Multiparametric Analysis of NK Cell Responses

To determine NK cell responses in blood, spleen and lung single-cell suspensions were stained as previously described, with minor modifications.^{71 (link),72 (link)} We incubated single-cell suspensions in 1.5 mL Eppendorf tubes for 2 h at 37°C in a medium containing brefeldin A. Cells were suspended in buffer and incubated with anti-CD16/CD32 mAb for 30 min at room temperature. The cells were then stained for surface markers, fixed, permeabilized, and intracellularly stained for cytokines, according to the manufacturer’s instructions. The antibodies and reagents used for flow cytometry were as follows: PE-anti-mouse CD49b (Cat. #108907), FITC-anti-mouse CD3 (Cat. #100204), PE-anti-mouse-CD8a (Cat. #100707), APC-anti-mouse-CD4 (Cat. #100412), anti-mouse CD16/32 (Cat. #101320), APC-anti-mouse-CD107a (Cat. #121613), APC anti-mouse FceRla (Cat. #134316) and PE/Cyanine7-anti-mouse IFN-γ (Cat. #505825, BioLegend). Stained cells were analyzed by flow cytometry using a Cytoflex-LX flow cytometer (Bekeman). Data were analyzed using FlowJo software (Tree Star).