Human monocyte isolation and culture

Peripheral blood samples were obtained in heparin from normal adult volunteers using protocols approved by the Institutional Review Board of the University of Utah and Veterans Affairs Salt Lake City Healthcare System (IRB no. 10-0398). Peripheral blood mononuclear cells (PBMC) were isolated by an adherent method previously described[34]. The PBMC were cultured in 96-well culture plates (2.5 x 10⁴ cells/0.3 cm²) with fat/PGZ or fat/ROS culture conditioned media diluted in half with SFM media (Gibco Life Technologies, ThermoFisher Scientific) supplemented with 20% serum. Conditioned media was collected from ROS-treated, PGZ-treated, or untreated (no drug) fat cultures. The conditioned media was diluted in monocyte media prior to use in the monocyte cultures. The monocytes were stimulated using LPS (10 ng/mL) (Sigma-Aldrich) for 24 hrs at 37 °C/5% CO₂ then the PBMC-conditioned media was collected and stored at −80°C. Media from PBMC without LPS stimulation served as controls. Media was assayed for tumor necrosis factor-α (TNFα) by ELISA following manufacturer’s instructions.