Mito tracker green

Fetal bovine serum (FBS) was purchased from AusGeneX (Molendinar, Australia, CAT NO. FBS500-S); DMEM basic (1X) was from Gibco Life Technologies (Shanghai, China); penicillin/streptomycin solution (100 U/mL and 100 mg/mL, respectively), 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and Cellular grade DMSO were from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); 3-(4,5-dimethyl-1,3-thiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide(MTT) was obtained from Biosharp Life Technology Co., Ltd. (Hefei, China); Hoechest 33342, Mito-Tracker Green, and Lyso-Tracker Green were from Beyotime Biotechnology Co., Ltd. (Shanghai, China). All cells were cultured in a CO₂ incubator (Wiggens, WCI-180, Beijing, China). All the in vitro fluorescence images experiments were obtained by using an Olympus IX73 + DP73 inverted microscope and laser confocal microscope (Carl Zeiss LSM 900, Jena, Germany). Hypoxic condition was created by coverslips (Citoglas, Hong Kong, China) and a hypoxia chamber (Stemcell Technologies, Cat 27310, Kent, WA, USA). Cytotoxicity assay was performed using an LED light (F&V, Z96kit, Fuijan, China) as irradiation source.

The treated cells were cultured on glass coverslips and fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized in 0.1% Triton X-100 in PBS for 4 min, blocked with 1% BSA/PBS for 1 h, and then incubated with Mito-Tracker Green (Beyotime, Nanjing, China) for 1 h at room temperature. The cell nuclei were counterstained with Hoechst 33342, and images were acquired using a fluorescence microscope.

A mitochondrial membrane potential assay kit with JC-1 was utilized to determine the ∆Ψm within fluoride-treated cells. The HL-7702 cells were incubated with 0, 0.1, 1.0, and 10 μg/L T-2 toxin for 24 h. After exposure, the cells were collected by trypsinization, re-centrifuged, and finally resuspended in complete DMEM at the concentration of 1 × 10⁵ cells/mL. The cells were kept in the 5% CO₂ incubator for 20 min after being mixed with the 1 mL JC-1 stain liquid. After washing, the cells were imaged by using a fluorescence microscope. Mitochondrial morphology was evaluated by staining with Mito-Tracker Green (Beyotime Biotechnology, Haimen, China). After 24 h treatment, the HL-7702 cells were washed and stained for 0.5 h with Mito-Tracker Green; then, the images were captured (Leica SP5, Leica Microsystems Inc., Wetzlar, Germany).

Mitochondrial superoxide in skin fibroblasts was detected with MitoSOX (5 μM, YEASEN, Shanghai, China) staining. MitoTracker Green (200 nM, Beyotime, Shanghai, China) was incubated for mitochondria colocation at 37°C for 20 min in the dark. The fluorescence was observed and photographed with a laser confocal microscope.

BMDMs were incubated with Mito-Tracker Green (#C1048, Beyotime) at 37 °C for 30 min and then analyzed using flow cytometry. Mitochondrial DNA copy number was determined by real-time PCR using specific probes against CytB and Ppia.

BMDMs were seeded in 6-well plates and treated with the fluorescent dyes DCFH-DA (#S0033, Beyotime) and MitoSOX Red (#M36008, Invitrogen) to measure the intracellular ROS and mitochondrial superoxide levels, respectively. Mito-Tracker Green was used to detect mitochondrial content. BMDMs were incubated with Mito-Tracker Green (#C1048, Beyotime) at 37 °C for 30 min and then analyzed by flow cytometry, using the Accuri C6 (BD) and subsequent analysis was performed using CFlow Plus.

Biochemical reagents, antibodies, and plasmids were purchased from companies indicated as follows: N-acetyl-p-aminophenol (APAP, Weikeqi Biotech, Chengdu, China); MitoTracker-Green, JC-1, LysoTracker-Red, Hoechst 33342, and FITC-conjugated goat anti-rabbit IgG (Beyotime, Shanghai, China); TMRE (Solarbio, Beijing, China); cytochalasin B and colchicine (Meilun, Dalian, China); Calcein-AM (Genesion, Guangzhou, China); Mito-Tempo (Sigma, St Louis, MO); pLP-VSVG (Clontech, Mountain View, CA); polyclonal rabbit anti-proteasome 20S α5 and anti-calnexin antibodies (Biosynthesis, Lewisville, TX); alanine transaminase (ALT); and aspartate transaminase (AST), glutathione (GSH), and glutathione disulfide (GSSG) test kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).