Mitosox red mitochondrial superoxide indicator

The chemical substances, antibodies, and reagents used herein were obtained from various sources. Dulbecco's modified Eagle medium (DMEM), phosphate buffer saline (PBS), Collagenase, fetal bovine serum (FBS), and Lipofectamine 3,000 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); Mitochondrial division inhibitor 1 and MitoSOX Red Mitochondrial Superoxide Indicator were purchased from MedChemExpress(Shanghai); TRIzol reagent and mRNA qRT-PCR Sybr Green Detection Kit were purchased from Invitrogen (USA). An Annexin V-FITC, Propidium Iodide (PI) Detection Kit,m and A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) kit were purchased from BD Biosciences (New Jersey, USA); The following primary antibodies were used in this experiment: anti-GAPDH (1:100, Cell Signaling Technology, USA), anti-UCP2 (1:5,000, R&D Systems, Inc, USA), anti-MMP9 (1:1,000, R&D Systems, Inc. USA), anti-TGF-beta (1:1,000, Abcam, Cambridge, Britain), and anti-p-DRP1 (1:1,000, Cell Signaling). MTT assay (Beyotime); LDH assay (Beyotime).

The MitoSOX Red mitochondrial superoxide indicator (MedChemExpress) was used for detecting mtROS. According to the protocol, MitoSOX Red was first diluted with serum-free culture medium at 1:1000 to a final concentration of 5 μmol/L. Cells were collected and suspended in diluted MitoSOX Red and incubated in a 37°C cell incubator for 20 min. They were mixed every 5 minutes to allow complete exposure to the probe. The cells were then centrifuged at 800 rpm for 3 min to obtain the precipitate. The cells were washed twice with a serum-free cell culture medium. Finally, flow cytometry (BD bioscience, FACSCalibur) was used for the assay.