Figure 1 shows typical results for NK assays. The dot plot shows a clear distinction between live and dead target cells, and a clear separation between effector and target cells. In this example, purified NK effectors mixed with painted K562 cells at a 1:1 ratio resulted in approximately 50% killing of the target cells.
Purified NK cells and CFSE-painted K562 cells were mixed at various effector-to-target ratios, incubated, and stained with 7-AAD. Results (data not shown) from the Guava Cell Toxicity Assay run on Guava PCA-96 and Guava EasyCyte Systems were comparable to results obtained with other similar assays and/or flow cytometry systems.
Daudi cells are known to be more resistant to NK activity than K562 cells. Figure 2 illustrates that the Guava Cell Toxicity Assay accurately reflects that difference in NK susceptibility, showing that many more K562 than Daudi cells were killed. Moreover, results were highly reproducible on two different Guava PCA-96 Systems.
Different mixtures of effector-to-target ratios were prepared using NK and K562 cells in 96-well plates. No bias of results over time or well location were observed, and the percentages of target cells killed for all samples were consistent over the time needed to acquire an entire plate. The %CVs for percent of target cells killed were under 10% at effector-to-target ratios that resulted in killing above background. Also, the %CVs for the MFIs were typically under 10%, showing that the populations stayed consistent over the plate.
Painted K562 cells were mixed with purified NK cells at various effector-to-target ratios and acquired at different times. The percent target cells killed and MFI values were similar for up to 24 hours for all ratios tested, indicating that samples kept at room temperature were stable up to 24 hours post-incubation.
Painted K562 cells were mixed with purified NK cells at various effector-to-target ratios and incubated in various vessels (i.e., microcentrifuge tubes and round- and flat-bottom 96-well plates). The most optimal NK activity was observed in round-bottom 96-well plates (data not shown). K562 cells were painted and mixed with purified NK cells at both a constant cell concentration and constant target concentrations, stained following the above protocol, and run on a Guava PCA-96 System. Identical results were obtained regardless of whether a constant total cell concentration or a constant target cell concentration was used.
Allogenically stimulated effector PBMCs were mixed with target cells from the same donor at various ratios and run on a Guava PCA-96 System. Figure 3 shows that the Guava Cell Toxicity Assay detected CMC, but, as expected, higher effector-to-target ratios were necessary than for the NK experiments.
IL-2 is known to induce a dose-dependent increase in NK activity. Painted K562 cells were mixed with purified NK cells and IL-2 was added at increasing concentrations. Results demonstrated that even at the lowest concentration, IL-2 caused a large increase in NK-mediated killing.