Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA300934

Local Sample ID:	1
Subject ID:	SU002913
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP002919
Sampleprep Summary:	In vitro metabolic flux experiments involved the media in confluent cells being changed just prior to the experiment. Three biological replicate samples of each cell line were pulsed with 10 μM U-glucose (13C6 99% purity) label from Cambridge Isotope (No. CLM-1396-1) or 4 μM U-glutamine (13C5, 15N2, 99% purity) label from Cambridge Isotope (No. CNLM-1275-H-0.5) for 2 h. Following the pulse, cells were spun down and washed with PBS. 1 mL of 80% UPLC-grade ice cold methanol was added to each pellet. Pellets were vortexed for 1 min and incubated at −80 °C to extract metabolites. Analysis of metabolites is described below. Frozen tumors were manually homogenized in liquid nitrogen using a mortar and pestle chilled by dry ice and liquid nitrogen. As the flank tumors were very large, an aliquot of tumor powder was weighed and incubated at −80 °C with 5 volumes of 80% ice-cold HPLC grade methanol to extract metabolites. Samples (both in vivo and in vitro) were centrifuged at 14,000× g rpm for 10 min at 4 °C, and the supernatants were transferred to glass insert liquid chromatography vials.

Sampleprep ID:

SP002919

Sampleprep Summary:

In vitro metabolic flux experiments involved the media in confluent cells being changed just prior to the experiment. Three biological replicate samples of each cell line were pulsed with 10 μM U-glucose (13C6 99% purity) label from Cambridge Isotope (No. CLM-1396-1) or 4 μM U-glutamine (13C5, 15N2, 99% purity) label from Cambridge Isotope (No. CNLM-1275-H-0.5) for 2 h. Following the pulse, cells were spun down and washed with PBS. 1 mL of 80% UPLC-grade ice cold methanol was added to each pellet. Pellets were vortexed for 1 min and incubated at −80 °C to extract metabolites. Analysis of metabolites is described below. Frozen tumors were manually homogenized in liquid nitrogen using a mortar and pestle chilled by dry ice and liquid nitrogen. As the flank tumors were very large, an aliquot of tumor powder was weighed and incubated at −80 °C with 5 volumes of 80% ice-cold HPLC grade methanol to extract metabolites. Samples (both in vivo and in vitro) were centrifuged at 14,000× g rpm for 10 min at 4 °C, and the supernatants were transferred to glass insert liquid chromatography vials.