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MB Sample ID: SA004285
| Local Sample ID: | SBEP_Metab_LPSactivated1 |
| Subject ID: | SU000103 |
| Subject Type: | Animal cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | RAW 264.7 |
| Cell Strain Details: | RAW 264.7 |
| Cell Primary Immortalized: | immortalized |
| Species Group: | Mammal |
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Sample Preparation:
| Sampleprep ID: | SP000099 |
| Sampleprep Summary: | Suspentions softly centrifuged, buffer removed, amonium bicarbonate added, metabolites extraxted with chloroform/methanol (2:1, v/v), vortexed, centrifuged, aqueous layer dried in vacuum concentrator, derivatization with methoxyamine in pyridine, N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and 1% trimethylchlorosilane (TMCS) |
| Sampleprep Protocol Comments: | Cell suspensions were softly centrifuged (230 × g for 5 min) and as much buffer as possible was removed. Then, 170 µL of 150 mM ammonium bicarbonate was added to the cell pellet and the cell suspensions were transferred to 2 mL micro-centrifuge tubes for extraction. Subsequently, the water soluble metabolites were extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v). After vortexing, the samples were centrifuged (12,000 × g for 5 min) and the upper (aqueous) layers containing water-soluble metabolites were transferred into glass vials, followed by drying in a vacuum concentrator. For the derivatization, 20 µL of methoxyamine in pyridine (30 mg/mL) were added to each sample, followed by incubation at 37°C with shaking for 90 min to protect carbonyl groups. Next, 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were added to each vial, followed by incubation at 37°C with shaking for 30 min to derivatize hydroxyl and amine groups. The samples were then allowed to cool to room temperature. |
| Processing Method: | Homogenization |
| Extraction Method: | The water soluble metabolites were extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v). After vortexing, the samples were centrifuged (12,000 × g for 5 min) and the upper (aqueous) layers containing water-soluble metabolites were transferred into glass vials, followed by drying in a vacuum concentrator. |
| Extract Concentration Dilution: | chilled (-20°C) chloroform/methanol (2:1, v/v) |
| Extract Enrichment: | Vacuum Concentrator |
| Sample Resuspension: | 20 µL of methoxyamine in pyridine (30 mg/mL) |
| Sample Derivatization: | 20 µL of methoxyamine in pyridine (30 mg/mL), 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS), |
| Cell Type: | Macrophage |
