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MB Sample ID: SA237322
| Local Sample ID: | LA-4 |
| Subject ID: | SU002460 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002460 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| LA-4 | SA237322 | FL029385 | LPS+ATP | Treatment |
Collection:
| Collection ID: | CO002453 |
| Collection Summary: | Mouse bone marrow was cultured in low glucose DMEM supplemented with 30% L929 cell-conditioned medium, 20% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin for 6-7 days until the cells reached confluence. BMDMs were then reseeded in culture dishes overnight in RPMI-1640 medium containing 1% Nutridoma-SP medium (Sigma-Aldrich). LPS-primed BMDMs were stimulated with 5 mM ATP in the presence or absence of 10 uM JX06 for 45 min. Unstimulated macrophages were used as control. |
| Sample Type: | Macrophages |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002472 |
| Treatment Summary: | Macrophages were treated with 300 ng/ml LPS for 3 h. LPS-primed BMDMs were stimulated with 5 mM ATP in the presence or absence of 10 uM JX06 for 45 min. Unstimulated macrophages were used as control. |
| Treatment Compound: | LPS, ATP, JX06 |
Sample Preparation:
| Sampleprep ID: | SP002466 |
| Sampleprep Summary: | Macrophages were lysed, and polar metabolites were extracted using methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich) (Liu et al., 2015). Extracts were dried in a vacuum concentrator at room temperature and stored at -80 freezer. |
| Processing Storage Conditions: | Described in summary |
| Extraction Method: | methanol and H2O |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003866 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Waters XBridge Amide (100 x 4.6mm,3.5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002864 |
| Chromatography Summary: | The HPLC (Ultimate 3000 UHPLC) is coupled to QE-MS (Thermo Scientific) for metabolite separation and detection. An Xbridge amide column (100 × 2.1 mm i.d., 3.5 μm; Waters) is employed for compound separation at room temperature. The mobile phase A is water containing 5 mM ammonium acetate (pH 6.8), and the mobile phase B is acetonitrile. The linear gradient used is as follows: 0 min, 85% B; 1.5 min, 85% B, 5.5 min, 35% B; 10 min, 35% B, 10.5 min, 35% B, 14.5 min, 35% B, 15 min, 85% B, and 20 min, 85% B. The flow rate was 0.15 mL/min from 0 to 10 min and 15 to 20 min and 0.3 mL/min from 10.5 to 14.5 min. The QE-MS is equipped with a HESI probe with related parameters set as below: heater temperature, 120 °C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.0 kV for the positive mode and 2.5 kV for the negative mode; capillary temperature, 320 °C; S-lens, 55; scan range (m/z): 70 to 900 for pos mode (1.31 to 12.5 min) and neg mode (1.31 to 6.6 min) and 100 to 1000 for neg mode (6.61 to 12.5 min); resolution: 70000; automated gain control (AGC), 3 × 106 ions. Customized mass calibration was performed before data acquisition. |
| Methods Filename: | The_HPLC_methods.docx |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Waters XBridge Amide (100 x 4.6mm,3.5um) |
| Flow Gradient: | The linear gradient used is as follows: 0 min, 85% B; 1.5 min, 85% B, 5.5 min, 35% B; 10 min, 35% B, 10.5 min, 35% B, 14.5 min, 35% B, 15 min, 85% B, and 20 min, 85% B. |
| Flow Rate: | 0.15 mL/min from 0 to 10 min and 15 to 20 min and 0.3 mL/min from 10.5 to 14.5 min. |
| Solvent A: | 100% water; 5 mM ammonium acetate, pH 6.8 |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003607 |
| Analysis ID: | AN003866 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | LC-MS peak extraction and integration were performed using commercial available software Sieve 2.2 (Thermo Scientific). The peak area was used to represent the relative abundance of each metabolite in different samples. |
| Ion Mode: | UNSPECIFIED |
