Biosensors are activated after direct NK cell recognition of tumor target cells. Caspase and granzyme biosensors are activated in tumor target cells after NK effector conjugation. NK92 cells were conjugated at 1:1, with each target cell line expressing biosensors, and RLU was measured every 3 minutes for 240 minutes. The mean datum point at every 6 minutes is plotted. Fold activation in signal is shown for (A) Jurkat cells and (B) K562 cells. For inhibitor studies, K562 cells were preincubated with Q-VD-OPH (10 μM) or diluent, and NK92 cells were preincubated with C20 (50 μM); then fold activation in signal was measured for: (C) K562-GLS.VGPD and (D) K562-GLS.DEVD. (E) NK92 cells were conjugated at 3:1 with a K562 clonal line (GLS.SGR.5) expressing the GrzA/K sensor with diluent (phosphate-buffered saline [PBS]) or Mg²⁺-EGTA in PBS (4 mM). (F) To test specificity of GLS.SGR, K562 cells were preincubated with Q-VD-OPH (10 μM) or diluent, and NK92 cells were preincubated with C20 (50 μM), nafamostat mesylate (NM, 3.5 μM) or diluent, before conjugation at 3:1. Only NM inhibited GLS.SGR activation (P < .0001). The fold change in signal from 3 independent reads at 96 minutes is indicated. Data are represented as mean ± standard deviation (SD). ***P < .001; ns, not significant by one-way ANOVA. Shown is a representative figure from at least 3 independent experiments.