Anti cd56 pe cy7

In 96-well plates (BD, Franklin Lakes, NJ, USA), 100,000 viable PBMCs/well of 6 Colombian HCs were resuspended in 200 µL RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS and were treated with calcidiol at 250 nM and calcitriol (active form of VitD) at 0.5 nM (both within the physiological range) or with 0.01% v/v EtOH as vehicle control.
Cells were harvested 48 h posttreatments in polypropylene tubes, centrifuged at 700 × g for 5 min and washed with PBS. Extracellular staining was done with fluorochrome-labeled antibodies purchased from eBioscience (Santa Clara, CA, USA): fixable viability dye eFluor 506, anti-CD3 PE-Cy7, anti-CD4 PE, anti-CD8 eFluor^® 450, anti-CD56 PE-Cy7, and anti-TIM-3 APC-Cy7. Samples were acquired on a BD-LSRFortessa™ flow cytometer, and data were analyzed in FACSDiva v.8.0.1 software. The gating strategies are shown in Figure S1 in Supplementary Material.

Ethical approval was obtained from the TCD School of Biochemistry and Immunology Research Ethics Committee for experiments involving PBMCs. Human peripheral blood mononuclear cells (PBMCs) from anonymous healthy donors were obtained by informed consent from buffy coats of blood packs from the Irish Blood Transfusion Service, using Lymphoprep (Axis-Shield) gradient centrifugation. For cell sorting to analyse constituent cells, PBMCs were stained with anti-CD14 APC, anti-CD19 PE, anti CD3 PE-Cy5.5 and anti-CD56 PE-Cy7 (eBiosciences). Cells were fixed and permeabilised using the FoxP3 staining buffer set (eBiosciences) and were stained with anti-MNDA FITC (Cell Signalling). Labelled cells were analysed on a FACSCanto II flow cytometer (BD Biosciences) and were evaluated with FlowJo software (TreeStar). Monocytes were isolated from PBMCs by positive selection using CD14 beads (Miltenyi Biotech), following the manufacturer’s instructions. Macrophages were generated by growing the monocytes in the presence of 50 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF, Sigma–Aldrich) or 20 ng/ml macrophage colony stimulating factor (M-CSF, Sigma–Aldrich), for 7 days, changing the media every 2–3 days.