Gsh gssg glo kit

Total and oxidized glutathione was measured by GSH/GSSG-Glo Kit (Promega, WI, USA) following the manufacturer’s protocol with the following modifications. First, plasma was loaded onto an Amicon Ultra 10 K centrifugal filter device (MilliporeSigma, MA, USA, 15,000 × g for 30 min at 4 °C) for filtration. Next, a mixture of 2.2 µL filtered plasma and 19.8 µL saline (22 µL total) was loaded onto a 96-well microtiter plate (ThermoFisher Scientific, MA, USA). Twenty-two microliter of total or oxidized glutathione reagent (100 mM of N-Ethylmaleimide was included in the oxidized glutathione reagent) supplied by the kit was then added onto each well. After five minutes of shaking the plate, 44 μL of luciferin generation reagent supplied by the kit was added onto each well. After thirty minutes of incubation, luciferin detection reagent supplied by the kit was added onto each well and the plate was equilibrated for 15 min. The intensity of chemiluminescence was measured using a plate reader (TECAN Infinite M200Pro, Tecan Group Ltd., Switzerland). The absolute concentrations of total and oxidized glutathione were calculated with the use of standard curves of glutathione supplied by the kit.

Cellular glutathione was quantified using the GSH/GSSG-Glo kit (Promega) according to the instructions provided by the manufacturer. Drug-treated samples were normalized to parallel cell viability measurements using the CellTiter-Glo assay (Promega).

The ratio between reduced and oxidized glutathione is a good indicator of oxidative stress in cells. Cells were plated in 96-well plates and allowed to attach overnight. Post treatments, growth media was removed cells were washed with PBS, and total glutathione and GSSG were each assayed in triplicate via GSH/GSSG-Glo^™ kit (Promega, Madison, WI, USA) following manufacturers instructions using SpectraMax M2e (Molecular devices).