Cell Meter™ Intracellular GSH Assay Kit *Optimized for Flow Cytometry with 405 nm excitation*

Protocol summary

1. Prepare cells with test compounds at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL
2. Prepare and add ThiolTrace^TM Violet 500 working solution to cells
3. Incubate at 37^oC for 20 to 30 minutes
4. Read fluorescence intensity at Ex/Em = 405/525 nm-Pacific Orange filter set

Important notes
Thaw at room temperature before starting the experiment.

ThiolTrace^TM Violet 500 stock solution (500X):
Add 200 µL of DMSO (Component C) into the vial of ThiolTrace^TM Violet 500, and mix well. Note: Aliquot and stored the unused ThiolTrace^TM Violet 500 stock solution at -20 ^oC.  Avoid repeated freeze/thaw cycles.

ThiolTrace^TM Violet 500 working solution (1X):
Add 2 µL of ThiolTrace^TM Violet 500 stock solution into 1 mL of Assay Buffer (Component B) or buffer of your choice, and mix well. Note: ThiolTrace^TM Violet 500 working solution can be prepared in the cell culture medium containing serum.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Treat cells with test compounds for a desired period of time. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to the incubation with ThiolTrace™ Violet working solution. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
   
   
2. Centrifuge the cells at 1000 rpm for 4 minutes, and wash cells in 1 mL of buffer of your choice (Optional).
   
   
3. Resuspend cells in 1 mL ThiolTrace^TM Violet 500 working solution and incubate them at 37^oC incubator for 20 to 30 minutes.
   
   
4. Monitor the fluorescence intensity with a flow cytometer using Pacific Orange filter set (Ex/Em = 405/525 nm). Gate on the cells of interest, excluding debris.