Assess changes in intracellular glutathione levels with the Intracellular GSH Assay. This whole cell glutathione assay employs a proprietary thiol-sensitive dye, ThioBright™ Green, to monitor relative changes in the concentration of the free form of glutathione, aka GSH. Analyze the fluorescent signal by flow cytometry.
Glutathione, γ-L-glutamyl-L-cysteinyl-glycine or GSH, is the most abundant non-protein thiol in cells. GSH is involved in the regulation of a number of essential biochemical processes within the cell. Primarily recognized as a key intracellular source of reducing power for combating the toxic accumulation of free radical byproducts, GSH is also involved with detoxification and removal of exo/endogenous toxins and alkylating agents. In its role as a cell signaling agent, GSH is involved in DNA synthesis and cell proliferation regulation. Due to the genetically conserved molecular processes by which cells die, intracellular levels of GSH can favor a cell death pathway via apoptosis (adequate intracellular GSH stores) or via necrosis or autophagy (depleted intracellular GSH stores). Detection of a drop in intracellular GSH concentration in an experimental cell population relative to a negative (healthy cell) control is often indicative of an apoptosis induction event. Due to the strong correlation between intracellular GSH depletion and the onset of the apoptotic process, ICT’s Intracellular GSH Assay provides a good screening option for assessing the potency of apoptosis inhibitor and activator reagents. The kit provides all the essential reagents and an easy to follow protocol to assess changes in intracellular GSH levels using a flow cytometry analysis method. The key assay reagent is a proprietary thiol-sensitive dye, ThioBright™ Green, which quickly penetrates cell membrane structures and accumulates primarily within the cytosol of living cells. In the presence of free-thiol-containing molecules such as GSH, the non-fluorescent ThioBright™ Green dye molecule binds covalently to the GSH target molecule and converts to the fluorescent form of the dye. During periods of oxidative stress or GSH depletion associated with cell death processes, cytosolic concentrations of the green fluorescent dye form become significantly diminished. The reduction in intracellular GSH concentration directly translates into an easily monitored reduction in the green fluorescence output within the dying or oxidatively stressed cell population.
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- Prepare samples and controls.
- Reconstitute ThioBright™ Green with 500 µL DMSO to produce a 200X stock concentrate.
- Add ThioBright Green to each sample at 1:200. For example, add 5 µL to 995 µL cultured cells.
- Incubate 15-30 minutes.
- Optional: Spin and pellet cells, remove supernatants, and gently resuspend in Assay Buffer or other isotonic buffer.
- If desired, label with additional stains, such as Hoechst, Propidium Iodide, 7-AAD, or an antibody.
- Analyze with a flow cytometer. ThioBright Green excites at 490 nm and emits at 525 nm.