Cell-mediated cytotoxicity measurements can be divided in two methods, depending on whether radioactive material is used or not. Natural Killer cell-mediated cytotoxicity is routinely measured with a short-term assay by labeling the target cells with radioactive chromium (51Cr). The advantages of this method are (1) highly sensitivity, (2) easy execution, (3) low spontaneous release, and (4) nontoxic markers. The disadvantages include short half-life of the label and handling and disposal of radioactive supplies. Many attempts have been made to adapt this cytotoxicity assay to abolish radioactivity while maintaining its high sensitivity. Consequently, various nonradioactive methods have been developed. One of these methods utilizing the release of enzymes (LDH) as a result of cytolysis or membrane dyes (PKH-26) is usually less sensitive than radioactive assays. MTT-based colorimetric/enzymatic assays are highly sensitive and easy to use but unfortunately works only with adherent tumor cell targets. Fluorescent dyes such as carboxy-fluorescein diacetate can easily accumulate in the cytoplasm of effector or target cells. After cytotoxicity, the release of the dyes into the supernatant or their retention in target cells is calculated. However, spontaneous release of these dyes can be quite high, causing false positive results leading to a decreased sensitivity and restricting their use in short-term assays. More recently, flow cytometric methods using fluorescent monoclonal antibodies such as anti-CD56 for effector and anti-CD33 for target cells have been defined.
1. Zamai L, Ahmad M, Bennett IM, Azzoni L, Alnemri ES, Perussia B. Natural killer (NK) cell-mediated cytotoxicity: Differential use of TRAIL and Fas ligand by immature and mature primary human NK cells. J Exp Med 1998;188(12):2375-80.
2. Sieni E, Cetica V, Mastrodicasa E, Pende D, Moretta L, Griffiths G, et al. Familial hemophagocytic lymphohistiocytosis: A model for understanding the human machinery of cellular cytotoxicity. Cell Mol Life Sci 2012;69(1):29-40.
3. Suck G, Branch DR, Smyth MJ, Miller RG, Vergidis J, Fahim S, et al. KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity. Exp Hematol 2005;33(10):1160-71.
4. Valiathan R, Lewis JE, Melillo AB, Leonard S, Ali KH, Asthana D. Evaluation of a flow cytometry-based assay for natural killer cell activity in clinical settings. Scand J Immunol 2012;75(4):455- 62.
5. Goldberg JE, Sherwood SW, Clayberger C. A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry. J Immunol Methods 1999; 224(1-2):1-9.
6. Ozdemir O, Savaşan S. Combinational IL-2/IL-15 induction does not further enhance IL-15-induced lymphokine-activated killer cell cytotoxicity against human leukemia/lymphoma cells. Clin Immunol 2005;115(3):240-9.
7. Zaritskaya L, Shurin MR, Sayers TJ, Malyguine AM. New flow cytometric assays for monitoring cell-mediated cytotoxicity. Expert Rev Vaccines 2010;9(6):601-16.
8. Kasatori N, Ishikawa F, Ueyama M, Urayama T. A differential assay of NK-cell-mediated cytotoxicity in K562 cells revealing three sequential membrane impairment steps using three-color flow-cytometry. J Immunol Methods 2005;307(1-2):41-53.
9. Ozdemir O, Ravindranath Y, Savaşan S. Cell-mediated cytotoxicity evaluation using monoclonal antibody staining for target or effector cells with annexinV/propidium iodide colabeling by fluorosphere-adjusted counts on three color flow cytometry. Cytometry A 2003;56(1):53-60.
10. Brunner KT, Mauel J, Cerottini JC, Chapuis B. Quantitative assay of the lytic action of immune lymphoid cells on 51-Crlabelled allogeneic target cells in vitro; Inhibition by isoantibody and by drugs. Immunology 1968;14(2):181-96.
11. King MA. Detection of dead cells and measurement of cell killing by flow cytometry. J Immunol Methods 2000;243:155-66.
12. Roder JC, Haliotis T, Klein M, Korec S, Jett JR, Ortaldo J, et al. A new immunodeficiency disorder in humans involving NK cells. Nature 1980;284(5756):553-5.
13. Morales A, Ottenhof PC. Clinical application of a whole blood assay for human natural killer (NK) cell activity. Cancer 1983;52(4):667-70.
14. Matzinger P. The JAM test: A simple assay for DNA fragmentation and cell death. J Immunol Methods 1991;145:185- 92.
15. Langhans B, Ahrendt M, Nattermann J, Sauerbruch T, Spengler U. Comparative study of NK cell-mediated cytotoxicity using radioactive and flow cytometric cytotoxicity assays. J Immunol Methods 2005;306(1-2):161-8.
16. Korzeniewski C, Callewaert DM. An enzyme-release assay for natural cytotoxicity. J Immunol Methods 1983;64(3):313-20.
17. Weidmann E, Brieger J, Jahn B, Hoelzer D, Bergmann L, Mitrou PS. Lactate dehydrogenase-release assay: A reliable, nonradioactive technique for analysis of cytotoxic lymphocytemediated lytic activity against blasts from acute myelocytic leukemia. Ann Hematol 1995;70(3):153-8.
18. van de Loosdrecht AA, Nennie E, Ossenkoppele GJ, Beelen RH, Langenhuijsen MM. Cell mediated cytotoxicity against U 937 cells by human monocytes and macrophages in a modified colorimetric MTT assay. A methodological study. J Immunol Methods 1991;141(1):15-22.
