Stock solutions preparation
1. Dissolve COL G in 1.5 ml sterile H2O and make 3 aliquots of 500 μl (each aliquot is the Solution A) and store at -20°C
2. Dissolve COL H in 3 ml sterile H2O and make 3 aliquots of 1 ml (each aliquot is the Solution B) and store at -20°C
3. Dissolve the Thermolysin in 1.5 ml sterile H2O and make 3 aliquots of 500 μl (each aliquot is the Solution C) and store at -20°C.
Buffers Preparation
1. Cardiomyocytes isolation buffer (CIB) 1 liter: Prepare a solution of NaCl 120 mM, KCl 5.4 mM, Na2HPO4*7H2O 0.33 mM, MgSO4*7H2O 0.5 mM, BDM 10 mM, HEPES 25 mM, Glucose 22 mM, Taurine 30 mM. Adjust the pH (7.2-7.4) with 5 M NaOH and filter for sterility. Add 3% of antibiotic pen/strep. Add 0.05 mU of insulin per litre of cardiomyocyte isolation buffer. CIB can be stored at 4°C.
2. Perfusion Buffer (135 ml): 0.4 mM EGTA in CIB.
3. Digestion buffer (70ml): Solubilize one aliquot of Solution A, one aliquot of Solution B + CaCl2 300 μM, in CIB. Immediately before use add one aliquot Solution C
4. Stop Buffer (25ml): CIB + EDTA 2mM. 5. Culture media: MEM media, 1% Glutamine, 1% pen/strep, 1.26 mM CaCl2.
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