RPMI medium supplemented with 10% (v/v) fetal bovine serum for 2 days before starvation
General protocol information
Sampling method:
collected manually
Quenching procedure:
Stimulated cells washed twice with 5% mannitl and incubated for 10 min in 1ml of methanol containing 25 mM each of the three internal standards (L-methionine sulphone, D-camphor-10-sulphonic acid and 2-(N-morpholin-o)ethanesulfonic acid).
Extraction technique:
chloroform-methanol
Sample analyzing method:
enzymatic, CE-MS
Methods description - Notes
Metabolome analysis - the stimulated cells were washed twice with 5% mannitol and incubated for 10 min in 1 ml of methanol containing 25 µM each of the three internal standards (L-methionine sulphone, D-camphor-10-sulphonic acid and 2-(N-morpholin-o)ethanesulfonic acid). Aft
... er 500 µl of Milli-Q purified water was added, 600 µl of the solution was taken and mixed well with 400 µl of chloroform, and then centrifuged for 15 min at 20 000 g and 4 ºC. The separated 400-µl aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter to remove any protein. The filtrate (320 µl) was lyophilised and dissolved in 50 ml of Milli-Q water containing reference compounds (200 µM each of trimesate and 3-aminopyrrolidine) and then injected into a CE-TOF-MS system (Agilent Technologies) [1,2]. To measure the amount of PYRex by CE-TOF-MS, 400 µl of methanol containing 50 µM each of five internal standards (L-methionine sulphone, D-camphor-10-sulphonic acid, 2-(N-morpholin-o)ethanesulfonic acid, trimesate and 3-aminopyrrolidine) was added to 100 µl of the medium. After 200 µl of Milli-Q water and 500 µl of chloroform were added, the samples were subjected to the same protocol that was used to analyse the intracellular metabolome. For the data analysis, the obtained intracellular and extracellular absolute amounts (nmol) were converted into intracellular and extracellular concentrations (mM), respectively, by dividing the absolute amounts by the total intracellular water content of hepatocytes (2.64x10-5 L per sample; [3]) and the total extracellular medium content (4 ml), respectively.
--------------References---------------
[1] Soga T, Baran R, Suematsu M, Ueno Y, Ikeda S, Sakurakawa T, KakazuY, Ishikawa T, Robert M, Nishioka T, Tomita M (2006). Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption. J Biol Chem 281: 16768–16776. http://doi.org/fch7nt [2] Soga T, Igarashi K, Ito C, Mizobuchi K, Zimmermann H-P, Tomita M (2009) Metabolomic profiling of anionic metabolites by capillary electrophoresis mass spectrometry. Anal Chem 81: 6165–6174. http://doi.org/ftfn6j [3] Le Cam A, Freychet P (1977) Neutral amino acid transport. Characterization of the A and L systems in isolated rat hepatocytes. J Biol Chem 252: 148–156.
RPMI medium supplemented with 10% (v/v) fetal bovine serum for 2 days before starvation
General protocol information
Sampling method: collected manually
Quenching procedure: Stimulated cells washed twice with 5% mannitl and incubated for 10 min in 1ml of methanol containing 25 mM each of the
three internal standards (L-methionine sulphone, D-camphor-10-sulphonic acid and 2-(N-morpholin-o)ethanesulfonic acid).
Extraction technique: chloroform-methanol
Sample analyzing method: enzymatic, CE-TOF-MS
Methods description - Notes
Metabolome analysis - the stimulated cells were washed twice with 5% mannitol and incubated for 10 min in 1 ml of methanol containing 25 µM each of the three internal standards (L-methionine sulphone, D-camphor-10-sulphonic acid and 2-(N-morpholin-o)ethanesulfonic acid). After 500 µl of Milli-Q purified water was added, 600 µl of the solution was taken and mixed well with 400 µl of chloroform, and then centrifuged for 15 min at 20 000 g and 4 ºC. The separated 400-µl aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter to remove any protein. The filtrate (320 µl) was lyophilised and dissolved in 50 ml of Milli-Q water containing reference compounds (200 µM each of trimesate and 3-aminopyrrolidine) and then injected into a CE-TOF-MS system (Agilent Technologies) [1,2]. To measure the amount of PYRex by CE-TOF-MS, 400 µl of methanol containing 50 µM each of five internal standards (L-methionine sulphone, D-camphor-10-sulphonic acid, 2-(N-morpholin-o)ethanesulfonic acid, trimesate and 3-aminopyrrolidine) was added to 100 µl of the medium. After 200 µl of Milli-Q water and 500 µl of chloroform were added, the samples were subjected to the same protocol that was used to analyse the intracellular metabolome. For the data analysis, the obtained intracellular and extracellular absolute amounts (nmol) were converted into intracellular and extracellular concentrations (mM), respectively, by dividing the absolute amounts by the total intracellular water content of hepatocytes (2.64 x10- 5 L per sample; [3]) and the total extracellular medium content (4 ml), respectively.
--------------References---------------
[1] Soga T, Baran R, Suematsu M, Ueno Y, Ikeda S, Sakurakawa T, KakazuY, Ishikawa T, Robert M, Nishioka T, Tomita M (2006). Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption. J Biol Chem 281: 16768–16776.
[2] Soga T, Igarashi K, Ito C, Mizobuchi K, Zimmermann H-P, Tomita M (2009) Metabolomic profiling of anionic metabolites by capillary electrophoresis mass spectrometry. Anal Chem 81: 6165–6174.
[3] Le Cam A, Freychet P (1977) Neutral amino acid transport. Characterization of the A and L systems in isolated rat hepatocytes. J Biol Chem 252: 148–156.
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