Catalase assay kit

Intracellular hydrogen peroxide levels and intracellular CAT activity were determined using a hydrogen peroxide assay kit (Beyotime, China) and catalase assay kit (Beyotime, China), respectively.
Briefly, for the determination of intracellular hydrogen peroxide, a 50-μl sample was added to a 96-well plate. Then, 100 μl hydrogen peroxide detection reagent was added and mixed well. Then, the mixture was incubated at RT for 30 minutes, and the OD546 was immediately measured. The concentration of intracellular hydrogen peroxide was calculated based on the standard curve.
For the determination of intracellular CAT, 2 μl sample, 38 μl catalase test buffer were added into the centrifuge tube, then with another 10 μl of 250 mM hydrogen peroxide solution, the mixture was incubated at 25° C for 1–5 min. Then, 450 μl catalase reaction stop solution was added to stop the reaction. Ten microliters of the above mixture was added to 40 μl catalase assay buffer. After mixing well, the mixture was added to color working solution in a 96-well plate. The cells were incubated at 25° C for at least 15 min, and then the OD520 was measured. Intracellular CAT activity was calculated based on the standard curve of CAT.

Conidia were spread on cellophane-coated solid plates containing 1 mM H₂O₂ and incubated for the indicated times. Fungal cells were collected along with cellophane and ground by liquid nitrogen. The resulted cell debris was dissolved in Catalase Assay Buffer (Beyotime, Shanghai, China) for catalase assay. The crude cell lysate was quantified by the total soluble protein using the Bradford assay. The crude cell lysate containing the protein at the concentration of 0.2 mg/mL was used to determine the enzyme activity using the Catalase Assay Kit (Beyotime, Shanghai, China). This assay is based on colorimetrically measuring the hydrogen peroxide substrate remaining after the action of catalase. The colorimetric method uses a substituted phenol (3,5-dichloro-2-hydroxybenzenesulfonic acid), which couples oxidatively to 4-aminoantipyrine in the presence of hydrogen peroxide and horseradish peroxidase, to give a red quinoneimine dye (N-(4-antipyryl)-3-chloro-5-sulfonatep-benzoquinone-monoimine) that absorbs at 520 nm. One unit of catalase activity was defined as the decomposition of 1 µmol of H₂O₂ per minute at pH 7.0 and 25 °C.

The flies (n ≥ 50, 25 days after eclosion) were homogenized thoroughly in cold RIPA buffer (Solarbio, #R0020) supplemented with cocktail protease inhibitor (bimake, #B14001) with tissue homogenizer (Next Advance) and incubated on ice for 30 min. Samples were centrifuged at 13800 g for 10 min at 4°C, and the supernatants were collected for the redox system analysis. The determination of the content of ROS, hydrogen peroxide, and MDA used the Tissue Reactive Oxygen Species Detection Kit (Bestbio, #BB‐470512), Hydrogen Peroxide Assay Kit (Beyotime, #S0038), and Lipid Peroxidation MDA Assay Kit (Beyotime, #S0131S), respectively, according to the manufacturer's instructions. The determination of the activity of SOD, catalase, GST, and Caspase‐3 used the Total Superoxide Dismutase Assay Kit with WST‐8 (Beyotime, #S0101S), Catalase Assay Kit (Beyotime, #S0051), GST Activity Assay Kit (Solarbio, #BC0355), and Caspase‐3 Activity Assay Kit (Beyotime, #C1116), respectively, according to the manufacturer's instructions.

Yeast cells in exponential phase were harvested and washed twice with cold sterile water followed by resuspension in lysis buffer containing acid-washed glass beads and 20 cycles of 10 s vortexing plus 20 s cooling [14 (link)]. After centrifugation at 12000 rpm for 15 min, the supernatants were collected and used for an enzyme activity assay and determination of intracellular H₂O₂ levels. The protein concentration was determined using the BCA protein Assay Kit (Sangon Biotech, Shanghai, China) following the manufacturer's instruction. The total superoxide dismutase (SOD) activity and the catalase activity were determined using a Total Cellular SOD Activity Kit (Dojindo Molecular Technologies, Rockville, MD, USA) and a Catalase Assay Kit (Beyotime Biotechnology, Shanghai, China), respectively. The intracellular H₂O₂ level was determined using an H₂O₂ Assay Kit (Beyotime Biotechnology, Shanghai, China).

The catalase activity was determined using a commercially available catalase assay kit (Beyotime) according to the manufacturer's instructions. BM-MSCs were seeded in 6-well plates (9.6 cm²/well, 50,000 cells/cm²) and treated with different concentrations of KGN. The cells were collected by washing with ice-cold phosphate-buffered solution (PBS) and lysed with cell lysis buffer (Beyotime). The concentration of total lysate proteins was quantified with a BCA protein assay kit (Beyotime). Each lysate was mixed with colorimetric assay substrate solution from the kit and incubated at room temperature for 15 min. Absorbance at 520 nm was measured using a microplate reader (BioTek), alongside a standard curve.