Cd158b ch l

Anti-human CD3 (OKT3), CD56 (MEM188), CD11a (HI111), ICAM-1 (HA58), NKG2D (5C6), CD2 (RPA-2.10), CD25 (BC96), Perforin (dG9), Granzyme B (GB11), CD16 (CB16), CD69 (FN50), and FAS (15A7) mAbs were purchased from eBioscience (San Diego, CA, USA). Anti-human 2B4 (clone 2–69), NKp44 (P44-8.1), CD122 (Mik-β3), CD132 (AG184), NKp30 (P30-15), NKp46 (9E2), DNAM-1 (DX11), KIR-NKAT2 (DX27), CD94 (HP-3D9), NKB1 (DX9), CD158a (HP-3E4), and CD158b (CH-L) mAbs were purchased from BD Pharmingen (San Jose, CA, USA). PE-conjugated anti-human HIF-1α (546-16) were purchased from BioLegend (San Diego, CA, USA). For surface staining, cells were stained with the indicated PE-, FITC-, APC-, or PerCP-conjugated mAbs in 100 μL of FACS buffer (BD Biosciences, Franklin Lakes, NJ, USA). To detect NKp44L, target cells were incubated with 20 μg/mL of NKp44-Ig fusion proteins (R&D Systems, Minneapolis, MN, USA). Cells were then incubated with Fcγ-fragment-specific conjugated affinity-purified F(ab′)₂ fragments of goat anti-human IgG (1/50; Jackson ImmunoResearch, West Grove, PA, USA). Flow cytometry was performed with a FACSCantoΙΙ (BD Biosciences) and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).