Demonstration of a Single-Parameter, Rapid Flow Cytometry Assay in Assessing Natural Killer Cell Cytotoxic Function

Assessing cytotoxic function of immune effector cells, such as natural killer (NK) cells, is necessary to determine their potency. Various cytotoxicity assays measuring target cell death have been reported, however these assays typically feature a number of limitations that curb their broad applicability. Quantitative assays of NK cell killing traditionally measure the release of chromium or a fluorescent dye from labelled target cells. These assays typically show low reproducibility and often underestimate cell death for many cell types. Recent strategies aimed at ensuring manufacture of high NK cell doses commonly include NK cell co-culture with antigen presenting cells as feeder cells for their expansion. Such expansion platforms result in a final NK cell product with a high content of dead or dying feeder cells. This end product also complicates the accurate measurement of target cell death in NK cell cytotoxicity assays. Here we report a rapid flow cytometry assay for quantifying target cell killing after single-color labeling of the target cells prior to co-incubation with NK cells. In this assay, target cells are first labeled with calcein-AM (CAM), a green fluorescent dye. CAM-labeled target cells are then co-incubated with feeder cell expanded-NK cells in 96-well plates using various NK cell-to-target ratios. After 4 hours of NK cell-target cell co-incubation, a fixed volume of the cell suspension was analyzed using a flow cytometer capable of reading 96-well plates. The live, CAM-labeled target cells were gated and quantitated for all the treatment conditions to determine the number of CAM-labeled target cells present per fixed volume.

Using this assay, we observed highly consistent cytotoxicity results against both suspension and adherent cell targets. The accuracy of this assay was also demonstrated by NK cell dose-dependent responses. Importantly, the presence of dead feeder cells in the NK cell suspension does not interfere with the analysis of this assay. In addition, the gating strategy is simple, consistent, and the analysis between treatment groups does not require cumbersome calculations or compensation that could introduce variability. Assessment of target cell death was also shown to be more reliable and consistent when compared with the measurement of CAM release using a microplate reader. The latter assay has previously been shown to be highly variable with inconsistent results depending on target cell and CAM dilution. Our flow cytometry assay consistently identifies live cells, avoiding errors due to variability in dye intake and dye release detection.