Table 5. Applications of cell-mediated cytotoxicity assays.
Listed are examples of published assays to analyze cytotoxic effects mediated by different effector cells, illustrating the various uses of the flow cytometry assay.
| Flow cytometry assay | ||||||
|---|---|---|---|---|---|---|
| Application | Effector cells/drug | Target cells | Staining | E:T ratio(s) | Incubation time (h) | Reference |
| T cell– mediated cytotoxicity | NY-ESO–specific T cells | Jurkat/A2K | CFSE, CMTMR, propidium iodide | 1.5:1 to 0.1:1 | 4 | 97 |
| CD8+ T cells | DoHH2, Karpas, Raji | CFSE, 7-AAD | 50:1 to 2.5:1 | 24 | 98 | |
| γδ T cells | THP-1 | CFSE, propidium iodide | 20:1 to 1:1 | 4 | 39 | |
| NK cell– mediated cytotoxicity | NK cells | K562 | CellTrace Violet Dye | 1:1 | 5 | 45 |
| NK cells | K562 | Annexin V, propidium iodide, BCECF | 1:1 to 1:10 | 1 to 3 | 99 | |
| NK cells | K562 | DiO, propidium iodide | 75:1 to 6.25:1 | 3 | 44 | |
| NK cells | K562 | DIOC18, 7-AAD | 20:1 to 1:1 | 4 | 100 | |
| Macrophage-mediated cytotoxicity | U937 | IGROV1 | CFSE, propidium iodide, CD89-phycoerythrin | 2:1 to 1:1 | 2.5 | 101 |
| Oncolytic virus–mediated cytotoxicity | PBMCs, adenovirus expressing BiTE (EGFR×CD3) | A431, A549 | GFP, 7-AAD | MOI = 20, E:T = 6:1 | 5 days | 95 |
| BiTE-mediated cytotoxicity | CD3+ T cells enriched from PBMCs, BiTE (CEA×CD3) | CHO, ASPC-1, BxPC3, HPAC, HPAF II, H727, LS174T, HT-29, MKN45, Pan0813, A549, PC3, BT474 | Vybrant DiO, propidium iodide | 10:1 to 1:2 | Up to 72 | 102 |
| ADCC | PBMCs, NK92MI, trastuzumab | BT-474, MCF-7, Hs578T | CFSE, FVD/7-AAD/propidium iodide | 1:1 to 1:20 | Overnight | 103 |
7-AAD, 7-amino-actinomycin D; ADCC, antibody-dependent cellular cytotoxicity; BCECF, 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein; BiTE, bispecific T-cell engager; CFSE, carboxyfluorescein succinimidyl ester; CMTMR, 5- and 6-([(4-chloromethyl)benzoyl]-amino) tetramethylrhodamine; E:T, effector to target; FVD, fixed viability dye; GFP, green fluorescent protein; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell; NK, natural killer.