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MB Sample ID: SA006098
Local Sample ID: | C4 |
Subject ID: | SU000134 |
Subject Type: | Animal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57BL/6J |
Age Or Age Range: | 13-wk-old |
Gender: | Male |
Species Group: | Mammal |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU000134 |
Subject Type: | Animal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57BL/6J |
Age Or Age Range: | 13-wk-old |
Gender: | Male |
Species Group: | Mammal |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
C4 | SA006098 | FL001578 | control | Treatment |
Collection:
Collection ID: | CO000118 |
Collection Summary: | Mitochondria were isolated from quadriceps muscle by differential centrifugation, as described previously (38). Briefly, quadriceps muscle samples were homogenized on ice using a motor-driven Potter-Elvehjem tissue grinder. After initial centrifugation, the supernatant containing the mitochondrial and sarcoplasmic fraction was transferred to a chilled microcentrifuge tube and centrifuged at 10,000 g for 2 min to pellet mitochondria. The supernatant containing sarcoplasmic fraction was frozen for further analysis. Mitochondrial pellet was washed twice by resuspending/centrifugation and finally suspended in a mitochondrial storage buffer. The levels of both the LCFa-CoA and sphingolipids in homogenates and various muscle fractions were normalized to total protein content, as measured by 660 nm Protein Assay (Thermo Scientific; Pierce Protein Biology Products, Rockford, IL). |
Sample Type: | Blood |
Treatment:
Treatment ID: | TR000136 |
Treatment Summary: | Control/Diabetic; insulin treated/Diabetic; insulin deprived |
Treatment Compound: | blank/Insulin/Insulin |
Treatment Route: | Skin implants |
Animal Anesthesia: | phenobarbital |
Animal Endp Euthanasia: | 5 weeks after treatment |
Animal Endp Tissue Coll List: | plasma, muscle, liver and skin |
Animal Endp Tissue Proc Method: | see attached reference publication PDF |
Sample Preparation:
Sampleprep ID: | SP000131 |
Sampleprep Summary: | Mitochondria were isolated from quadriceps muscle by differential centrifugation, as described previously (38). Briefly, quadriceps muscle samples were homogenized on ice using a motor-driven Potter-Elvehjem tissue grinder. After initial centrifugation, the supernatant containing the mitochondrial and sarcoplasmic fraction was transferred to a chilled microcentrifuge tube and centrifuged at 10,000 g for 2 min to pellet mitochondria. The supernatant containing sarcoplasmic fraction was frozen for further analysis. Mitochondrial pellet was washed twice by resuspending/centrifugation and finally suspended in a mitochondrial storage buffer. The levels of both the LCFa-CoA and sphingolipids in homogenates and various muscle fractions were normalized to total protein content, as measured by 660 nm Protein Assay (Thermo Scientific; Pierce Protein Biology Products, Rockford, IL). / Plasma free fatty acid concentrations were measured by liquid chromatography/mass spectrometry (LC/MS), as described previously (51). Briefly, 50 ?l of plasma was spiked with heptadecanoate internal standard (ISTD) and analyzed with Applied Biosystems (Foster City, CA) API5000 mass spectrometer coupled with a Cohesive (Franklin, MA) TX2 liquid chromatography system. Concentration of individual FFA was measured against a six-point standard curve prepared for each analyte. Both the ISTD and individual FFA standard curves were prepared in 2% fatty acid-free human albumin solution. All analytes were monitored as their [M ? H]? ions. plasma LCFa-CoA esters were estimated using the LC-MS/MS method (9). After extraction in the presence of internal standard (20 ng of heptadecanoyl-CoA), samples were analyzed by UHPLC-ESI-MS/MS operating in multiple reaction monitoring mode [Waters Acquity UHPLC, C8 UPLC BEH column 2.1 × 150 mm, 1.7 ?m (Waters, Milford, MA) and TSQ Quantum Ultra triple-quad mass spectrometer (Thermo Fisher Scientific, Waltham, MA)]. All standard curves were prepared using chemicals from Avanti Polar Lipids. |
Sampleprep Protocol Filename: | PMID-24368672-Zabielski-Nair-AJPEM-2014.pdf |
Sampleprep Protocol Comments: | Pubmed ID: 24368672 |
Combined analysis:
Analysis ID | AN000195 | AN000196 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | ||
Chromatography system | ||
Column | ||
MS Type | ESI | ESI |
MS instrument type | Triple quadrupole | Triple quadrupole |
MS instrument name | ABI Sciex API 5000 QQQ | Thermo TSQ Quantum Ultra |
Ion Mode | NEGATIVE | POSITIVE |
Units | uM | uM |
Chromatography:
Chromatography ID: | CH000129 |
Chromatography Summary: | Plasma free fatty acid concentrations were measured by liquid chromatography/mass spectrometry (LC/MS), as described previously (51). Briefly, 50 ?l of plasma was spiked with heptadecanoate internal standard (ISTD) and analyzed with Applied Biosystems (Foster City, CA) API5000 mass spectrometer coupled with a Cohesive (Franklin, MA) TX2 liquid chromatography system. Concentration of individual FFA was measured against a six-point standard curve prepared for each analyte. Both the ISTD and individual FFA standard curves were prepared in 2% fatty acid-free human albumin solution. All analytes were monitored as their [M ? H]? ions. LCFa-CoA esters were estimated using the LC-MS/MS method (9). After extraction in the presence of internal standard (20 ng of heptadecanoyl-CoA), samples were analyzed by UHPLC-ESI-MS/MS operating in multiple reaction monitoring mode [Waters Acquity UHPLC, C8 UPLC BEH column 2.1 150 mm, 1.7 ?m (Waters, Milford, MA) and TSQ Quantum Ultra triple-quad mass spectrometer (Thermo Fisher Scientific, Waltham, MA)]. All standard curves were prepared using chemicals from Avanti Polar Lipids. |
Methods Filename: | PMID-24368672-Zabielski-Nair-AJPEM-2014.pdf |
Chromatography Comments: | Pubmed ID: 24368672 |
MS:
MS ID: | MS000158 |
Analysis ID: | AN000195 |
Instrument Name: | ABI Sciex API 5000 QQQ |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | Briefly, 50 ul of plasma was spiked with heptadecanoate internal standard (ISTD) and analyzed with Applied Biosystems (Foster City, CA) API5000 mass spectrometer coupled with a Cohesive (Franklin, MA) TX2 liquid chromatography system. Concentration of individual FFA was measured against a six-point standard curve prepared for each analyte. Both the ISTD and individual FFA standard curves were prepared in 2% fatty acid-free human albumin solution. All analytes were monitored as their [M - H]- ions. |
Ion Mode: | NEGATIVE |
MS ID: | MS000159 |
Analysis ID: | AN000196 |
Instrument Name: | Thermo TSQ Quantum Ultra |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | LCFa-CoA esters were estimated using the LC-MS/MS method (9). After extraction in the presence of internal standard (20 ng of heptadecanoyl-CoA), samples were analyzed by UHPLC-ESI-MS/MS operating in multiple reaction monitoring mode [Waters Acquity UHPLC, C8 UPLC BEH column 2.1 × 150 mm, 1.7 um (Waters, Milford, MA) and TSQ Quantum Ultra triple-quad mass spectrometer (Thermo Fisher Scientific, Waltham, MA)]. All standard curves were prepared using chemicals from Avanti Polar Lipids. |
Ion Mode: | POSITIVE |