Disruption of lipid raft microdomains inhibits NK cell IFN-γ production in response to immobilized IgG and IL-12. (A) Purified human NK cells from a healthy donor were pretreated with MβCD (a cholesterol chelator) and then plated onto flat-bottom wells that were precoated overnight with 100 μg/mL human IgG (ie, immobilized Ab) in medium containing 10 ng/mL IL-12. Control conditions consisted of NK cells cultured with medium alone (Medium), immobilized IgG alone (IgG), or IL-12 alone (IL-12). NK cells were cultured with brefeldin-A for 12 hours and analyzed for IFN-γ production by intracellular flow cytometry. In another arm of the same experiment, NK cells that had been pretreated with MβCD were reconstituted with cholesterol prior to use in the immobilized IgG assay. The percentage of NK cells actively producing IFN-γ is shown within each dot plot. Results from a representative donor are shown (n = 4 donors tested). (B) NK cells pretreated with MβCD were used in an immobilized IgG assay with 10 ng/mL IL-12, as described in panel A. Cell culture supernatants were harvested after 24 hours and analyzed for IFN-γ content by ELISA. Results depict the mean plus or minus SEM of 4 donors tested. *P < .005 versus mock pretreatment (Mock Pre-Tx) control for the same stimulation condition.