Cd14 fitc antibody clone hcd14

Peripheral blood mononucleated cells (PBMCs) were obtained from normal blood donors of the Etablissement Français du Sang (EFS), Toulouse, France. Informed written consent was obtained from all blood donors, and the study protocol was approved by the INSERM-EFS ethical committee. Briefly, CD14+ monocytes were purified from PBMC isolated on Pancoll (Pan Biotech, Aidenbach, Germany) by negative sorting using Dynabeads (Dynabeads Untouched Human Monocytes, Invitrogen, Oslo, Norway). Purity was measured by flow cytometry (FC-500 Beckman Coulter, Brea, CA, USA) using a CD14-FITC antibody (clone HCD14, Bio Legend, San Diego, CA, USA). Data were analyzed using FlowJo (Tree Star). Purity was >90%.

CD14+ monocytes were purified by positive magnetic sorting (Affymetrix), from PBMC isolated on Pancoll (Pan Biotech). Purity was measured by flow cytometry (MACSQuant 10, Miltenyi), using a CD14-FITC antibody (clone HCD14, BioLegend). Sample purity was routinely ≥95%. Macrophages were derived from monocytes (MDM). Monocytes (0.5 × 10⁶/ml) were differentiated into macrophages in the presence of recombinant M-CSF (50 ng/ml; BioLegend) for 5 days. Cells were cultivated at 37°C/5% CO₂ in RPMI medium 1640 + Glutamax (Gibco), supplemented with 10% fetal calf serum (Gibco), Penicillin G (Gibco), and Streptomycin (Gibco).