Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA332539

Local Sample ID:	Heart504
Subject ID:	SU003192
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Sample Preparation:

Sampleprep ID:	SP003198
Sampleprep Summary:	All tissue sample was first homogenized in liquid nitrogen and then 10-20 mg tissues were weighed into a new 1.7 ml Eppendorf tube. Ice cold extraction solvent (400 ul 80% methanol/water) and 10 ul metformin-d6 (50 ng/ul in water) was added to each sample. Geno/Grinder homogenizer was used (1500 rpm, 1 to 2 min) to further break down the tissue chunk and form an even suspension. 200 ul supernatant containing polar metabolites was removed after centrifugation at 20,000 g at 4 °C for 10 min and transferred to LC vial for polar metabolite analysis using LC-HRMS. The rest samples (200 ul solvent and insoluble pellet) were briefly homogenized using Geno/Grinder again (1500 rpm, 30 sec) to loosen the bottom pellet and better mix with extraction solvent. 480 ul MTBE and 10 ul internal standard solution were added. After rigorous vortexing, 120 ul water was added to initiate phase separation. All samples were centrifuged at 20,000 g at 4 °C for 10 min. The supernatant containing lipids was transferred to a new Eppendorf tube and dried using speed vacuum. Dry pellets were stored in -80 °C freezer until ready for LC-HRMS analysis. To extract metabolites and lipids from mouse plasma, 10 ul plasma was mixed with 10 l water containing internal standards (50 ng/ul metformin-d6, 5 mM [U-13C]-glucose and [U-13C]-lactate, and 80 ul ice cold methanol was added. After vortex for 1 min, the mixture was centrifuged with a speed of 20,000 g at 4 °C for 10 min, and 20 ul was transferred directly to LC vial without solvent evaporation, followed by LC-HRMS analysis (injection volume, 3 ul) of polar metabolites such as glucose and metformin. To the rest samples (80 l solvent and pellet), 192 ul MTBE and 10 ul lipid internal standard solution were added. After rigorous vortexing, 48 ul water was added to initiate phase separation. All samples were centrifuged at 20,000 g at 4 °C for 10 min. The supernatant containing lipids was transferred to a new Eppendorf tube and dried using speed vacuum. The dry pellets of lipids from mouse tissues or plasma were reconstituted into (300 ul for tissue and 50 ul for plasma) sample solvent (isopropanol: ethyl acetate, 1:1, v/v), and 3 ul was injected to LC-HRMS for lipidomics analysis.

Sampleprep ID:

SP003198

Sampleprep Summary:

All tissue sample was first homogenized in liquid nitrogen and then 10-20 mg tissues were weighed into a new 1.7 ml Eppendorf tube. Ice cold extraction solvent (400 ul 80% methanol/water) and 10 ul metformin-d6 (50 ng/ul in water) was added to each sample. Geno/Grinder homogenizer was used (1500 rpm, 1 to 2 min) to further break down the tissue chunk and form an even suspension. 200 ul supernatant containing polar metabolites was removed after centrifugation at 20,000 g at 4 °C for 10 min and transferred to LC vial for polar metabolite analysis using LC-HRMS. The rest samples (200 ul solvent and insoluble pellet) were briefly homogenized using Geno/Grinder again (1500 rpm, 30 sec) to loosen the bottom pellet and better mix with extraction solvent. 480 ul MTBE and 10 ul internal standard solution were added. After rigorous vortexing, 120 ul water was added to initiate phase separation. All samples were centrifuged at 20,000 g at 4 °C for 10 min. The supernatant containing lipids was transferred to a new Eppendorf tube and dried using speed vacuum. Dry pellets were stored in -80 °C freezer until ready for LC-HRMS analysis. To extract metabolites and lipids from mouse plasma, 10 ul plasma was mixed with 10 l water containing internal standards (50 ng/ul metformin-d6, 5 mM [U-13C]-glucose and [U-13C]-lactate, and 80 ul ice cold methanol was added. After vortex for 1 min, the mixture was centrifuged with a speed of 20,000 g at 4 °C for 10 min, and 20 ul was transferred directly to LC vial without solvent evaporation, followed by LC-HRMS analysis (injection volume, 3 ul) of polar metabolites such as glucose and metformin. To the rest samples (80 l solvent and pellet), 192 ul MTBE and 10 ul lipid internal standard solution were added. After rigorous vortexing, 48 ul water was added to initiate phase separation. All samples were centrifuged at 20,000 g at 4 °C for 10 min. The supernatant containing lipids was transferred to a new Eppendorf tube and dried using speed vacuum. The dry pellets of lipids from mouse tissues or plasma were reconstituted into (300 ul for tissue and 50 ul for plasma) sample solvent (isopropanol: ethyl acetate, 1:1, v/v), and 3 ul was injected to LC-HRMS for lipidomics analysis.