Multiplexing laser bead technology

Plasma adipokines, cytokines, and chemokines were evaluated using Multiplexing LASER Bead Technology (Eve Technologies, Calgary, AB). Plasma testosterone, 17β-E2, and 17α-E2 were evaluated by LC/MS/MS (29 (link)).

THP-1 human acute monocytic leukemia cells (American Type Culture Collection) were cultured in RPMI 1640 medium (Corning) with 10% FBS and differentiated into HMDMs using phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich), as described previously (32 (link), 109 (link)). For activation of TLR4, HMDMs were cultured with a medium containing 100 ng/mL of LPS from E. coli O111:B4 (Sigma-Aldrich) for 12 h as described previously (32 (link)). TLR7 KO THP-1 cell lines, whose TLR7 expression is completely depleted, were previously generated using a CRISPR/Cas9 approach (32 (link)). Before transfection, the cells were primed with 100 units/mL of interferon γ (Thermo Fisher Scientific) for 18 to 24 h (42 (link)). To deliver RNAs to endosomes, we used the cationic liposome DOTAP (Sigma-Aldrich) as previously described (32 (link), 110 (link), 111 (link)). In brief, 230 pmol of synthetic RNAs or Resiquimod (R848, InvivoGen) were mixed with 60 µL of HBS buffer and 15 µL of DOTAP reagent and incubated for 15 min. The RNA-DOTAP solution was then added to 1 mL RPMI 1640 medium with 2% FBS, followed by incubation of the cells for 16 h. Cytokine concentrations of the cultured medium of HMDMs were measured by Multiplexing LASER Bead Technology (Eve Technologies) or ELISA kit (R&D Systems), as previously described (32 (link)).