Anti cd14 coated microbeads

Human peripheral blood mononuclear cells (PBMC) from buffy coat of healthy donors were isolated by Lymphoprep density-gradient centrifugation (Nycomed Pharma, Oslo, Norway). Cells were washed 3 times in phosphate buffered saline (PBS), pH 7.4, and were isolated by density-gradient separation (Lympholyte; Cedarlane, Hornby, Ontario, Canada). CD14+ monocytes were purified by incubation with anti-CD14–coated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by sorting with a magnetic device (MiniMacs Separation Unit; Miltenyi Biotec), according to the manufacturer's instructions.
The purity of the isolated monocytes was evaluated by staining with a fluorescein isothiocyanate (FITC)–conjugated anti-CD14 antibody against monocytes and analyzing stained cells by flow cytometry. The purity was higher than 95% CD14+. The viability of the monocytes was up to 99%, using the Trypan blue staining.
Before in vitro experiments, monocytes were cultured for 24 h, in RPMI 1640, containing 2 mM L-glutamine, 100 units/ml of penicillin, 100 mg/ml of streptomycin, and 250 pg/ml of Fungizone (Gibco, Grand Island, NY), in the absence of antioxidant agents, at 37°C in a humified atmosphere, containing 5% CO₂.

Recombinant human (rh) macrophage colony-stimulating factor (M-CSF) and rh interleukin (IL)-10 were from R&D Systems (Minneapolis, MN, USA). Interferon (IFN)-γ, anti-CD14-coated microbeads, phycoerytrin (PE)-CD163 monoclonal antibody (mAb) and fluorescein isothiocyanate (FITC)-CD206 mAb were from Miltenyi Biotec (Gladbach, Germany). Allophycocyanin (APC)-CD16, APC-Alexa Fluor 750-human leukocyte antigen-D region-related (HLA-DR) and APC-Alexa Fluor 700-CD36, PE-Cyanine dye Cy7 (PE-Cy7)-CD197 (CCR7) mAbs were from Beckman Coulter (San Jose, CA, USA). PE-CD1a and FITC-CD14 mAbs were from PharMingen (San Diego, CA, USA). Fetal bovine serum (FBS) was from Hyclone Laboratories (Logan, UT, USA). RPMI 1640 was from GIBCO (Paisley, UK). Sytox^® Blue dead cell stain and 2-7-dichlorodihydrofluorescein diacetate (H2DCF-DA) were purchased from Molecular Probes (Carlsband, CA, USA). Propranolol, isoproterenol, lipopolysaccharide (LPS; from Escherichia coli strain 0111:B4), trypan blue solution and FITC-dextran were from Sigma-Aldrich (Milan, Italy). ML385, a novel and specific Nrf2 inhibitor, was purchased from Selleck Chemicals (Cologne, Germany).

Human peripheral blood mononuclear cells were isolated from buffy coats (obtained from Skåne University Hospital, Lund, Sweden) by Lymphoprep (ρ = 1.077 g/ml; Axis-Shield, Norway) density centrifugation at 700 × g for 20 min. Cells from the interphase were washed three times with phosphate-buffered saline (PBS) and monocytes were purified using anti-CD14-coated microbeads (Miltenyi Biotec, Germany) according to the manufacturer’s instruction (monocyte purity was >96%). To obtain M1 or M2 macrophages, cells were cultured for 6 days in RPMI-1640-GlutaMAX-I medium (Life Technologies, UK), 10% (v/v) FBSi, 1% (v/v) AA, and 5 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF; R&D Systems, UK) or 12.5 ng/ml macrophage colony-stimulating factor (M-CSF; R&D Systems) as described previously (18 (link)).