Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA057696

Local Sample ID:	10_25_2016_islet_OHE_20
Subject ID:	SU001003
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6N
Age Or Age Range:	4 weeks old (young) or 10-15 months old (old)
Gender:	Male and female
Animal Animal Supplier:	Charles River Labs
Animal Feed:	standard chow diet (PicoLab Rodent Diet 20 pellets, #5053

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Subject:

Subject ID:	SU001003
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6N
Age Or Age Range:	4 weeks old (young) or 10-15 months old (old)
Gender:	Male and female
Animal Animal Supplier:	Charles River Labs
Animal Feed:	standard chow diet (PicoLab Rodent Diet 20 pellets, #5053

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
10_25_2016_islet_OHE_20	SA057696	FL010378	Old (10-15 months)	Age
10_25_2016_islet_OHE_20	SA057696	FL010378	16.8mM Glucose (60 min)	Treatment

Collection:

Collection ID:	CO000997
Collection Summary:	Pancreata were digested using Liberase TL, and islets were purified using a Histopaque gradient followed by hand-picking under a dissecting microscope. Islets were allowed to recover overnight in RPMI 1640 media supplemented with 8 mM glucose, 10% FBS, 2 mM L-glutamine, 100 U/mL Pen/Strep, 1 mM sodium pyruvate, 10 mM HEPES, and 0.25 mg/mL amphoterecin B.
Collection Protocol Filename:	Methods.pdf
Sample Type:	Pancreatic Islets

Treatment:

Treatment ID:	TR001017
Treatment Summary:	Islets were starved for 1 hour in KRBH supplemented with 2.8 mM glucose as for GSIS assays. Islets were then transferred to petri dishes containing U-13C-labeled glucose as indicated and immediately seeded into wells of non-treated 12-well plates at a density of 100 size-matched islets per replicate in a final volume of 500 μl KRBH containing basal (2.8 mM) or stimulatory (16.8 mM) concentrations of labeled glucose. Tracing was performed for the indicated durations at 37oC with 5% CO2. Following the trace, islets were rapidly chilled by swirling plate to resuspend islets then transferring entire contents of each well to an Eppendorf tube on ice. Islets were then pelleted by centrifugation at 500 x g for 2 min at 4oC, washed in 1 mL of ice cold 0.9% NaCl, centrifuged as before, then pellets were frozen in a dry ice/ethanol bath and stored at -80oC.

Treatment ID:

TR001017

Treatment Summary:

Islets were starved for 1 hour in KRBH supplemented with 2.8 mM glucose as for GSIS assays. Islets were then transferred to petri dishes containing U-13C-labeled glucose as indicated and immediately seeded into wells of non-treated 12-well plates at a density of 100 size-matched islets per replicate in a final volume of 500 μl KRBH containing basal (2.8 mM) or stimulatory (16.8 mM) concentrations of labeled glucose. Tracing was performed for the indicated durations at 37oC with 5% CO2. Following the trace, islets were rapidly chilled by swirling plate to resuspend islets then transferring entire contents of each well to an Eppendorf tube on ice. Islets were then pelleted by centrifugation at 500 x g for 2 min at 4oC, washed in 1 mL of ice cold 0.9% NaCl, centrifuged as before, then pellets were frozen in a dry ice/ethanol bath and stored at -80oC.

Sample Preparation:

Sampleprep ID:	SP001010
Sampleprep Summary:	Metabolites were extracted from pellets using a bligh and dyer-based methanol/chloroform/water extraction with inclusion of norvaline as a polar internal standard. Briefly, 250 μl MeOH, 250 μl CHCL3, 100 μl water containing norvaline were added to pellets. This was vortexed for 10 minutes followed by centrifugation at 10,000 g for 5 minutes at 4°C. The upper phase was separated and dried under vacuum at 4°C until dry. Polar metabolites were derivatized in 2% (w/v) methoxyamine hydrochloride in pyridine and incubated at 45°C for 60 minutes. Samples were then silylated with N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide (MtBSTFA) with 1% tert-butyldimethylchlorosilane (tBDMCS) at 45°C for 30 minutes. Polar derivatives were analyzed by GC-MS using a DB-35MS column (30m x 0.25 mm i.d. x 0.25 μm) installed in an Agilent 7890B gas chromatograph (GC) interfaced with an Agilent 5977A mass spectrometer (MS) with an XTR EI source using the following temperature program: 100 °C initial, increase by 3.5 °C/min to 255 °C, increase by 15 °C/min to 320 °C and hold for 3 min.

Sampleprep ID:

SP001010

Sampleprep Summary:

Metabolites were extracted from pellets using a bligh and dyer-based methanol/chloroform/water extraction with inclusion of norvaline as a polar internal standard. Briefly, 250 μl MeOH, 250 μl CHCL3, 100 μl water containing norvaline were added to pellets. This was vortexed for 10 minutes followed by centrifugation at 10,000 g for 5 minutes at 4°C. The upper phase was separated and dried under vacuum at 4°C until dry. Polar metabolites were derivatized in 2% (w/v) methoxyamine hydrochloride in pyridine and incubated at 45°C for 60 minutes. Samples were then silylated with N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide (MtBSTFA) with 1% tert-butyldimethylchlorosilane (tBDMCS) at 45°C for 30 minutes. Polar derivatives were analyzed by GC-MS using a DB-35MS column (30m x 0.25 mm i.d. x 0.25 μm) installed in an Agilent 7890B gas chromatograph (GC) interfaced with an Agilent 5977A mass spectrometer (MS) with an XTR EI source using the following temperature program: 100 °C initial, increase by 3.5 °C/min to 255 °C, increase by 15 °C/min to 320 °C and hold for 3 min.

Combined analysis:

Analysis ID	AN001578
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890B
Column	Agilent DB5-MS (15m × 0.25mm)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5977A
Ion Mode	POSITIVE
Units	Total ion counts

Chromatography:

Chromatography ID:	CH001107
Chromatography Summary:	Polar derivatives were analyzed by GC-MS using a DB-35MS column (30m x 0.25 mm i.d. x 0.25 μm) installed in an Agilent 7890B gas chromatograph (GC) interfaced with an Agilent 5977A mass spectrometer (MS) with an XTR EI source using the following temperature program: 100 °C initial, increase by 3.5 °C/min to 255 °C, increase by 15 °C/min to 320 °C and hold for 3 min.
Instrument Name:	Agilent 7890B
Column Name:	Agilent DB5-MS (15m × 0.25mm)
Chromatography Type:	GC

MS:

MS ID:	MS001456
Analysis ID:	AN001578
Instrument Name:	Agilent 5977A
Instrument Type:	Single quadrupole
MS Type:	EI
Ion Mode:	POSITIVE