RAW 264.7 Macrophage Cell Culture and Conditioning

All studies in this work were performed using RAW 264.7 murine immortalized macrophages (ATCC TIB-71™). Macrophages were expanded, maintained, and cultured in basal macrophage medium, which is comprised of DMEM (Thermo Fisher Scientific; 12430062), 10% FBS (Thermo Fisher Scientific; 26140079), and 1% antibiotic/antimycotic (Sigma-Aldrich; A5955). Cells were cultured to 70% confluence before conditioning began. Cells were conditioned by addition of medium with lipopolysaccharide (LPS; Sigma-Aldrich; L2880 and Invitrogen; 00-4976-93), interferon gamma (IFN-γ; R&D Systems; 485-MI), or interleukin (IL)-4 (PeproTech; 214-14) as indicated. RAW 264.7 macrophages were conditioned with LPS or IFN-γ alone to quantify individual stimulus dynamic response, with LPS or IFN-γ sequentially to recover iNOS expression via orthogonal input, or with LPS or IFN-γ simultaneously to quantify supra-additivity and model predictive control strategy response. Pre-treatment, 24 h of 100 ng/mL IL-4 prior to addition of LPS or IFN-γ, was used to induce an anti-inflammatory, non-naïve state for experiments involving hysteretic effects.