3-Measurement of GSH Content

Changes in the cellular content of GSH were measured after monobromobimane (mBrB) labeling as previously described (De Jesus Ferreira et al., 2005) (Baxter et al., 2015). For this, C6 rat glioma cells were seeded in 96-well culture plates. When reaching 80% confluence, the cells were treated with BSO (10 µM) or SFN (10 µM) or ACV (100 µM) for 24 h. The culture medium was then replaced by the extracellular medium containing 50 µM mBrB. The extracellular medium comprised 124 mM NaCl, 3.5 mM KCl, 25 mM NaHCO₃, 1.25 mM NaH₂PO₄, 1 mM CaCl₂, 2 mM MgSO₄, 10 mM D-glucose, and 10 mM HEPES (pH: 7.4). Incubation with mBrB lasted for 30 min, and then the cells were washed with the extracellular medium to remove unbound mBrB. Fluorescence was then measured in cellulo with a plate reader (Tecan “Spark” 20M) at 527 nm after excitation at 380 nm. Background fluorescence was obtained from mBrB unlabeled cells. For data processing, the background fluorescence was subtracted to all fluorimetric signals which were further normalized to mBrB fluorescence recorded in control cells. An experimental determination was performed eight times per experiment. The data are expressed as percentages of at least three distinct experiments performed on different cell cultures.