Mitochondria isolation kit

Mitochondrial isolation from cultured Kupffer cells was performed using a commercial Mitochondria Isolation Kit (Solarbio, cat. no.: SM0020, Beijing, China), as per the manufacturer’s protocol. Briefly, a total of 1 mL of precooled lysis buffer was used to resuspend Kupffer cells collected by trypsinization. In an ice bath, the cell suspensions were ground 30 times in a small-volume glass homogenizer. After centrifugation at 1000× g at 4 °C for 5 min, performed twice, the supernatants were further centrifuged at 12,000× g at 4 °C for 10 min to obtain the crude mitochondrial precipitates. The mitochondrial precipitates were resuspended in 50 μL of wash buffer, then centrifuged at 4 °C for 5 min at 1000× g. The supernatants were centrifuged at 12,000× g for 10 min at 4 °C to obtain mitochondrial precipitates of high purity. These obtained mitochondrial precipitates were resuspended in store buffer or used immediately.

SK-N-SH cells were cultured in MEM supplemented with 10% fetal bovine serum and antibiotics in a humidified atmosphere of 5% CO₂ at 37 °C. At least 10⁷ SK-N-SH cells were used per experiment. The mitochondria were prepared with a Mitochondria Isolation Kit (Solarbio) for cultured cells. Freshly isolated mitochondria were resuspended in ice cold buffer (10 mM HEPES, 2 mM K₂HPO₄, 10 mM succinate, 250 mM sucrose, 1 mM ATP, 0.08 mM ADP, 1 mM DTT, pH 7.5)^{56 (link)}. Aliquots were incubated with FL- or CT-α-syn at 37 °C for 1 h. After incubation, the mixture was centrifuged at 13000 g at 4 °C for 10 min. An equal volume of the supernatant was subjected to SDS-PAGE and the band transferred to a nitrocellulose membrane. The membrane was blocked in TBS-T (20 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 8.0) with 5% skim milk overnight at 4 °C prior to the adding anti-cyto c monoclonal antibody (Abcam, Cat.ab133504, 1:1000 dilution) and incubating for 1 h at room temperature. Following three washes with TBS-T, the membrane was incubated with goat anti-rabbit-HRP secondary antibody in blocking buffer (Beijing Biodragon, Cat.BF03008, 1:5000 dilution) in TBS-T with 5% skim milk for 1 h at room temperature. The membrane was visualized by enhanced chemiluminescence reagents (BioRad) after another three washes.

Mitochondria were extracted using the Mitochondria isolation kit (Solarbio Beijing, Beijing, China). The 1/2LC₅₀ and LC₅₀ (24 h) of C6 were achieved after adding the DMSO solution containing C6 to the reaction system. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as the positive control. The mitochondrial membrane potential was measured according to the procedure indicated on the mitochondrial membrane potential kit (Solarbio Beijing, Beijing, China), and DMSO was set as the blank control.