GSH/GSSG-Glo™ Assay

The GSH/GSSG-Glo™ Assay is a luminescence-based system for the detection and quantification of total glutathione (GSH +GSSG), GSSG and GSH/GSSG ratios in cultured cells. A change in GSH levels is important in the assessment of toxicological responses and is an indicator of oxidative stress, potentially leading to apoptosis or cell death. The 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-d...

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The GSH/GSSG-Glo™ Assay is a luminescence-based system for the detection and quantification of total glutathione (GSH +GSSG), GSSG and GSH/GSSG ratios in cultured cells. A change in GSH levels is important in the assessment of toxicological responses and is an indicator of oxidative stress, potentially leading to apoptosis or cell death. The 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. Determination of total glutathione and 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.

Features - Benefits

• Physiologically Relevant GSH/GSSG Ratios: Actual levels of total glutathione and GSSG are measured directly in cell-culture wells, minimizing the loss of GSH and GSSG, compared to conventional assays that require upfront sample preparation and indirect GSSG calculation.
• More Robust Performance: Bioluminescent technology and a simple protocol minimize sample handling, reducing variability.
• Simplified Protocol: Assay reagents are added directly to cells cultured in multiwell plates. The homogeneous add-mix-read format eliminates time-consuming sample deproteination and centrifugation steps required of conventional assays.
• Greater Sensitivity: Fewer cells are required in these assays than in conventional assays because of the enhanced sensitivity.
• Faster Results: The homogeneous add-mix-read protocol minimizes hands-on time, and the bioluminescence technology minimizes incubation time.
• Adaptable to Automation: The glow-type signal is stable, with a half-life greater than two hours, and the protocol is adaptable to automation in 96- and 384-well plates.
• No Fluorescence Interference: Using luminescence readout eliminates the fluorescent interference between reagents and test compounds sometimes seen in fluorescence assays. Such overlap can confound analysis and present misleading or irrelevant data.

Applications

• Measure total glutathione and GSSG levels in cells as an indicator of cell viability.
• Measure the GSH/GSSG ratios in cells as an indicator of oxidative stress.
• Profile drugs and new chemical entities for modulation of total GSH and GSSG levels in cells.

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