Anti cd107a alexa fluor 647 antibody

Human peripheral blood mononuclear cells (PBMC) were isolated from the blood of 14 healthy donors using Hypaque-Ficoll (GE Healthcare, Amersham, UK) density centrifugation, with informed consent and full ethical approval (REC ref 06/Q1701/120). 3 × 10⁵ PBMCs were stimulated overnight with 1 ng/mL recombinant human IL-15. Peptide pulsed 721.174 targets were prepared as for the stabilization assays. Target cells were resuspended with PBMCs at an effector-to-target (E:T) ratio of 5:1 in fresh R10 medium containing peptide and anti-CD107a-Alexa-fluor-647 antibody (eBioscience, Hatfield, UK). Cells were incubated for 1 h at 26°C, then 6 μg/mL Golgi-Stop^™ (BD Biosciences) was added, and incubated for a further 3 h at 26°C. Cells were washed in wash buffer (1% BSA/0.1% NaN₃) and blocked with blocking buffer (10% human serum in PBS) for 30 min and then stained with the following antibodies: anti-CD3-PerCP (Biolegend, San Diego, US), anti-human CD56-PE and anti-human CD158b-FITC (both BD Biosciences). Cells were fixed in 1% PFA and analyzed by flow cytometry. Individual assays for each donor were performed once in duplicate and the mean value used for subsequent analysis.