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MB Sample ID: SA015894
| Local Sample ID: | Hypo407 |
| Subject ID: | SU000378 |
| Subject Type: | mouse |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57Bl/6 |
| Age Or Age Range: | 5.5 mo |
| Weight Or Weight Range: | 29.1 g-49.2 g |
| Gender: | male |
| Animal Animal Supplier: | JAX |
| Animal Housing: | 4/cage |
| Animal Feed: | Purina chow |
| Species Group: | Mammal |
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Collection:
| Collection ID: | CO000372 |
| Collection Summary: | Brain tissue samples were homogenized in 50:50 Acetonitrile:Water (10 µL to every 1 mg of tissue) with washed ceramic beads on a MagNA Lyser, using two 30 sec pulses at 2,000 rpm with a 1 min chilling step in between pulses. Samples were vortexed on a multi-tube vortexer for 2 min at 5,000 rpm and centrifuged at room temperature for 4 min at 16,000 rcf. A 50 µL aliquot of each experimental brain tissue homogenized supernatant was transferred to a labeled 2.0 mL Lo-Bind eppendorf tube. Analytical pooled samples were created by combining 15 µL aliquot from each experimental sample in a 2.0 mL Lo-Bind eppendorf tube. The QC pooled sample was vortexed for 30 sec and 50 µL aliquots were transferred to 5 labeled 2.0 mL Lo-Bind eppendorf tubes. Next, 350 µL of acetonitrile was added to study sample and QC pooled sample tubes. The samples were vortexed on a multi-tube vortexer for 2 min at 5,000 rpm, and centrifuged at room temperature for 4 min at 16,000 rcf. The supernatants were transferred to new labeled tubes and dried on a lyophilizer overnight. Dried samples were reconstituted in 50 µL of 95:5 Water:Methanol, mixed on an analog vortex mixer for 1 min with speed set at 10. Then, the samples were centrifuged at room temperature for 4 min at 16,000 rcf, and the supernatants were transferred to autosampler vials for data acquisition. UPLC-MS Methods: UPLC-MS spectra were collected for all samples. UPLC was performed on a Waters Acquity UPLC with an Acquity HSS T3 column (2.1x 100mm x 1.8 µm) at 50 °C using the reversed-phase separation. Water with 0.1% formic acid (mobile phase A) and methanol with 0.1% formic acid (mobile phase B) were used as mobile phases and the metabolites were chromatographically separated using a gradient separation: (see the 3. Sheridan-Mice-Hypothalamus_RP-Metadata and Analytical Metadata.xlsx file for the flow gradient). Mass spectroscopy analysis was performed using a Synapt G2 Si ESI-Q-TOF using a 10 µL injection volume. UPLC-MS data were collected over 70-1000 m/z in both positive and negative modes. |
| Sample Type: | brain homogenate |
