Mitochondria isolation kit

Icariin (purity 98.75%) and JC-1 dye were purchased from MedChem Express (MCE, NJ, USA). The dihydroethidium (DHE) probe and mitochondria isolation kits were obtained from Beyotime Biotechnology (Jiangsu, China). The in situ cell death detection kit (Roche) was obtained from Sigma-Aldrich (St. Louis, MO, USA); and the primary antibodies against Sirt3, Sirt1, PGC-1α, Mfn2, Cyt-b, and β-actin, as well as the goat anti-rabbit and goat anti-mouse secondary antibodies were obtained from Abcam Biotechnology (Cambridge, MA, USA). The primary antibody against Apelin was obtained from Signalway Antibody (SAB, USA). Empty adenoviral vectors (Ad-EV) and recombinant adenoviral vectors expressing Apelin (Ad-Apelin), Sirt3 (Ad-Sirt3) or Apelin-specific small hairpin RNA (Ad-sh-Apelin), as well as Sirt3-specific small hairpin RNA (Ad-sh-Sirt3), were purchased from Hanbio Technology Ltd. (Shanghai, China). The titer of the adenoviruses was approximately 1.2×10¹⁰ PFU/mL.

Mitochondria were isolated from GAS muscles or myotubes using Mitochondria Isolation Kits (Beyotime, China) according to the manufacturer’s instructions. The activities of complexes I, II, and IV were determined in freshly isolated mitochondrial homogenates using commercial assay kits from Abcam (Cambridge, MA, USA) following the manufacturer’s protocol.

Mitochondria were isolated using mitochondria isolation kits (Beyotime Institute of Biotechnology, c3606 for tissue and c3601 for cell) according to the manufacturer's instructions. Briefly, fresh mouse hearts (6 to 8 mice per group) were minced in the centrifuge tube. The pellet was homogenized in the trypsin-EDTA (8 μL/mg) solution and centrifuged at 600g for 20 s. After washing, the pellet was resuspended with 8 μL/mg mitochondria separation reagent B combined with PMSF and transferred into a prechilled glass homogenizer. The suspension was homogenized for 20–30 times on the ice and centrifuged at 600g for 5 min. Mitochondria were pelleted from the supernatant via centrifugation at 11,000g for 10 min. The final pellet was either resuspended in 40 μL/mg mitochondria store liquid for intact function assay or lysed for Western blot analysis.
For cellular mitochondria isolation, H9c2 cardiomyocytes at a density of 5 × 10⁷ cells were harvested and resuspended in a 2.5 mL mitochondria isolation buffer. The suspension was homogenized for 15–20 times in the glass homogenizer and centrifuged at 600g for 10 min. Then, the supernatant was centrifuged at 11,000g for 10 min. The final mitochondrial pellet was lysed for Western blot analysis.