Cell Meter™ Intracellular GSH Assay Kit *Optimized for Flow Cytometry*

Protocol summary

1. Prepare cells with test compounds at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL
2. Add 5 µL of 200X Thiolite™ Green into 1 mL of cell solution
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 15 to 30 minutes
4. Pellet the cells and resuspend the cells in 1 mL of growth medium
5. Analyze cells using flow cytometer with FL1 channel

Important notes
Thaw all the kit components at room temperature before starting the experiment.

1. Thiolite™ Green stock solution (200X):
Add 500 µL of DMSO (Component C) into the vial of Thiolite™ Green (Component A) and mix well to make 200X Thiolite™ Green stock solution. Protect from light.

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

1. For each sample, prepare cells in 1 mL warm medium or buffer of your choice at a density of 5×10⁵ to 1×10⁶ cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
   
   
2. Treat cells with test compounds for a desired period of time to induce apoptosis.
   
   
3. Add 5 µL of 200X Thiolite™ Green stock solution into the treated cells.
   
   
4. Incubate the cells in a 37°C, 5% CO₂ incubator for 15 to 30 minutes. 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 Thiolite™ Green dye-loading solution. The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
   
   
5. Optional: Centrifuge the cells at 1000 rpm for 4 minutes and then re-suspend cells in 1 mL of Assay Buffer (Component B) or buffer of your choice.
   
   
6. Monitor the fluorescence intensity using a flow cytometer with FL1 channel (Ex/Em = 490/525 nm). Gate on the cells of interest, excluding debris.