Anti cd16 pc5

EDTA whole blood samples from 5 healthy subjects, 10 patients with mild and 10 patients with severe disease were stained with anti-HLA-DR PE and ECD, anti-CD3 ECD, anti-CD4 PE, anti-CD8 FITC, anti-CD19 PC7, anti-CD14 FITC, anti-CD16 PC5, anti-CD15 PE, anti-CD57 FITC, anti-CD56 PE, anti-CD11c PCP, anti-CD123 PE, anti-CD83 PE, anti-CD38 PE, anti-CD23 ECD and isotype controls (all from Beckman Coulter) for 20 minutes in the dark at +4°C. Samples were analyzed on the flow cytometer Cytomics FC500 (Beckman Coulter). Data were processed by FlowJo V.10.

Peripheral blood mononuclear cells (PBMC) from healthy blood donors were separated by Ficoll-Hypaque (P.A.A. GmBH, Germany) density gradient centrifugation, washed in Ca²⁺ and Mg²⁺ free HBSS buffered with 10 mM HEPES (Invitrogen). Cells were frozen in heat-inactivated AB⁺ human serum with 10% DMSO (Sigma-Aldrich) at a final concentration of 15 × 10⁶ cells/ml and stored in liquid nitrogen until use. The phenotype of the fresh PBMCs and monocyte (MN) sub-populations were monitored by flow cytometry. MNs were isolated from PBMC with a human monocyte enrichment kit, without depletion of CD16 cells, according to manufacturer’s instructions (EasySep^®, StemCell Technologies). The phenotypic analysis was performed with 10⁶ cells incubated in 100 μl of PBS-5% FCS (FACS buffer) in the presence of 0.1 mg/ml of anti-CD14-PE and anti-CD16-PC5 (Beckman Coulter) for 30 min at 4 °C in the dark. At least 5.10⁴ cells were acquired and then analysed by flow cytometry (FACSCalibur, BD Biosciences).