Glutathione measurement
After sampling, the cell culture fluid was directly quenched using a -20°C ethanol bath and kept cold in a CoolRack® (Corning) during the sample preparation. A volume containing 107 cells was then centrifuged at 1000g, 1 min, -5°C. Cell pellets were washed twice using cold 0.9% NaCl solution and frozen at -80°C. The day of the analysis, the cell pellet was resuspended in a 10mM EDTA solution (pH 8) and a 13C-labelled glutathione internal standard (Buchem) was added. Cells were lysed using a sonifier (Brandson) during for 30 seconds (pulse mode [15sec on / 5sec off]) with a 10% amplitude. In order to measure total glutathione, a DTT solution was added to the lysate and incubated 30 min at room temperature to reduce glutathione disulfide (GSSG). After the 30 minutes, N-ethylmaleimide stock solution was added and incubated 40 min at room temperature, protected from light. Proteins were then precipitated by addition of -20°C acetone. After precipitation, the sample were centrifuged at 17000g for 5min and the supernatant was transferred in a new tube for overnight evaporation (Speedvac, Supplier). The sample pellet was reconstitute in mobile phase A (0.1% formic acid:water) using sonication. The sample was analyzed by LC-MS using a tandem quadrupole detector (Waters). The liquid chromatography was performed on a HSST 3 column of 10 cm (Waters) at 45°C and under a gradient of mobile phase A and B (0.1%formic acid:acetonitrile). GSH quantity were determined by comparison with the internal standard and normalized by the cell quantity.