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MB Sample ID: SA184054

Local Sample ID:	3
Subject ID:	SU002030
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002030
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
3	SA184054	FL022654	Wild-type	Genotype

Collection:

Collection ID:	CO002023
Collection Summary:	All samples extracted from mice were collected in accordance with regulations and established guidelines for humane treatment of research animals and were reviewed and approved by an Institutional Animal Care and Use Committee, Project Code HD-EX00066. Primary hepatocytes were isolated from male mice between 12-18 weeks of age by the two-step collagenase perfusion method. Mice were fasted 16 hours before the ex-periments. After isolation, cells were plated in M199 media with 10% FBS for 4 hours in 6-well plates pre-coated with collagen I. After cells attached to the plates, they were washed with glucose output media (GOM) (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.2 mM CaCl2, 20 mM NaCO3, 25 mM HEPES pH 7.4, and 0.025% BSA), and incubated in fresh GOM for 2 hour. GOM media was replaced with fresh, pre-warmed GOM media and cellular treatments initiated. Hepatocytes were treated with 5mM unlabeled glutamine for 60 min. Hepatocytes were washed with ice cold PBS twice and immediately frozen in liquid nitrogen.
Sample Type:	Liver

Treatment:

Treatment ID:	TR002042
Treatment Summary:	All samples extracted from mice were collected in accordance with regulations and established guidelines for humane treatment of research animals and were reviewed and approved by an Institutional Animal Care and Use Committee, Project Code HD-EX00066. Primary hepatocytes were isolated from male mice between 12-18 weeks of age by the two-step collagenase perfusion method. Mice were fasted 16 hours before the ex-periments. After isolation, cells were plated in M199 media with 10% FBS for 4 hours in 6-well plates pre-coated with collagen I. After cells attached to the plates, they were washed with glucose output media (GOM) (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.2 mM CaCl2, 20 mM NaCO3, 25 mM HEPES pH 7.4, and 0.025% BSA), and incubated in fresh GOM for 2 hour. GOM media was replaced with fresh, pre-warmed GOM media and cellular treatments initiated. Hepatocytes were treated with 5mM unlabeled glutamine for 60 min. Hepatocytes were washed with ice cold PBS twice and immediately frozen in liquid nitrogen.

Treatment ID:

TR002042

Treatment Summary:

All samples extracted from mice were collected in accordance with regulations and established guidelines for humane treatment of research animals and were reviewed and approved by an Institutional Animal Care and Use Committee, Project Code HD-EX00066. Primary hepatocytes were isolated from male mice between 12-18 weeks of age by the two-step collagenase perfusion method. Mice were fasted 16 hours before the ex-periments. After isolation, cells were plated in M199 media with 10% FBS for 4 hours in 6-well plates pre-coated with collagen I. After cells attached to the plates, they were washed with glucose output media (GOM) (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.2 mM CaCl2, 20 mM NaCO3, 25 mM HEPES pH 7.4, and 0.025% BSA), and incubated in fresh GOM for 2 hour. GOM media was replaced with fresh, pre-warmed GOM media and cellular treatments initiated. Hepatocytes were treated with 5mM unlabeled glutamine for 60 min. Hepatocytes were washed with ice cold PBS twice and immediately frozen in liquid nitrogen.

Sample Preparation:

Sampleprep ID:	SP002036
Sampleprep Summary:	Metabolites were extracted on dry ice with 80:20 methanol:water, vortexed, centrifugation at 14,000 g at 4 °C for 15 minutes, dried under nitrogen gas, and reconstitution in 35:40:25 acetonitrile:methanol:water for injection.

Combined analysis:

Analysis ID	AN003177
Analysis type	MS
Chromatography type	Unspecified
Chromatography system	Dionex UltiMate 3000 RSLC
Column	HILICpak VT50 2D (150 mm x 2.0 mm,5um particle size,Shodex,Japan)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap
Ion Mode	NEGATIVE
Units	Peak area

Chromatography:

Chromatography ID:	CH002349
Chromatography Summary:	Liquid chromatography separation was achieved on a HILICpak VT50 2D column (150 mm x 2.0 mm, 5 µm particle size, Shodex, Japan). Buffer A consists of 90% acetonitrile, 10% water, containing 20 mM Triethylamine : Formic acid at pH 9.18; Buffer B consists of 5% acetonitrile, 95% water containing 54 mM Triethyl-amine : Formic acid at pH 3.03. Flow rate is 0.2mL/min from 0 to 5 minutes, then 0.3 mL/min from 5.1 to 58 min, and reduced again to 0.2mL/min from 58.1 to 60 min. The gradient starts with 0%B from 0 to 10 min, then increases linearly from 0 to 16%B from 10 to 27 min, up to 65%B at 32 min, 87%B at 34 min, 100%B hold from 34.1 to 47 min, then 0%B from 47.1 to 60 min.
Instrument Name:	Dionex UltiMate 3000 RSLC
Column Name:	HILICpak VT50 2D (150 mm x 2.0 mm,5um particle size,Shodex,Japan)
Chromatography Type:	Unspecified

MS:

MS ID:	MS002955
Analysis ID:	AN003177
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The Mass Spec parameters are set as Source Fragmentation: None; Sheath gas flow rate: 45; Aux gas flow rate: 15; Sweep gas flow rate: 3; Spray voltage: 3.00 kV; Ca-pillary temp: 310°C; S-lens RF level: 50; Aux gas heater temp: 350°C; For Full MS: Scan range: 65.0 to 975.0 m/z; Resolution: 140,000; Polarity: Negative; AGC target: 3e6; Max-imum IT: 500 ms.
Ion Mode:	NEGATIVE