Anti nkg2c pe clone 134591

Cells were stained in 96-well round-bottom plates, as described elsewhere [24 (link)]. Briefly, cells were blocked with FcR Blocking Reagent (Miltenyi Biotech) and stained with fluorophore-labeled antibodies for surface markers, including viability marker (Fixable Viability Stain 700; BD Biosciences) in FACS buffer. Cells were washed in FACS buffer, fixed, and permeabilized using Cytofix/Cytoperm Kit (BD Biosciences). Cells were then stained for intracellular markers with further FcR blocking, washed again, resuspended in FACS buffer, acquired using a BD LSRII flow cytometer and FACSDiva software, and analyzed using FlowJo V10 (Tree Star). FACS gates were set using unstimulated cells or Fluorescence Minus One (FMO) controls, a minimum cutoff was determined as the frequency of responding NK cells in the presence of FCS alone [21 (link)], and samples with <100 NK cell events were excluded from the analysis.
The fluorophore-labeled antibodies used were anti-CD3-V500 (clone UCHT1) (BD Biosciences), anti-CD56-BV605 (clone HCD56), anti-IFN-γ-BV785 (clone 45.B3) (Biolegendanti-CD16-APC (clone CB16), anti-CD57-e450 (clone TB01) (eBiosciences), and anti-NKG2C-PE (clone 134591) (R&D Systems).