false,"given":"Rosalia","family":"Bus\u00e0","sequence":"additional","affiliation":[{"name":"Department of Research, I.R.C.C.S.-ISMETT, 90127 Palermo, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luca","family":"Cicero","sequence":"additional","affiliation":[{"name":"Istituto Zooprofilattico Sicilia, IZS, 90127 Palermo, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9290-6103","authenticated-orcid":false,"given":"Albert","family":"Comelli","sequence":"additional","affiliation":[{"name":"Fondazione Ri.MED, 90133 Palermo, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0154-6270","authenticated-orcid":false,"given":"Ester","family":"Badami","sequence":"additional","affiliation":[{"name":"Fondazione Ri.MED, 90133 Palermo, Italy"},{"name":"Department of Research, I.R.C.C.S.-ISMETT, 90127 Palermo, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,10,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1906","DOI":"10.3389\/fimmu.2019.01906","article-title":"CD8(+) T Lymphocyte and NK Cell Network: Circuitry in the Cytotoxic Domain of Immunity","volume":"10","author":"Uzhachenko","year":"2019","journal-title":"Front. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/0022-1759(77)90163-6","article-title":"A simplified isotope release assay for cell-mediated cytotoxicity against anchorage dependent target cells","volume":"18","author":"Timonen","year":"1977","journal-title":"J. Immunol. Methods"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"159","DOI":"10.2220\/biomedres.32.159","article-title":"One-step simple assay to determine antigen-specific cytotoxic activities by single-color flow cytometry","volume":"32","author":"Nakagawa","year":"2011","journal-title":"Biomed. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1126\/science.7123245","article-title":"Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication","volume":"218","author":"Gratzner","year":"1982","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/S1046-2023(03)00123-3","article-title":"The flow cytometric PKH-26 assay for the determination of T-cell mediated cytotoxic activity","volume":"31","author":"Fischer","year":"2003","journal-title":"Methods"},{"key":"ref_6","first-page":"9","article-title":"Amine-reactive dyes for dead cell discrimination in fixed samples","volume":"9","author":"Perfetto","year":"2010","journal-title":"Curr. Protoc. Cytom."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2049","DOI":"10.1038\/nprot.2007.296","article-title":"Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester","volume":"2","author":"Quah","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1053","DOI":"10.1097\/CAD.0b013e32825209f6","article-title":"5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells","volume":"18","author":"Masterson","year":"2007","journal-title":"Anticancer Drugs"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1002\/bit.20059","article-title":"Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells","volume":"86","author":"Ignowski","year":"2004","journal-title":"Biotechnol. Bioeng."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1513\/pats.200507-067DS","article-title":"Bioluminescence imaging","volume":"2","author":"Sadikot","year":"2005","journal-title":"Proc. Am. Thorac. Soc."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Karimi, M.A., Lee, E., Bachmann, M.H., Salicioni, A.M., Behrens, E.M., Kambayashi, T., and Baldwin, C.L. (2014). Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0089357"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2421","DOI":"10.1182\/blood.V76.12.2421.2421","article-title":"Biology and clinical relevance of human natural killer cells","volume":"76","author":"Robertson","year":"1990","journal-title":"Blood"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1038\/nprot.2006.458","article-title":"Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D","volume":"2","author":"Schmid","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"471408","DOI":"10.1155\/2010\/471408","article-title":"Timing of imaging after d-luciferin injection affects the longitudinal assessment of tumor growth using in vivo bioluminescence imaging","volume":"2010","author":"Inoue","year":"2010","journal-title":"Int. J. Biomed. Imaging"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e1","DOI":"10.1182\/blood-2015-03-632273","article-title":"Live cell evaluation of granzyme delivery and death receptor signaling in tumor cells targeted by human natural killer cells","volume":"126","author":"Vrazo","year":"2015","journal-title":"Blood"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1038\/nri3839","article-title":"Perforin and granzymes: Function, dysfunction and human pathology","volume":"15","author":"Voskoboinik","year":"2015","journal-title":"Nat. Rev. Immunol."}],"container-title":["Journal of Imaging"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2313-433X\/7\/11\/222\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:22:06Z","timestamp":1760167326000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2313-433X\/7\/11\/222"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,23]]},"references-count":16,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2021,11]]}},"alternative-id":["jimaging7110222"],"URL":"https:\/\/doi.org\/10.3390\/jimaging7110222","relation":{},"ISSN":["2313-433X"],"issn-type":[{"value":"2313-433X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,10,23]]}}}