Nkg2c pe

Peripheral blood mononuclear cells (PBMCs) from 47 healthy controls and 151 MS patients were isolated from blood samples collected in EDTA tubes using Ficoll–Hypaque density gradient centrifugation and cryopreserved in fetal calf serum with 10% dimethyl sulfoxide until analysis. Sample staining for flow cytometry analysis was performed using anti-NKG2A (clone Z199) indirectly labeled with a secondary goat anti-mouse PE-Cy7 (Biolegend) and the following fluorochrome-conjugated antibodies: anti-CD3-PerCP (BD Pharmigen), anti-CD56-APC (Biolegend), antiCD16-eFluor450 (eBioscience), and NKG2C-PE (R&D Systems). For the analysis of FcRγ (MS, n = 139; controls n = 47) and PLZF expression (MS, n = 86; controls n = 26), cells were treated with a fixation/permeabilization kit (BD Biosciences) followed by incubation with anti-FcRγ-FITC (Millipore) and anti-PLZF-PE CF594 (BD Biosciences). Samples were acquired in LSRFortessa (BD Biosciences) and data were analyzed using FlowJo software (Tree Star, Oregon, USA), using the gating strategy shown in Figure 1.

The following antibodies were used: Biolegend (NKG2D-APC/Cy7, NKp46-FITC, NKp30-PE, CD158-FITC, CXCR6-PE, CD54-FITC), Novus Biologics (c-myc- BB421), BD Biosciences (NKP44-BB515, CD57-BV421, DNAM-1-PE, 2B4-BV421, Ki67- BB786, p-STAT3-Percp-cy5.5) and R&D systems (NKG2C-PE, NKG2A-alexa-488).
Intracellular staining for the phenotyping assay was done using the “Transcription Factor Phospho Buffer Set” (BD Biosciences). Instructions were followed according to protocol specified by the manufacturer. For all stains, 50–200 μL of cells at a concentration of 2.5 × 10⁶ cells/mL were resuspended in a 96 well V-bottom plate. First, cell surface makers were stained at optimized concentrations of fluorescent antibodies in PBS (3% FBS) at room temperature in the dark, for 20–30 min. Second, cells were fixed using 100 μL fix/perm solution for 40 min at 4 °C. Third, pre-cooled (−20 °C) Perm III buffer was added to the cells and cells were incubated at 4 °C for 15 min. Finally, the cells were stained with intracellular antibodies in perm/wash buffer at 4 °C for 40 min. 2 washes with perm/wash buffer was performed after each step. The cells were resuspended in 3% FBS/PBS at the end of the stain and data was acquired using the BD Fortessa. The analyses were done using FlowJo.

Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stored in liquid nitrogen. NK cell purification was performed with the Dynabeads Untouched Human NK cells kit (Life Technologies, Paisley, UK). Antibodies used were: CD56-PECy7, CD3-PerCP, CD16-APC-Cy7, CD57-Pacific Blue (all BioLegend, San Diego, USA), CD56-fluorescein isothiocyanate (FITC) (BD Biosciences, Oxford, UK), CD3-Pacific Blue (eBioscience, Hatfield, UK), CD158a,h-PE, CD158b-FITC, CD158b1/b2,j-PE (all Beckman Coulter, Marseille, France), NKp30-APC, NKp46-APC, NKG2D-APC (all Miltenyi Biotec, Gladbach, Germany), NKG2C-PE, NKG2C-PErCP, NKG2C-APC (all R&D Systems Europe, Oxford, UK), rabbit STAT4 (Invitrogen, Paisley, UK) with goat anti rabbit secondary-APC (Abcam, Cambridge, UK).