Gsh glo kit

Cells were plated in 96-well plates and treated with erastin for 24 hours. Intracellular glutathione was determined using GSH-Glo kit (Promega) following manufacturer's instructions. Values were normalized to cell number.

As indicative of oxidative stress, GSH levels were measured using GSH-Glo Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. This test quantifies reduced glutathione based on the luciferase conversion reaction. For this test, only two MeHg concentrations were used: 0.1 and 5 µM MeHg, which represent non-toxic and toxic values, respectively, under the experimental conditions of the present study. After 24 h of exposure to MeHg, medium was removed and GSH-Glo reagent (luciferin-NT + glutathione S-transferase + GSH-Glo reaction buffer) was added to the samples and incubated for 30 min at room temperature. Thereafter, 100 µl of luciferin detection reagent were added. The samples were equilibrated at room temperature for 15 min and the luminescence signal was read using the Glomax multidetector system (Promega). The luminesce signal were used as an indicative of reduced glutathione on each well and was normalized by the number of viable cells. All experimental steps are summarized in Fig. 1.

MitoSOX Red (catalog no. M36008, Thermo Fisher Scientific) was used to measure mitochondrial superoxide. Total ROS levels were measured using a ROS-Glo Kit (catalog no. G8820, Promega) and glutathione levels were measured using a GSH Glo kit (catalog no. V6911, Promega, Wisconsin, USA).

Polyacrylic acid (PAA, MW 1,800), polyvinylpyrrolidone (PVP, MW 29,000), potassium permanganate (KMnO₄), L-glutathione reduced (GSH), manganese chloride tetrahydrate (MnCl₂·4H₂O), 2',7'-dichlorofluorescein diacetate (DCF-DA), and cobalt (II) chloride hexahydrate (CoCl₂·6H₂O) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). A thiol detection assay kit was purchased from Abnova (Taipei City, Taiwan). Biotech cellulose ester dialysis membrane (Spectra/Por®, molecular weight cut off 100 kDa) was obtained from Spectrum Labs (Laguna Hills, CA, USA). A CCK-8 cell viability assay kit was obtained from Dojindo (Kumamoto, Japan). A GSH-Glo kit was purchased from Promega (Madison, WI, USA). Cyclin B1 and cyclin D1 antibodies were purchased from Cell signaling Technology (Danvers, MA, USA). GAPDH antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). All chemicals and solvents were used without further purification.

The intracellular levels of GSH were measured with the GSH-Glo™ kit (Promega, WI, USA). Briefly, 8000 cells were incubated in a 96-well plate. The next day, the medium was removed, rinsed twice with PBS and glucose-free medium was added. A total of 100 μL of 1 × GSH-Glo™ reagent were then added into the 96-well plate followed by the removal of the medium 12 h later. With slight shaking, the well was incubated for 30 min at room temperature. Equal volumes of reconstituted luciferin-detection reagent were then added into each well and samples were incubated for an additional 15 min at room temperature. Luminescence detection in a micro-well reader was then conducted. The intracellular levels of NADPH, total NADP (NADPH + NADP⁺), and ATP were measured with the NADP/NADPH-Glo^TM kit and the CellTiter-Glo^® kit (Promega, WI, USA), respectively, according to the manufacturer’s instructions.