Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA164207

Local Sample ID:	mouse_CSF_buffer C_2
Subject ID:	SU001838
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Sample Preparation:

Sampleprep ID:	SP001844
Sampleprep Summary:	CSF was collected by puncture of the cisterna magna with a glass capillary [39] and flash-frozen for further analysis. Per condition, 3 µl of precleared CSF were extracted by brief sonication in 200 µl of the indicated extraction buffers. After centrifugation for 10 min at maximum speed on a benchtop centrifuge (Eppendorf) the cleared supernatant was dried using a nitrogen dryer and reconstituted in 30 µl water by brief sonication. Extracted metabolites were spun again and cleared supernatant was transferred to LC-MS micro vials. A small amount of each sample was pooled and serially diluted 3- and 10-fold to be used as quality controls throughout the run of each batch. Extraction buffer “A”: 40:40:20 of acetonitrile:methanol:water, supplemented with 0.1M formic acid and isotopically-labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). Extraction buffer “B”: 80% LC-MS-grade methanol, 20% 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water and supplemented with isotopically labeled internal standards (17 amino acids and isotopically labelled reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). Extraction buffer “C” and “C + Ellman’s”: Solution 1: 100% LC-MS Methanol Solution 2: 25mM Ammonium Acetate and 2.5mM Na-Ascorbate in LC-MS water supplemented with isotopically labelled reduced glutathione and isotopically labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). Ellman’s reagent (5,5′-Dithiobis(2-nitrobenzoic acid),D8130, Sigma Aldrich): 20 mM in “Solution 2”. Final composition is 80% solution 1 and 20% solution 2.

Sampleprep ID:

SP001844

Sampleprep Summary:

CSF was collected by puncture of the cisterna magna with a glass capillary [39] and flash-frozen for further analysis. Per condition, 3 µl of precleared CSF were extracted by brief sonication in 200 µl of the indicated extraction buffers. After centrifugation for 10 min at maximum speed on a benchtop centrifuge (Eppendorf) the cleared supernatant was dried using a nitrogen dryer and reconstituted in 30 µl water by brief sonication. Extracted metabolites were spun again and cleared supernatant was transferred to LC-MS micro vials. A small amount of each sample was pooled and serially diluted 3- and 10-fold to be used as quality controls throughout the run of each batch. Extraction buffer “A”: 40:40:20 of acetonitrile:methanol:water, supplemented with 0.1M formic acid and isotopically-labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). Extraction buffer “B”: 80% LC-MS-grade methanol, 20% 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water and supplemented with isotopically labeled internal standards (17 amino acids and isotopically labelled reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). Extraction buffer “C” and “C + Ellman’s”: Solution 1: 100% LC-MS Methanol Solution 2: 25mM Ammonium Acetate and 2.5mM Na-Ascorbate in LC-MS water supplemented with isotopically labelled reduced glutathione and isotopically labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). Ellman’s reagent (5,5′-Dithiobis(2-nitrobenzoic acid),D8130, Sigma Aldrich): 20 mM in “Solution 2”. Final composition is 80% solution 1 and 20% solution 2.