| MS Description
|
Equal amounts of cells (10 ml cell culture … Equal amounts of cells (10 ml cell culture with A730 = 1.0) were harvested by rapid filtration, and metabolites were extracted using a previously described method (Soga, 2007; Yoshida et al., 2008), with modifications. Briefly, the harvested cells were immediately filtered, and then the intermediate metabolites were quenched and extracted in a 1.2 ml solvent mixture (CHCl3 : CH3OH : H2O, 2.5:2.5:1, v/v/v) containing 10 μg l−1 of D-(+)-camphor-10-sulfonic acid as an internal standard for semiquantitative analysis. After centrifugation at 13 000 r.p.m. (15 000 g) at 4°C for 5 min, 400 μl of the upper phase was transferred to a new tube and vacuum-dried. The intracellular metabolites were quantified by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LCMS-8040 triple quadrupole LC/MS/MS spectrometer; Shimadzu, Kyoto, Japan) as described previously (Luo et al., 2007). s described previously (Luo et al., 2007).
|
