Gsh assay kit

HUVECs monolayers that were 80% confluent were organized in groups and received the following treatments: no treatment (control), RTV (7.5 μM) only, ginsenoside Rb1 (20 μM) only, and RTV (7.5 μM) after pretreatment for one hour with ginsenoside Rb1 (20 μM). The cells were harvested by scrapping the monolayer in PBS after being washed 2 times with 1x cold PBS. Cellular oxidative stress was indirectly determined by measuring glutathione levels with a GSH assay kit (Promega, Madison, WI), as per the manufacturer’s instructions.

Sub-confluent cultures of cells were transfected with 4Ub-FL or FL plasmid using Lipofectamine 2000 reagent (Life Technologies). Cells were seeded at 10,000 cells/well in 96-wells plate 48 hr post transfection and incubated with compounds or vehicle (DMSO) at the doses and times indicated. Luciferase activity in cell lysate was determined with a luciferase assay kit (Promega) according to the manufacturer’s instructions. Bioluminescence was measured by using a Glomax Multidetection system (Promega). Glutathione was assayed using the Promega GSH assay kit (V6611).

GSH activity was measured with a commercially available GSH assay kit according to the manufacturer’s instructions (Promega) requiring flow stoppage, incubation period, and generation of discrete data. Briefly, after treatment with compounds, cells were washed with PBS, and 30 μL of GSH-Glo reagent 1X with luciferin-NT substrate and glutathione S-transferase were added in each well. After 30 min, the lysed cells were transferred in a 96-well opaque white luminometer plate and added the same volume of reconstituted luciferin detection reagent for 20 min. GSH activity was measured using a luminometer. This assay is specific for GSH; other thiols do not cause interference in the assay. Cellular GSH concentration was calculated as fold change over vehicle controls.