Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA322128

Local Sample ID:	IS07_serum
Subject ID:	SU003071
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Sample Preparation:

Sampleprep ID:	SP003077
Sampleprep Summary:	A modified Bligh-Dyer protocol was used to extract lipids from the brain and spinal cord tissue. Brain homogenates (either 300 mg/ml or 50 mg/ml) were prepared with ice cold water using a Bead Mill homogenizer (Bead Ruptor Elite, Omni international, USA). Spinal cord homogenates were prepared at 65 mg/ml in cold water. Immediately after homogenization, the brain homogenates were diluted with cold water (150 µL of 300 mg/ml brain homogenate was mixed with 800 µl of cold water. For the 50 mg/ml brain homogenates, 1 mL of homogenate was used directly. Spinal cord homogenates were diluted 10-fold (100 μl of the spinal cord homogenates with 900 μl of cold water). For all samples, 10 μl of the diluted homogenate was removed and stored at -80°C for protein quantification using a BCA assay. The diluted tissue homogenates were combined with a mixture of chloroform (containing 0.01% butylated hydroxytoluene, BHT): methanol: water (3:2:1) in glass tubes. After shaking and vortexing thoroughly, the mixture was centrifuged (Sorvall ST 40R Centrifuge, Thermo Fisher Scientific) at 1300 rpm for 10 minutes. The lower layer was carefully removed and saved in a glass tube. The remaining top layer was further extracted twice with 1.25 ml chloroform with 0.01% BHT; the lower layers were carefully removed and combined. The combined lower layer was then washed with 300 μl of 1 M KCl followed by 300 μl of water and vacuum dried completely (Savant SpeedVac SPD130DLX vacuum concentrator, Thermo Fisher Scientific, USA) to obtain the dried lipid extract. Serum (3 μl) was added directly to a vial containing 1.2 mL of chloroform: methanol: 300 mM ammonium acetate in water (300:665:35). The contents were mixed thoroughly, and the vials were centrifuged for 5 min at low speed in a clinical centrifuge to pellet proteins before submitting samples to the mass spectrometer.

Sampleprep ID:

SP003077

Sampleprep Summary:

A modified Bligh-Dyer protocol was used to extract lipids from the brain and spinal cord tissue. Brain homogenates (either 300 mg/ml or 50 mg/ml) were prepared with ice cold water using a Bead Mill homogenizer (Bead Ruptor Elite, Omni international, USA). Spinal cord homogenates were prepared at 65 mg/ml in cold water. Immediately after homogenization, the brain homogenates were diluted with cold water (150 µL of 300 mg/ml brain homogenate was mixed with 800 µl of cold water. For the 50 mg/ml brain homogenates, 1 mL of homogenate was used directly. Spinal cord homogenates were diluted 10-fold (100 μl of the spinal cord homogenates with 900 μl of cold water). For all samples, 10 μl of the diluted homogenate was removed and stored at -80°C for protein quantification using a BCA assay. The diluted tissue homogenates were combined with a mixture of chloroform (containing 0.01% butylated hydroxytoluene, BHT): methanol: water (3:2:1) in glass tubes. After shaking and vortexing thoroughly, the mixture was centrifuged (Sorvall ST 40R Centrifuge, Thermo Fisher Scientific) at 1300 rpm for 10 minutes. The lower layer was carefully removed and saved in a glass tube. The remaining top layer was further extracted twice with 1.25 ml chloroform with 0.01% BHT; the lower layers were carefully removed and combined. The combined lower layer was then washed with 300 μl of 1 M KCl followed by 300 μl of water and vacuum dried completely (Savant SpeedVac SPD130DLX vacuum concentrator, Thermo Fisher Scientific, USA) to obtain the dried lipid extract. Serum (3 μl) was added directly to a vial containing 1.2 mL of chloroform: methanol: 300 mM ammonium acetate in water (300:665:35). The contents were mixed thoroughly, and the vials were centrifuged for 5 min at low speed in a clinical centrifuge to pellet proteins before submitting samples to the mass spectrometer.