Lipopolysaccharide

To M1 macrophage polarization, monocytes culture medium was removed and replaced with RPMI + L-Glutamine medium (Gibco, 21127, MA, USA) with 10% Fetal Bovine Serum (ATCC, 30-2020, VA, USA), 1% antibiotic (Pen Strp. Gibco15140, MA, USA), supplementing with interferon-gamma (INFɣ) 20 ng/mL (Millipore, IF002, MA, USA) lipopolysaccharide 100 ng/mL (Sigma, L4391-1 MG, MA, USA) maintaining the standard conditions of 36.5 °C and 5% CO₂ for 24 h.
M1 macrophages were further stimulated with a single dose of LDL (10 µg/mL) and the transcriptome was evaluated at different times (0, 24, 48, and 72 h) in each study group. RPMI 1640 supplemented with 5% FBS, and IFN-γ (20 ng/mL) lipopolysaccharide 100 ng/mL (Sigma, L4391-1MG, MA, USA) maintaining the standard conditions of 36.5 °C and 5% CO₂. The confluence in each technical replicate was 200,000 cells per well and they were performed in triplicate for each volunteer. Cell counts were performed using the TC20™ automated cell counter (Bio-Rad, 145-0102, CA, USA).

CD4⁺ T cells (1 × 10⁵, 100 μL) were co-cultured with autologous B cells (4 × 10⁵, 100 μL) in U-bottom 96 well plates (5 × 10⁵, 200 μL volume/well) as described previously [46 (link)]. Anti-CD11a mAb, 1 μg/mL, or mouse isotype control IgG1 was used to observe the LFA-1/ICAM blocking role when CD4⁺ T cells and B cells were incubating. B cells alone, B cells plus lipopolysaccharide (Sigma, St Louis, MO, USA), and B cells plus lipopolysaccharide and anti-CD11a were cultured as controls. After incubation in RPMI 1640/10% FBS/penicillin/streptomycin at 37°C/5% CO₂ for 8 days, 50 μL fresh media was added to each well. On the fourth day after that, supernatants (200 μL) were harvested and stored at 4°C.