Uquantmicroplate spectrophotometer

Day 3 WT adult fruit flies were maintained on 1% agar containing 0.01 M C14-SPH. Ethanol was used as a control for 12 h before H₂O₂ levels were measured. Every 12 h, H₂O₂ was recorded till the 60th hour. H₂O₂ Assay Kit (Beyotime Biotechnology, Shanghai) was used to assay the H₂O₂ concentration. The H2O2 concentration determination was achieved by the oxidation of divalent iron ions. Their oxidation produced ferric ions and formed a purple product with xylenol orange in a particular solution. Tissue samples were homogenized at a ratio of 100 µl in lysis buffer from the kit per 5 mg of tissue. The lysis buffer could be substituted by phosphate-buffered saline (PBS, containing 135 mM NaCl, 4.7 mM KCl, 10 mM Na₂HPO₄, 2 mM NaH₂PO₄, pH 7.4) buffer. The samples were centrifuged at about 12,000 g for 5 min at 4°C and the supernatant was used for H₂O₂ measurement. All of the above operations need to be performed at 4°C or on ice. All tests were done in triplicate. H2O2 detection reagent was thawed on ice or on an ice water bath. The test was performed using 50 µl of samples and standard curve was set up by 50 µl of 1, 3, 10, 30, and 100 µM H2O2 standard solution. The absorbance at 560 nm was recorded on a uQuantMicroplate spectrophotometer (Biotek Instruments), and the concentration of H2O2 in the sample was calculated based on the standard curve.

Day 3 adult flies were transferred to cornmeal medium containing 10 mmol/L paraquat for CAT activity detection. After feeding for 24 h, approximately 20 mg flies were used for each replication and homogenized in 100 µl lysis buffer from the kit with 1% Triton. Supernatant of the homogenization was transferred to a new test tube for CAT analysis with the CAT activity assay kit (Beyotime Biotechnology, Shanghai, China). Briefly, under the catalysis of peroxidase, H2O2 residuals could oxidize the chromogenic substrate. Absorbance of the developed red color product, N-4-antipyryl-3-chloro-5-sulfonate-pbenzoquinonemonoimine, was detected at 520 nm on a uQuantMicroplate spectrophotometer (Biotek Instruments, Winooski, VT) after incubation for 15 min at 25°C. To calculate CAT enzyme activity, a standard curve was made using H2O2 as standard at the concentrations of 0, 0.625, 1.25, 2.5, 3.75, and 5 mM. Protein concentration of each sample was determined using a bicinchoninic acid (BCA) protein determination kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions.