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Exposure of Dynorphin 2-17 to free fatty acids. FFA preparations were thawed and briefly warmed immediately before addition to cell culture media. FFA were added to final concentrations of 50, 100, and 150 mM in RPMI 1640 media (containing 10% FBS, 0.3 mg/mL l-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin) and allowed to complex with the albumin present in the FBS at 37 °C for 1 h. The final FFA/albumin molar ratio was approximately 2:1. For individual experiments, U937 macrophages were treated in media containing 0–150 μM FFA, and were subsequently maintained under standard cell culture conditions for 4–72 h.
Generation of conditioned media and cytokine protein quantification. Macrophages were maintained under control conditions or treated with 150 μM PA for 48 h, washed and subsequently incubated in fresh, PA-free media for 6–48 h. Conditioned media were collected from courtship behavior cultures at the appropriate time points, then aliquoted and snap-frozen to ?80 °C. Aliquots were thawed only once prior to analysis or use on cultured cells. IP-10 protein concentrations in conditioned cell culture media were analyzed by ELISA (R&D Systems) following manufacturer’s instructions. Samples outside the range of the standard curve were diluted in ELISA sample buffer and re-analyzed.