Human cd14 magnetic beads

Peripheral blood mononuclear cell (PBMC) was isolated from the peripheral blood using density gradient method. CD14⁺ monocytes and naïve CD4⁺ T cells were isolated using human CD14 magnetic beads (Miltenyi Biotec, Germany) and human naïve CD4 T Cell Isolation Kit II (Miltenyi Biotec, Germany) according to the manufacturer's protocol. The isolated cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. In induced differentiation experiment, Mo-DCs were differentiated from CD14⁺ monocytes using IL-4 (20 ng/mL; Thermo Fisher, USA) and GM-CSF (20 ng/mL; Thermo Fisher, USA) for 7 days, followed by treatment with LPS (20 ng/mL; Sigma, USA) for 3 days. The phenotype of Mo-DC was identified by flow cytometry. In cocultured experiment, naïve CD4⁺ T cells were cocultured with Mo-DCs in a 1 : 5 ratio.

Peripheral blood mononuclear cells (PBMCs) were isolated from DM2 + CVD subjects and healthy controls by Ficoll density gradient centrifugations (Axis-Shield) as described in detail previously [86 (link)]. In brief, after washing PBMCs, CD14⁺ monocytes were isolated using human CD14 magnetic beads and MACS® cell separation columns according to manufacturer’s instructions (Miltenyi Biotec, Leiden, The Netherlands). Then, these CD14⁺ monocytes were ex vivo treated with IFNγ and/or apabetalone, and phenotyped for gene expression (NanoString) and protein secretion (Millipore Milliplex® Human Cytokine/Chemokine Array).

Trypticase soy broth was procured from Fisher Scientific (Hampton, NH, USA). Fetal bovine serum (FBS) and gentamicin (catalog no. 15750060) were purchased from Gibco-BRL (Grand Island, NY, USA). α-Minimal essential medium (α-MEM), anti-α-actin (A2228) antibody, rosiglitazone, polyinosinic-poly(C) [poly(I·C)], and lysostaphin (L7386) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Macrophage colony-stimulating factor (M-CSF), in the form of CMG 14-12 supernatant, and glutathione S-transferase RANKL (GST-RANKL) were prepared as previously described (33 (link)). Human M-CSF and Ficoll histopaque were purchased from Invitrogen (Carlsbad, CA, USA). Human CD14 magnetic beads were obtained from Miltenyi Biotec (Auburn, CA, USA). Anti-NFATc1 (7AG) antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-histone H3 (96C10) was obtained from Cell Signaling Technology (Beverly, MA, USA). Murine recombinant IL-4 and IFN-γ were purchased from Peprotech (Rocky Hill, NJ, USA).