Simulated ischemia/reperfusion (SI/R)

To simulate ischemic conditions, the culture medium was replaced with a hypoxic solution containing in mM: NaCl 119, KCl 5.4, MgSO₄ 1.3, NaH₂PO₄ 1.2, HEPES 5, MgCl₂ 0.5, CaCl₂ 0.9, Na-lactate 20, BSA 0.1% pH 6.4. To induce hypoxia, the cells were then placed into a three-gas incubator, gassed through with a mixture of 95% N₂ and 5% CO₂ for 4 h at 37 °C (simulated ischemia). Normoxic control cells were then covered with normoxic solution containing in mM: NaCl 125, KCl 5.4, NaH₂PO₄ 1.2, MgCl₂ 0.5, HEPES 20, MgSO₄, 1.3, CaCl₂ 1, glucose 15, taurine 5, creatine-monohydrate 2.5 and BSA 0.1%, pH 7.4 and cells were kept in normoxic incubator. After simulated ischemia or normoxia, the cells were placed to normoxic incubator; hypoxic or normoxic medium was then replaced by culture medium (simulated reperfusion) for 2 h at 37 °C.