Series wj 8000

Ventricular myocytes were freshly isolated from the left ventricle (LV) of the mice as previously described (Xu et al., 2002 (link)). Langendorff perfusion with Ca²⁺-free Tyrode solution (mmol/L: 135 NaCl, 4.0 KCl, 0.33 NaH₂PO₄, 1.0 MgCl₂ · 6H₂O, 10 HEPES, 10 glucose, and 10 BDM, pH 7.2 with NaOH) for 5 min, followed by 10 min perfusion with 0.3 mg/ml of collagenase B (Sigma, St. Louis, MO) and 0.6% bovine serum albumin (Promega, Mannheim, Germany). LV was separated, minced and incubated in a shaking bath for 5–10 min in collagenase-containing solutions. Cells were then harvested, washed twice, and stored in a high-K⁺ storage solution (mmol/L: 30 KCl, 10 KH₂PO₄, 70 glutamic, 0.5 MgCl₂, 15 tourine, 10 HEPES, 0.5 EGTA, 10 glucose, pH7.4 with KOH) at 4 °C. Only rod-shaped ventricular myocytes showing clear cross striations were used for the following experiments. For hypoxic exposure, acutely isolated ventricular myocytes were placed in a hypoxic cell culture chamber (Thermo Scientific Series WJ 8000), and were kept at 37 °C for 30 min with a constant stream of water-saturated 92% N₂, 5% CO₂, and 3% O₂.