Phenotypic and functional characterization of human blood NK cells. (A-E) NK cells were identified using a CD45⁺ lymphocyte gate, doublets were excluded by FSC-H versus FSC-A gating, and dead cells were excluded via the use of a viability dye. NK cells were defined as CD56⁺CD3⁻. (A) The frequency of CD56^bright NK cells was determined by plotting CD56 against CD16 and identifying CD56^bright CD16^dim cells. (B-E) The analysis was performed on total NK cells with no discrimination between CD56^bright and CD56^dim, with positive gates set using fluorescence minus 1 with isotype controls. (B) Percentage of NKG2C⁺ NK cells. (C) Percentage of DNAM1⁺ NK cells. (D) Percentage of CD94⁺ NK cells. (E) Percentage of CXCR3⁺ NK cells. (F) Degranulation of NK cells from control NPC1^+/+ and affected NPC1^−/− blood samples in response to K562 cells in the presence of monensin was calculated according to the following equation: (% of CD107a⁺ NK cells with stimuli) − (% of CD107a⁺ unstimulated NK cells). (G) Intracellular production of IFN-γ in NK cells from control NPC1^+/+ and affected NPC1^−/− blood samples in response to IL-12 and IL-18 simulation was calculated according to the following equation: (% of IFN-γ⁺ NK cells with stimuli) − (% of IFN-γ⁺ unstimulated NK cells). Circles represent control NPC1^+/+, diamonds represent NPC1^+/− carriers, and squares represent affected NPC1^−/− blood samples. Each symbol indicates an individual sample with the line at the median; *P < .05 and **P < .01 calculated by the Mann-Whitney U test, using GraphPad Prism v4. het, heterozygous carriers.