Description
Valuable Measurement of Glutathione (GSH-to-GSSG) Ratios
The GSH/GSSG-Glo™ Assay is a luminescence-based system that detects and quantifies total glutathione (GSH + GSSG), GSSG and GSH-to-GSSG ratios in cultured cells. Reduced glutathione (GSH) and oxidized glutathione (GSSG) both exist in healthy cells, the majority of which is in reduced (GSH) form. A change in GSH and GSSG levels are important when assessing toxicological responses and an indicator of oxidative stress, potentially leading to apoptosis or cell death. The GSH and GSSG detection assay provides a simple, rapid multiwell-plate format where stable luminescent signals are correlated with either the total GSH or the GSSG concentration of a sample directly in culture wells.
Both total glutathione and GSSG determinations are based on the reaction where GSH-dependent conversion of a GSH probe, Luciferin-NT, to luciferin by a glutathione-S-transferase enzyme is coupled to a firefly luciferase reaction. Light from luciferase is dependent on the amount of luciferin formed, which is in turn dependent on the amount of GSH present. This makes the luminescent signal proportional to the amount of GSH. Determining the total glutathione and amount of GSSG are performed in parallel reactions. In one configuration, the assay reagents measure total glutathione using a reducing agent that converts all the glutathione, GSH and GSSG in a cell lysate to the reduced form, GSH. In a second configuration, the assay reagents are used to measure only the oxidized form, GSSG. In this case, a reagent is added that blocks all the GSH while leaving the GSSG intact. This blocking step is followed by a reducing step that converts the GSSG to GSH for quantification in the luminescent reaction. Because the assays are performed directly on cells in culture wells, loss of GSH or GSSG is minimized, reducing variability.
GSH/GSSG Ratio Determination in Different Cell Lines
The GSH/GSSG-Glo™ Assay. GSH-dependent conversion of a GSH probe, Luciferin-NT, to luciferin by a glutathione S-transferase enzyme is coupled to a firefly luciferase reaction.
