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MB Sample ID: SA308628
| Local Sample ID: | KO_Liver_MR14 |
| Subject ID: | SU002963 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
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Sample Preparation:
| Sampleprep ID: | SP002969 |
| Sampleprep Summary: | Metabolites were extracted by a two-step liquid extraction adapted from Sellik et al.[1], 50 mg of frozen tissue was used and 500 µL of prechilled methanol (-80 °C) was added. For quantification and data normalization, 25 µL of internal standard solution containing 13C6-L-Arginine, 13C5 L Valine, 13C2-Citric acid, 2H4-Succinic acid and 13C6-Fructose-6-phosphate at a concentration of 100 µM was added. This results in a concentration of 10 µM in the final extract. The sample was homogenized by a tissue slicer and the extract was vortexed for 2 min until no larger cell clusters were visible. Afterward, the sample was sonicated for 2 min in a chilled (0 °C) ultrasonic bath. The cells were kept at -80 °C for 5 min and subsequently thawed. The thawed cells were vortexed and sonicated for 2 min each and centrifuged at 3000 x g for 5 min. The supernatant was collected and 250 µL of water acidified with 0.1 % acetic acid (LC-MS grade) was added. The sample was vortexed and sonicated for 2 min and subject to the same freezing-thawing cycle described before. The samples were centrifuged at 3000 x g for 5 min and the supernatant was collected. The combined supernatant was dried in a vacuum-centrifuge for 45 min. The residue was resuspended in 250 µL acetonitrile/water (50/50; v/v) by sonicating and vortexing for 2 min each. To remove remaining proteins or other cell debris the extracts have been centrifuged at 12,000 rpm for 2 min and filtered using Nanosep 3KDa Omega centrifugal filters (Pall, Port Washington, USA). After the extraction pooled QC samples were generated for the whole sample set as well as for each investigated sample group and analyzed along the study samples to monitor the analytical quality. Ref: [1] (Sellick CA, Hansen R, Stephens GM, Goodacre R, Dickson AJ. Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling. Nat Protoc. 2011 28;6(8):1241-9. doi: 10.1038/nprot.2011.366) |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
