GSH-Glo Glutathione Assay

• Versatile: Measure reduced glutathione and/or total glutathione levels from cells, tissue, blood...
• Fast and Easy to Use: Measure GSH levels directly from cell culture wells; no deproteinization step required.
• Excellent Sensitivity: Able to measure GSH levels from as few as several hundred cells. Easily scalable to 384-well plates.
• Robust: No interference by oxidized GSH or reducing agents.
• Proven Luminescent Technology: Assay powered by Ultra-Glo™ Luciferase. No interference by fluorescent compounds.

Schematic of the GSH-Glo™ Glutathione Assay. The assay is performed in two steps. In the 1st step, cells are lysed in the presence of the luciferin-NT substrate and glutathione S-transferase. Glutathione in the cells drives the formation of luciferin. In the 2nd step, Luciferin Detection Reagent is added to produce light that is directly proportional to the amount of GSH in the reaction.

Measurement of GSH Depletion and Recovery in Treated Cells. HeLa cells (5000 cells/well in 96 well format) were exposed to L-Buthionine-sulfoximine (BSO). BSO inhibits GSH synthesis thus reducing cellular GSH level.

Legend: 20hr BSO- cells treated with BSO and assayed for GSH after 20 hours. 40hrs BSO- cells treated with BSO, washed at 20 hours and fresh media plus BSO added. Cells assayed for GSH after 40 hours. BSO+Recovery- cells treated with BSO for 20 hours. After 20 hours cells were washed 2X with PBS and covered with fresh media without BSO. Cells assayed for GSH after 40 hours. All samples assayed for GSH using the GSH-Glo™ Glutathione Assay according to the protocol outlined in technical bulletin TB369.

Titration data using the GSH-Glo™ Glutathione Assay. Panel A. A standard curve was generated by serial twofold dilutions of a glutathione 10X solution in the wells of a 384-well plate. Panel B. Serial twofold dilution of HepG2 cells were plated and allowed to attach overnight. The cells were assayed using the protocol outlined in technical bulletin TB369.

GSH depletion by acetaminophen in (APAP) rat hepatocytes. GSH levels in lysates from adherent cells in 24 well plates were measured with the GSH-Glo Glutathione Assay. GSH concentrations were determined by interpolation from a GSH standard curve generated using the bioluminescent system. Exposure to 5mM acetaminophen for three hours substantially reduced GSH only after treatment for two days with a P450 inducer, 30 μM pregnenolone 16a-carbonitrile (PCN).