Anti cd14 mab conjugated magnetic microbeads

Immature dendritic cells were generated from peripheral blood mononuclear cells, as described (Sallusto and Lanzavecchia, 1994 (link)). Briefly, peripheral blood mononuclear cells were obtained from 30 ml of leukocyte-enriched buffy coat from healthy donors by centrifugation on a Ficoll-Hypaque plus (GE Healthcare) through density gradient. Monocytes were purified by positive selection using anti-CD14 mAb-conjugated magnetic microbeads (Miltenyi Biotec). CD14⁺ cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum (Invitrogen), and 2 mM glutamine (Invitrogen) containing 50 ng/ml GM-CSF (granulocytes monocytes-colony stimulating factor) and 250 ng/ml IL-4 (Immunotools). Cells were cultured for 5–7 days in 5% CO₂ atmosphere to obtain a population of iDCs. Purity of generated DCs was checked by flow cytometry, using Phycoerythrin (PE) conjugated anti-CD1a antibody (Becton Dickinson) on a FACScalibur flow cytometer (Becton Dickinson). DC populations were used when CD1a expression was > 95%. Written informed consent was obtained from each donor at the time of venous peripheral blood donation, in accordance with the Declaration of Helsinki, as approved by Azienda Ospedaliera Universitaria Federico II. All the experiments done by using blood donations were performed and analyzed anonymously, without any biographical reference to donors.

Human monocytes were purified from peripheral blood lymphocytes were isolated by using Ficoll gradients (lympholyte-H; Cedarlane, Burlington, Ontario). CD14 cells were purified by positive sorting through anti-CD14 mAb-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in 75 cm² flasks (Costar, Corning Life Sciences, Tewksbury, MA) in RPMI 1640 medium (Life Technologies Invitrogen), supplemented with heat-inactivated 10% lipopolysaccharide-free FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin (all from EuroClone, Milan, Italy) and 0.05 mM 2-βmercaptoethanol (Sigma Chemicals; St. Louis, MO) in the presence of human recombinant GM-CSF (25 ng/ml; R&D Systems, Minneapolis, MN) and IL-4 (25 ng/ml; R&D Systems) [21] (link). After 6 d, immature MDDC were washed and analyzed by cytofluorimetry for the expression of the surface markers CD1a, CD14, CD83 and CD38. MDDC were used in the experiments if >80% CD1a and <10% CD14 positive cells.