Amplex red reagent

All reagents and solvents were purchased at the highest purity available. Cadaverine dihydrochloride, 2HP dihydrochloride, horseradish peroxidase (HRP), ethanol, 4-vinylpyridine, ammonium citrate, and citric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Boric acid, sodium borate, and N-acetyl-3,7-dihydroxyphenoxazine (Amplex™ Red Reagent) were obtained from Thermo Fisher Scientific (Lenexa, KS, USA). Optima™ LC/MS-grade acetonitrile, water, and formic acid were purchased from Fisher Scientific (Thermo Fisher Scientific). Sequencing grade trypsin, chymotrypsin, and glycerol-free peptidyl-N-glycosidase F (PNGase F) were purchased from Promega (Madison, WI, USA) and New England BioLabs (Ipswich, MA, USA), respectively. Deionized water with a resistivity of 18 MΩ∙cm was purified using a Millipore Direct-Q3 Water Purification System (Billerica, MA, USA).

The reactions were set in 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 2 mM MgCl₂, 1 mM NAD(P)H, 50 μM Amplex red, with or without cytoplasmic extract prepared from NHDF. Horseradish peroxidase and Amplex red reagent were purchased from ThermoFisher Scientific. Each test (e.g., control or an experimental drug) was run in 5 independent wells, in 384-well plates (Nunc, Clear Polystyrene Plates) sealed with adhesive PCR film (ThermoFisher Scientific). All reaction mixes were prepared on ice and dispensed into cold plate (18 μl, with automatic pipette Rainin, EDP3, 0.1–1.2 ml) before addition of drug candidates (2 μl, with multichannel automatic micro-dispenser, Rainin, EDP3, 1–10 μl) or H₂O₂. The excitation at 544nm and emission at 590 nm were used to detect the resorufin fluorescence on Synergy H1, Hybrid reader (BioTek).

Cells were incubated with 50 μM Amplex Red reagent (Thermo Fisher) at 37 °C for 30 min. Fluorescence was detected at ex/em 550/590 nm using a Gemini XPS Microplate Reader (Molecular Devices, San Jose, CA, USA). To account for H₂O₂-specific fluorescence, PEG-CAT was used as described above. Intracellular production of H₂O₂ was estimated using a sensitive assay based on the aminotriazole-mediated inactivation of CAT, as previously described by Wagner et al. [40 (link)]. Cells at 70–90% confluence in 100 mm tissue culture dish were treated with BET for 6 h, and 20 mM 3-aminotriazole (3-AT) was then added and incubated at 37 °C for 0, 5, 10, 15, 30, and 45 min. Cells were rinsed twice and harvested with ice-cold 50 mM phosphate-buffered saline (PBS), and pH 7.4 and pellets were collected. H₂O₂ concentration was calculated by kinetic analysis of the rate of decrease in CAT activity [41 (link)]. Spectrophotometric CAT activities were initiated by addition of 30 mM H₂O₂ and the loss of absorbance at 240 nm at 25 °C was monitored on a Beckman DU-600 UV–Vis spectrophotometer (Beckman–Coulter, Fullerton, CA, USA). Initial CAT activities were calculated by fitting experimental data to the first-order kinetics and expressed as catalase k mU/mg cell protein.

For the quantification of the anthocyanin content, approximately 40–70 mg of leaf tissue was homogenized in 1 ml of propanol:HCl:water (18:1:81) and further extracted in a boiling-water bath for 3 min. The mixture was centrifuged at 5000 g for 40 min. The absorbance of the supernatant was measured at 535 and 650 nm, and referred to the fresh weight as described (Laureano-Marín et al., 2016 (link)).
To quantify hydrogen peroxide, 50 mg of leaf tissue was ground in liquid nitrogen with 250 μl of 50 mM phosphate buffer, pH 7.4, vortexed and shaken continuously at room temperature for 30 min. Samples were centrifuged at 4 °C at 12 000 g for 10 min. Fluorescence quantification of H₂O₂ was performed in the supernatant after incubation with Amplex Red reagent (Thermo Fisher Scientific) and horseradish peroxidase using 560 nm excitation and 590 nm emission filters. Standard calibration curves were obtained with known H₂O₂ concentrations.
To quantify the total Cys and glutathione contents, thiols were extracted, reduced with NaBH₄, and quantified by reverse-phase HPLC after derivatization with monobromobimane (Thermo Fisher Scientific) as described previously (Dominguez-Solis et al., 2001 (link)).

Approximately 50 mg of liver tissue was homogenized in 1 ml of homogenization buffer containing 25 mM Hepes pH7.4, 1 mM EDTA, 0.25 M sucrose, 2 mM MgCl₂, 1 μM butylated hydroxytoluene (BHT), 1:200 dilution of Sigma P8340 and 1 μM diethylenetriaminepentaacetic acid (Sigma-Aldrich). The homogenates were centrifuged at 500 × g for 5 min to pellet nuclei and debri. The resultant supernatant was centrifuged at 10,000 × g for 10 min at 4 °C to obtain mitochondria. The pellets were resuspended in 150 μl homogenization buffer. About 20 μL solution was saved for measuring protein concentration.
Mitochondrial H₂O₂ was detected using Amplex Red assay as described39 (link). The working solution was prepared using 100 μL of 10 mM Amplex Red reagent (Thermo Fisher Scientific), 2 μL of 1000 U/ml horseradish peroxidase (HRP) and 10 ml of 50 mM potassium phosphate (pH7.7) with 0.5 mM diethylenetriaminepentaacetic acid (Sigma-Aldrich). 50 μL of sample or standard was pipetted into 96 plates, followed by addition of 50 μl of the Amplex Red reagent/HPR working solution. The reaction mixture was protected from light and incubated at room temperature for 30 min. The fluorescence was measured with excitation in the range of 530–560 nm and emission at 590 nm. Mitochondrial H₂O₂ levels were normalized to protein levels.

The colorimetric measurement of total phenolic, flavonoid and tannin levels was conducted according to a recent study [18 (link)]. Standards, namely, gallic acid (GA) for phenolics, rutin (RU) for flavonoids and tannic acid for tannins, were used to explain the results. The antiperoxidase activity was measured by monitoring the formation of the highly fluorescent resorufin, produced by the interaction of Amplex Red® reagent (Thermo Fischer Scientific, Monza, Italy) with H₂O₂ in the presence of horseradish peroxidase (Sigma Aldrich), as described by Vargas and colleagues [19 (link)].