Gst activity assay 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.

Glutathione S‐transferase activity was quantified with a GST Activity Assay Kit (Solarbio) according to the manufacturer's instructions. Briefly, a total of 1 × 10⁶ cells were treated with 1 ml of activity detection reagent 1 and homogenized in an ice bath, and the precipitate was discarded. The supernatant (20 μl) was mixed with 180 μl of reagent 2 and 20 μl of reagent 3 in wells of a 96‐well plate. Activity was detected via increased absorbance at 340 nm in a plate reader.

Five canonical characteristics of ferroptosis were detected, including cellular concentrations of iron and malondialdehyde (MDA), GPx and GST activities, and mitochondrial morphology. For the iron, MDA and GSH-oxidized glutathione (GSSG) content assay, an Iron Colorimetric Assay Kit (Elabscience, E-BC-K139-S), a Lipid Peroxidation MDA Assay Kit (Beyotime, S0131S), and a GSH and GSSG Assay Kit (Beyotime, S0053) were used according to the manufacturer protocols. GPx and GST activity assays were performed using a glutathione peroxidase (GSH-PX) assay kit (Nanjing Jiancheng Bioengineering Institute, A005-1-2) and GST activity assay kit (BC0355, Solarbio, Beijing, China) following manufacturer protocols. Morphological observations of mitochondria were conducted using a transmission electron microscope (TEM) FEI Tecnai F20 (FEI, Netherlands) as previously described (57 (link)).