Pure nitrogen

Mouse cardiac endothelial cells (MCEC) (Cedarlane, Ontario, Canada), were cultured to confluency in Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, CA) in a 37 °C incubator with humidified room air and 5% CO2. Upon achieving confluency MCEC were exposed to simulated cold storage, and reperfusion injury, as previously described.^{9 (link)} In brief, media was replaced with ice cold University of Wisconsin solution (Bridge to Life, SC), augmented with either rapamycin, cyclosporine (CSA), FK506 (tacrolimus; TAC), or mycophenolate mofetil (MMF) at three different doses; 1, 10 and 100 ng mL⁻¹. Cells were rendered hypoxic in a hypoxic chamber (Billups-Rothenberg Inc, CA), flushed with pure nitrogen (Airgas, PA), and subjected to hypoxic condition for 6 hours at 4 °C. After cold hypoxic ischemia, cells underwent 24 hours of simulated reperfusion by removing the preservation solution and reintroducing warm culture media, and under normal culture conditions. To assess the impact of immunosuppressive regimes on pro-inflammatory cytokine release and cell injury, supernatants were removed to assay for KC release by standard ELISA techniques (BD Biosciences, NJ) and MTS Assay (Sigma Aldrich, MO), respectively.