2.9. Cell culture and cytokine analysis

Raw264.7 cells were obtained from ATCC (Manassas, VA). Cells were cultured in 50% DMEM with 10%FBS/50% AIMV media (Gibco). Cells were added to serially diluted compounds in AIMV media at 3×10⁵ cells/well. Peripheral blood samples were collected from healthy adult donors. The samples were collected after approval by the University of Montana Institutional Review Board, and signed written informed consent was obtained from each donor. PBMCs were isolated from peripheral blood using Ficoll-Paque. Cells were added at 5×10⁶ cells/well in RPMI with 5% human plasma to the serially diluted formulations. Supernatants were harvested from treated cells 18–24 h post-cell application. Supernatants were analyzed using either a DuoSet ELISA (R&D Systems, Minneapolis, MN) for human IL6 or a Luminex multiplex panel for analytes TNFα, IL-1β, IL-6, IFNγ, IL12p70 and IL-23 (R&D Systems) per the manufacturer’s instructions. Multiplex analysis was performed using a Luminex 200 instrument (Luminex Corporation). ELISAs were read on a SpectraMax^® M5 Multi-Mode Microplate Reader at 450 nm and raw OD plotted directly.