Tissue reactive oxygen species detection kit

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.

Total ROS was measured with a ROS Assay Kit (Beyotime) on an Accuri C6 flow cytometer (BD Biosciences). Flow cytometry data in FCS format, generated by BD Accuri C6 Software v1.0.264.21 (BD Biosciences), were analyzed utilizing FlowJo v10.0.7 (BD Biosciences). Flow cytometric analyses were performed utilizing a uniform gating strategy, details of which are graphically delineated in the Supplementary Information. In parallel, mitochondrial ROS was detected by a Cell Meter Fluorimetric Mitochondrial Superoxide Activity Assay Kit Optimized for Microplate Reader (AAT Bioquest, Sunnyvale, CA, USA) according to the kit instructions. ROS levels in colon tissue were detected using the Tissue Reactive Oxygen Species Detection Kit (BestBio, Shanghai, China). A Varioskan Multimode Microplate Reader (Thermo Fisher Scientific) was used to measure the fluorescence intensity.