19. van de Loosdrecht AA, Beelen RH, Ossenkoppele GJ, Broekhoven MG, Langenhuijsen MM. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Methods 1994;174(1-2):311-20.
20. Ewen C, Kane KP, Shostak I, Griebel PJ, Bertram EM, Watts TH, et al. A novel cytotoxicity assay to evaluate antigen-specific CTL responses using a colorimetric substrate for Granzyme B. J Immunol Methods 2003;276(1-2):89-101.
21. Ozdemir O. Evaluation of human mast cell-mediated cytotoxicity by DIOC18 target cell labeling in flow cytometry. J Immunol Methods 2007;319(1-2):98-103.
22. Fischer K, Mackensen A. The flow cytometric PKH-26 assay for the determination of T-cell mediated cytotoxic activity. Methods 2003;31(2):135-42.
23. Hopkinson K, Williams EA, Fairburn B, Forster S, Flower DJ, Saxton JM, et al. A MitoTracker Green-based flow cytometric assay for natural killer cell activity: Variability, the influence of platelets and a comparison of analytical approaches. Exp Hematol 2007;35(3):350-7.
24. von Zons P, Crowley-Nowick P, Friberg D, Bell M, Koldovsky U, Whiteside TL. Comparison of europium and chromium release assays: Cytotoxicity in healthy individuals and patients with cervical carcinoma. Clin Diagn Lab Immunol 1997;4(2):202-7.
25. Lecoeur H, Février M, Garcia S, Rivière Y, Gougeon ML. A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity. J Immunol Methods 2001;253(1-2):177-87.
26. Lee-MacAry AE, Ross EL, Davies D, Laylor R, Honeychurch J, Glennie MJ, et al. Development of a novel flow cytometric cellmediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide. J Immunol Methods 2001;252(1-2):83-92.
27. Jang YY, Cho D, Kim SK, Shin DJ, Park MH, Lee JJ, et al. An improved flow cytometry-based natural killer cytotoxicity assay involving calcein AM staining of effector cells. Ann Clin Lab Sci 2012;42(1):42-9.
28. Thakur A, Zaman A, Hummel J, Jones K, Hortelano G. Singlecolour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells. Biotechnol Lett 2012;34(3):447-53.
29. Cholujová D, Jakubíková J, Kubes M, Arendacká B, Sapák M, Ihnatko R, et al. Comparative study of four fluorescent probes for evaluation of natural killer cell cytotoxicity assays. Immunobiology 2008;213(8):629-40.
30. Zimmermann SY, Esser R, Rohrbach E, Klingebiel T, Koehl U. A novel four-colour flow cytometric assay to determine natural killer cell or T-cell-mediated cellular cytotoxicity against leukaemic cells in peripheral or bone marrow specimens containing greater than 20% of normal cells. J Immunol Methods 2005;296(1-2):63-76.
31. Ozdemir O. Flow cytometric mast cell-mediated cytotoxicity assay: A three-color flow cytometric approach using monoclonal antibody staining with annexin V/propidium iodide co-labeling to assess human mast cell-mediated cytotoxicity by fluorosphereadjusted counts. J Immunol Methods 2011;365(1-2):166-73.
32. Savaşan S, Buck S, Ozdemir O, Hamre M, Asselin B, Pullen J, et al. Evaluation of cytotoxicity by flow cytometric drug sensitivity assay in childhood T-cell acute lymphoblastic leukemia. Leuk Lymphoma 2005;46(6):833-40.
33. Ozdemir O. Flow cytometric cell-mediated cytotoxicity assay. J Immunol Methods 2007;318(1-2):158-9; author reply 160-1.
34. Godoy-Ramirez K, Franck K, Gaines H. A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multiparameter flow cytometry. J Immunol Methods 2000;239(1-2): 35-44.
35. Kim GG, Donnenberg VS, Donnenberg AD, Gooding W, Whiteside TL. A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4h 51Cr-release assay. J Immunol Methods 2007;325(1-2):51-66.
36. Johann S, Blümel G, Lipp M, Förster R. A versatile flow cytometry-based assay for the determination of short- and long-term natural killer cell activity. J Immunol Methods 1995;185(2):209-16.
37. Shabrish S, Gupta M, Madkaikar M. A modified NK cell degranulation assay applicable for routine evaluation of NK cell function. J Immunol Res 2016;2016:3769590.
38. Mace EM. Requirements for human natural killer cell development informed by primary immunodeficiency. Curr Opin Allergy Clin Immunol 2016;16(6):541-8.
39. Chahroudi A, Silvestri G, Feinberg MB. Measuring T cellmediated cytotoxicity using fluorogenic caspase substrates. Methods 2003;31(2):120-6.
40. La Muraglia GM, O’Neil MJ, Madariaga ML, Michel SG, Mordecai KS, Allan JS, et al. A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry. J Immunol Methods 2015;427: 85-93.
41. Somanchi SS, McCulley KJ, Somanchi A, Chan LL, Lee DA. A novel method for assessment of natural killer cell cytotoxicity using image cytometry. PLoS One 2015;10(10):e0141074.
42. Jin Q, Jiang L, Chen Q, Li X, Xu Y, Sun X, Zhao Z, Wei L. Rapid flow cytometry-based assay for the evaluation of γδ T cellmediated cytotoxicity. Mol Med Rep 2018;17(3): 3555-62.
43. Kandarian F, Sunga GM, Arango-Saenz D, Rossetti M. A flow cytometry-based cytotoxicity assay for the assessment of human NK cell activity. J Vis Exp 2017;(126).