Preparation of Perfusion Buffer

We prepared the perfusion buffer before the isolation experiments. To prepare perfusion buffer I, 8.0 g of NaCl, 0.122 g of NaH₂PO₄ ^* 2H₂O, 0.724 g of Na₂HPO₄ ^* 12H₂O, 0.4 g of KCl, 0.35 g of NaHCO₃, 2.38 g of HEPES, 0.19 g of EGTA, 0.991 g of C₆H₁₂O₆ ^* H₂O, and 12,500 U of heparin sodium were weighed and dissolved in 1,000 ml of pure water; the solution was adjusted to pH 7.4 with dilute hydrochloric acid and sodium hydroxide. For perfusion buffer II, 0.029 g of CaCl₂ ^* 2H₂O, 1 ml of fetal bovine serum, and 0.03 g of type IV collagenase were measured and dissolved in 50 ml of high glucose DMEM medium. Finally, the above solution was filtered through a 0.22 μm filter and stored at 4°C for the subsequent experiments.