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MB Sample ID: SA079572
| Local Sample ID: | 5 |
| Subject ID: | SU001210 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL6 |
| Age Or Age Range: | 2-3 months |
| Weight Or Weight Range: | 28-46 |
| Gender: | Female |
| Animal Animal Supplier: | Jackson Laboratory |
| Animal Housing: | Single House |
| Animal Light Cycle: | 12:12 |
| Animal Feed: | CHOW diet (Envigo Teklad 6% fat 7002, Madison, WI) and Western (HFD; high-fat, high-sugar; 21% milk fat and 34% sucrose (Envigo TD.08811) |
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Sample Preparation:
| Sampleprep ID: | SP001218 |
| Sampleprep Summary: | Approximately 20 mg of muscle tissue was homogenized in a glass homogenizer with 1.5 ml of 2:1 chloroform:methanol and then brought to 4 ml using the same ratio. The mixture was poured through a 2V grade qualitative 12.5 cm Whatman filter into a clean 10 ml glass tube. The volume in the tube was again brought up to 4 ml with the same 2:1 solution as above. One ml of water was added to the tube, vortexed for 20 seconds, and then centrifuged for 10 minutes at 2500 rpm. The top non-lipid portion was removed and the lower lipid-containing layer was dried under nitrogen.Lipid extracts were suspended in 100 uL of 2:1 Chloroform:Methanol. Injections were normalized such that equal amounts of lipid were analyzed for each sample, regardless of total lipid content of diet. 3 μL of extract was injected twice (n=2 replicates) onto a Waters Acquity UPLC system in discrete, randomized blocks. Next samples were separated using a Waters Acquity UPLC CSH Phenyl Hexyl column (1.7 µM, 1.0 x 100 mm), using a gradient from solvent A (water, 0.1% formic acid) to solvent B (Acetonitrile, 0.1% formic acid). Injections were made in 100% A, held at 100% A for 1 min, ramped to 98% B over 12 minutes, held at 98% B for 3 minutes, and then returned to starting conditions over 0.05 minutes and allowed to re-equilibrate for 3.95 minutes, with a 200 µL/min constant flow rate. The column and samples were held at 65 °C and 6 °C, respectively. The column eluent was infused into a Waters Xevo G2 TOF-MS with an electrospray source in positive mode, scanning 50-2000 m/z at 0.2 seconds per scan, alternating between MS (6 V collision energy) and MSE mode (15-30 V ramp). Calibration was performed using sodium iodide with 1 ppm mass accuracy. The capillary voltage was held at 2200 V, source temp at 150 °C, and nitrogen desolvation temp at 350 °C with a flow rate of 800 L/hr. |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | On ice |
