Anti cd107a mab h4a3

Human PBMC were stained using directly conjugated mAb against human CD3 (BW264/56), CD56 (REA196), CD57 (TB03), from Miltenyi Biotec (San Diego, CA, USA), Tim-3 (F38-2E2) from BioLegend and NKG2C (134591) from R&D Systems. Cells were stimulated with 1 μg anti-CD16 mAb (3G8; BioLegend) per 10⁶ PBMC and prepared for intracellular staining by adding brefeldin A (Sigma-Aldrich) 1 h after the start of incubation to a final concentration of 10 μg/mL and continuing the incubation for an additional 4 h. NK cell degranulation was detected by introducing directly conjugated anti-CD107a mAb (H4A3; BioLegend) at a 0.25 μg per 10⁶ PBMC at the time of brefeldin A addition. Cells were fixed and permeabilized after 5 h incubation using the Inside Stain Kit (Miltenyi Biotec) as per manufacturer's instructions and then stained with directly conjugated polyclonal Ab against human FcRγ from MilliporeSigma (Burlington, MA, USA) and anti-human IFN-γ mAb (4S.B3) from eBioscience (San Diego, CA, USA). Non-viable cells were excluded by fixable live/dead stain (Invitrogen) as per manufacturer's instructions. Data were acquired using a MoFlo Astrios EQ flow cytometer and data analyses and illustration performed using Kaluza software (both Beckman Coulter, Brea, CA, USA).