Intracellular GSH Assay Kit

1. Prepare samples and controls., 2. Dilute 10X Binding Buffer 1:10 WITH DiH2O., 3. Wash cells twice in ice-cold culture medium or PBS and resuspend in ice-cold 1X Binding Buffer at 5 x 105 – 1 x 106 cells/mL., 4. Reconstitute Bovine Annexin V-Fluorescein in 1 mL Annexin Reconstitution Buffer, 1X., 5. Dilute reconstituted Annexin V-Fluorescein 1:5 in PBS., 6. Add diluted Annexin V-Fluorescein to each sample at ~1:10., 7. Dilute PI 1:10 in PBS., 8. Add diluted PI to each sample at ~1:20., 9. Incubate 15 minutes on ice and protected from light., 10. Add 400 µL 1X Binding Buffer to each sample., 11. Analyze with a flow cytometer. Annexin V-Fluorescein excites at 494 nm and emits at 521 nm. PI excites at 536 nm and emits at 617 nm.

γ-L-glutamyl-L-cysteinyl-glycine (GSH; glutathione) 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 by- products, 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 this 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 reagent in the assay is a proprietary thiol-sensitive dye. This thiol-reactive dye 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 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. Because the GSH-bound form of the dye has the fluorescence properties of fluorescein (Ex/Em = 490/525 nm), it is perfectly suited to most standard flow cytometry instrument optical packages. Background fluorescence issues are minimized due to the non-fluorescent nature of the free form of the thiol-sensitive dye. Intracellular GSH Assay requires minimal procedural steps and hands-on time to complete. Experimentally treated suspension or trypsin-EDTA disassociated adherent cells are ready for analysis after briefly staining (15-30 minutes) with the GSH quantitation dye. Since the unbound reagent is non-fluorescent, subsequent wash steps are not required, thus simplifying the assay procedure. Each kit will enable the assessment of up to 100 (1 mL) samples via flow cytometry. This flow cytometer assay can be adapted for use with a fluorescence microscope or plate reader equipped with FITC/FAM dye-compatible excitation/emission optics. Use and optimization of these alternative fluorescence analysis methods will require additional modifications to this flow cytometer based protocol.