Mitochondrial membrane potential regulates cytokine production in cytokine-activated NK cells. (A-C) Mitochondrial mass was assessed by MitoTracker green and membrane potential (Δψm) by TMRM incorporation in freshly sorted NKBr and NKDim cells (empty bars) and cultured for 18 hours in presence of IL-15 ± IL-12/18 (filled bars). (C) TMRM/Mitotracker ratio (data are mean ± SEM; data summarized from 4 independent experiments with 2 donors each). (D) Primed or activated NKBr and NKDim cells were cultured for 18 hours with the following inhibitors: 10 μM of RO, 10 μM of AA, and 2 nM of oligomycin A. Expression of IFN-γ and percentage of cells producing IFN-γ were monitored by fluorescence-activated cell sorting (FACS; data from 3 independent experiments with at least 2 donors each). (E) TMRM+ and TMRM− NKBr and NKDim cells were sorted and cultured with IL-15 or IL-15/12/18 for 18 hours. Expression of IFN-γ and percentage of positive cells in the different conditions were measured by FACS (data from 4 HDs). P > .05 was considered not significant (ns). *P < .05, **P < .01, ***P < .001, ****P < .0001 using 1-way analysis of variance with Tukey’s correction.