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MB Sample ID: SA187832
| Local Sample ID: | 1400w-1 |
| Subject ID: | SU002088 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002088 |
| 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 |
|---|---|---|---|---|
| 1400w-1 | SA187832 | FL023153 | wild type C57BL/6 | Mouse_strain |
| 1400w-1 | SA187832 | FL023153 | Expansion stage | Culture_stage |
| 1400w-1 | SA187832 | FL023153 | 5 | Time_point(days) |
| 1400w-1 | SA187832 | FL023153 | 1400w | Treatment |
Collection:
| Collection ID: | CO002081 |
| Collection Summary: | Unfractionated wild-type bone marrow cells were cultured in regular stress erythropoiesis expansion medium (SEEM). At indicated time point, stress erythroid progenitor cells were harvested, washed with PBS, and re-cultured in SEEM supplemented with 25 mM uniformly labeled [13C]-Glucose (Sigma-Aldrich) for 24 hrs. Cells were counted, washed with PBS and snap frozen in liquid nitrogen. |
| Sample Type: | Stem cells |
Treatment:
| Treatment ID: | TR002100 |
| Treatment Summary: | To assess the role of iNOS in progenitor metabolism, SEEM cultures were treated with 10 μM 1400w or vehicle (DMSO) at day 3 for 48 hrs, and then cells were re-cultured in SEEM with U-[13C]-Glucose for 24 hrs. |
| Treatment Compound: | 1400w |
| Treatment Dose: | 10 μM |
| Treatment Vehicle: | DMSO |
| Cell Media: | SEEM |
Sample Preparation:
| Sampleprep ID: | SP002094 |
| Sampleprep Summary: | Cell pellets were extracted with 1 ml pre-chilled 50:50 HPLC-grade water:methanol (v/v) containing 1 µM chlorpropamide as the internal standard. The samples were vortexed briefly followed by thorough homogenization. The samples were then snap frozen with liquid nitrogen and immediately thawed at room temperature. This step was repeated for three times followed by centrifuging for 10 min at 12,000 x g and 4 °C. The supernatants were transferred into fresh microfuge tubes. The remaining cell pellets were re-extracted with 0.5 ml 50% methanol containing 1 µM chlorpropamide, homogenized, frozen and thawed three times, spun down, and the supernatants were combined with the first extraction. Metabolites-containing supernatants were concentrated to dryness at room temperature in a SpeedVac concentrator and re-dissolved in 100 µl 97:3 water:methanol (v/v). After centrifuging for 10 min at 13000 × g and 4°C, 70 µl of supernatants were transferred into autosampler vials for LC-MS analysis. Two types of control were prepared in triplicates to run in concert with the experimental samples: the process blank control, and the pooled control containing an equal volume from each experimental sample. |
Combined analysis:
| Analysis ID | AN003270 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Waters XSelect HSS C18 (250 x 4.6mm) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Exactive Plus Orbitrap |
| Ion Mode | NEGATIVE |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002414 |
| Chromatography Summary: | The sample run order was randomized to reduce bias from instrument drift. 10 µl sample was subjected to LC-MS analysis on a Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). Reversed-phase chromatography mode was used to separate compounds on a Xselect C18 HSS column (Waters) with solvent A (97:3 water:methanol (v/v), 10 mM tributylamine, and 15 mM acetic acid ) and solvent B (methanol). The flow rate was 200 µl/min, and the total run time was 25 min. The gradient was 0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B. The mass spectrometer was operated in a negative-ion mode at a resolution of 140,000 at m/z 200 and with a scan range of 85 to 1000 m/z. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Waters XSelect HSS C18 (250 x 4.6mm) |
| Flow Gradient: | The gradient was 0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B. |
| Flow Rate: | 200 ul/min |
| Solvent A: | 97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003042 |
| Analysis ID: | AN003270 |
| Instrument Name: | Thermo Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Raw data files were converted to .mzML file format using the ProteoWizard software, and they were analyzed by the MS-DIAL software. Metabolites were identified by comparison to an in-house reference library of pure metabolite standards which included mass-to-charge ratio (m/z) and retention time. For quantification of metabolite abundance, peak areas of identified metabolites were first normalized to the internal standard chlorpropamide, and then normalized to cell numbers from each sample. Data were analyzed using R and Cytoscape software. |
| Ion Mode: | NEGATIVE |
