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.