Mgcl2

Dynamic analysis of cellular redox was carried out using four different live cell imaging dyes, that detect specific molecular species: cytoplasmic superoxide is analysed using DHE (Life Technologies, D11347); mitochondrial superoxide is detected with mitoSOX™ Red (Life Technologies, M36008); global cellular hydrogen peroxide (H₂O₂) and peroxyl radicals (HO₂) are measured using DCFDA (Sigma, D6883) while unbound reduced glutathione (GSH) is assayed with MCB (Life Technologies, M-1381MP). Cells were cells co-transfected with an appropriate fluorophore that would not interfere with the fluorescence of the dye. All dyes were administered to the recording medium (RM) onto cells plated on coverslips, within Attafluor® metal cell chambers (Molecular Probes™, Thermo fisher, A-7816). RM composition is: 5.6 mMKCl (Sigma, P9333), 10 mM D-(+)-Glucose (Sigma, G7528), 10 mM HEPES (Sigma, H4304), 4.2 mM NaHCO₃ (Sigma, 56297), 138 mM NaCl (Sigma, S5886), 2.6 mM CaCl₂ (Sigma, C7902), 1.2 mM NaH₂PO₄ (BDH,1024940), 1.2 mM MgCl₂ (BDH, 10149), pH 7.4.
DHE (10 μM), mitoSOX (10 μM), and DCFDA (15 μM) were diluted in RM and imaged onto a Zeiss LSM510 confocal microscope. MCB was used at final concentration of 2.5 μM in RM. Brightness controlling settings were maintained consistently within the experiment for all techniques.

Farrerol (>99.8%) was purchased from Chengdu DeSiTe Biological Technology Co., Ltd. (Chengdu, China). Rat liver microsomes were prepared in the Department of Pharmaceutical Analysis of the School of Pharmacy, Hebei Medical University. β-Nicotinamide adenine dinucleotide phosphate (β-NADPH), uridine 5′-diphosphoglucuronic acid trisodium salt (UDPGA), alamethicin, MgCl₂, and phosphate buffer saline (PBS, pH 7.4) were obtained from BD Biosciences (Woburn, MA, USA). HPLC-grade acetonitrile was obtained from Fisher Scientific (Waltham, MA, USA). HPLC-grade formic acid was supplied by Fisher Scientific (Fair Lawn, NJ, USA). Purified water was procured from Wahaha Group Co., Ltd. (Hangzhou, China). The needles were supplied by Suzhou Suhang Technology Equipment Co., Ltd. (Suzhou, China). The metabolic cages were acquired from Shenzhen Biovano Technology Co., Ltd. (Shenzhen, China). The centrifugation and the ultrasound were performed with Eppendorf Centrifuge 5424 R (Eppendorf AG, Hamburg, Germany) and an E180H ultrasonic cleaner (Elma, Singen, Germany), respectively.