Glutathione colorimetric detection kit

The concentrations of glutathione, cysteine, and ROS were measured in glioma cells using Glutathione Colorimetric Detection Kit (Cat # K261-100, BioVision, Milpitas, CA, USA), Cysteine Assay Kit (Cat # MAK255-1KT, Sigma), and CM-H2DCF-DA (Thermo Fisher Scientific, H₂O₂ detection) following the standard protocols, respectively.

To assay total cellular glutathione (GSH), tumor and normal cells were exposed to PL and N-acetyl-L-cysteine (3 mM, NAC, Sigma-Aldrich). Cells (1 × 10⁶) were collected, centrifuged and lysed in 100 μL ice-cold lysis buffer for 10 min. The lysate was centrifuged for 10 min and the supernatant was used to assay GSH with a glutathione colorimetric detection kit (BioVision Inc., Milpitas, CA, USA). The total amount of GSH was measured using a fluorescence plate reader (Molecular Devices) at excitation (ex)/emission (em) = 380/460 nm. Quantification of glutathione disulfide (GSSG) was performed using a GSH/GSSG detection kit (ABcam, Cambridge, MA, USA). The drug-treated cells were collected in ice-cold buffer, homogenized and sonicated in icy water. GSH quencher (10 μL) was added and incubated for 10 min to quench GSH at room temperature. The samples were centrifuged and the supernatant was used to determine the GSSG concentration according to the manuscript protocol using a fluorescence plate reader. The change in GSSG levels in PL-treated samples compared to DMSO-treated control samples was expressed as the fold change.