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MB Sample ID: SA057694
Local Sample ID: | 10_25_2016_islet_OHC_18 |
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 |
Select appropriate tab below to view additional metadata details:
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_OHC_18 | SA057694 | FL010378 | Old (10-15 months) | Age |
10_25_2016_islet_OHC_18 | SA057694 | 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. |
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. |
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 |