Subtilisin a protease from bacillus licheniformis

HCF media was replaced with 4 mL of 4 °C homogenisation buffer (300 mM sucrose, 10 mM HEPES-KOH, 1 mM EGTA-KOH, pH 7.4) containing 2 mg Subtilisin A protease from Bacillus licheniformis (Sigma-Aldrich) and incubated at room temperature for 5 min. Digested cells were transferred to a 15-mL tube on ice and digested for an additional 15 min before filtration through a 10 μm mesh filter (PluriSelect) saturated with cold homogenisation buffer. Mitochondria were collected by centrifugation at 9,500 RCF at 4 °C for 5 minutes and washed 3 times in cold homogenisation buffer before resuspension in culture media. Mitochondrial number was determined by using a Multisizer 4e Coulter Counter (Beckman-Coulter) and corroborated by hemocytometry. Isolated mitochondria were incubated with HCFs or iPS-CMs for 0 to 4 h.

Mitochondria were isolated from HEK293 cells as previously described [18 (link)]. Briefly, 60 cm plates of HEK293 cells were collected by adding 5 mL of homogenization buffer (300 mmol/L sucrose, 10 mmol/L HEPES-KOH, 1 mmol/L EGTA-KOH, pH 7.4) with 1 mg Subtilisin A protease from Bacillus licheniformis (Sigma-Aldrich). A detailed protocol for mitochondria isolation can be found in our previous published manuscript [18 (link)].