Mitochondrial membrane potential kit with jc 1

The mitochondrial membrane potential (ΔΨm) was detected by mitochondrial membrane potential kit with JC-1 (Beyotime Biotechnology, China) under the manufacturer’s protocol. JC-1 is a cationic dye that accumulates in energized mitochondria. In healthy mitochondria, due to high mitochondrial membrane potential, JC-1 aggregates yielding a red fluorescence. On the other hand, in dysfunctional mitochondria with low mitochondrial membrane potential, JC-1 is predominantly a monomer that yields green fluorescence. MDA-MB-231 cells were seeded into 6-well plates with a concentration of 1 × 10⁶ cells/mL and then treated with different concentrations of α-mangostin (0, 1, 2, 4 µM). After 24 h, cells were digested by trypsin, resuspended in 1× binding buffer at a concentration of 1 × 10⁶ cells/mL and then were washed three times with cold PBS. After that, cells were stained with 25 µM JC-1 and incubated at 37 °C for 30 min. Finally, cells were analyzed with a flow cytometer. The fluorescence shift of JC-1 from red to green was measured with the green channel (488 nm/525 nm) to assess the mitochondrial dysfunction.

Berberine was purchased from Sigma Aldrich (St. Louis, MO, USA). In trials it was dissolved in dimethyl sulfoxide (DMSO) (DMSO final concentration < 0.5%) and then immediately diluted with the cell culture medium prior to use. Fetal bovine serum (FBS) was purchased from Life Technologies Co. (Shanghai, China) and high-glucose Dulbecco's Modified Eagle's Medium (DMEM) was obtained from Thermo Fisher Scientific Inc. (Beijing, China). Cell Counting Kit-8 (CCK-8) was acquired from Dojindo Laboratories (Shanghai, China). Cell cycle assay kit and Annexin V-FITC/PI Kit were obtained from Becton Dickinson (Franklin Lakes, New Jersey, USA). Reactive Oxygen Species Assay Kit and mitochondrial membrane potential kit with JC-1 were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Low melting agarose (LMA) was supplied by Amresco Corporation (Shanghai, China). Phenol, dimethyl sulfoxide (DMSO), vinorelbine ditartrate salt hydrate (VNB), H₂O₂, and cisplatin were purchased from Sigma Aldrich (St. Louis, MO, USA). Gel-Green was purchased from Biotium Inc. (Investment Blvd., Hayward, US). All the other materials were of analytical grade, including NaCl, Na₂-EDTA, Tris, Sodium N-lauroylsarcosine (SLS), NaOH, 1% Triton-100, Tris-base, HCl, Na₂HPO₄, KH₂PO₄, and KCl.