General Information
- Sample ID
- OES153959
- Sample Name
- chemical standards with S9 fraction incubation
- Subject type
- Human
Sample information
- Organism
- Homo sapiens
- Tissue
- Liver S9 fraction
- Protocol
- We experimentally incubated a mixture of 46 common metabolites (46std_mix) with the human liver S9 fractions for 24 h. The 46std_mix solution was prepared and stored at -80 °C before incubation. The incubation followed the previously reported protocol45 with minor modification. Before the experiment, 50 μL of pooled human liver S9 fraction solution (H0610.S9) was diluted into 500 μL using water. The NADPH regenerating system was reconstituted with the addition of 3.5 mL of water to make a final volume of 5 mL. The 4x cofactor stock was freshly prepared with the following composition: 10 mM UDPGA, 2 mM GSH, 2 mg/ml PAPS, 0.1 mM acetyl-CoA, and NADPH regenerating system (1 mM NADP, 5 mM glucose-6-phosphate, 1 unit glucose-6-phosphate dehydrogenase). The incubation was performed in the 1.5 mL of Eppendorf centrifuge tube. In each tube, 30 μL of S9 fraction, 30 μL of Tris buffer (0.2 M; pH 7.5; 2 mM MgCl2) and 30 μL of 46std_mix solution were first pooled. To start the reaction, 30 μL of 4× cofactor stock was added, and the incubation was carried out at 30 C for 24 h. 360 μL of MeOH:ACN (1:1, v:v) were added to terminate the reaction and extract the metabolites. For metabolite extraction, the sample was incubated at -20 °C for 1 hour to facilitate protein precipitation. After the incubation, samples were centrifuged at 13000 rpm and 4 °C for 15 min. The supernatant was taken out and evaporated to dryness at 4 °C. The samples were reconstituted with 120 μL of ACN/H2O (v:v, 1:1) and vortexed for 30 s and sonicated for 10 min at 4 °C water bath. Finally, the samples were centrifuged for 15 min at 17,000 xg and 4 °C. The supernatant was taken into the sample vial for LC-MS experiment.
- Projects
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1
Project ID Project name Submitter Date OEP003284 Unknown annotation in in-vitro metabolism experiment with KGMN Zhu, Zheng-Jiang 2022-04-06 - Experiments
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1
Experiment ID Experiment name Experiment type Experiment protocol Date OEX016598 OEX_Zheng-Jiang_2204061557 Metabolomic N/A 2022-04-06 - Runs
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1
Run ID Run Name Project Experiment Sample Data Date OER253320 OER_Zheng-Jiang_2204061614 OEP003284 OEX016598 OES153959 24 2022-04-06
Author Information
- Create Date
- 2022-04-06
- Submission
- Zheng-Jiang Zhu,