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MB Sample ID: SA094724
| Local Sample ID: | CI_6 |
| Subject ID: | SU001388 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male |
| Cell Primary Immortalized: | Islets isolated from pancreata of adult C57BL/J6 male mice |
| Cell Counts: | 100 islets |
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Subject:
| Subject ID: | SU001388 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male |
| Cell Primary Immortalized: | Islets isolated from pancreata of adult C57BL/J6 male mice |
| Cell Counts: | 100 islets |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CI_6 | SA094724 | FL013515 | Islet | Cell type |
| CI_6 | SA094724 | FL013515 | inorganic arsenic (sodium arsenite) | Treatment |
Collection:
| Collection ID: | CO001383 |
| Collection Summary: | Pancreatic islets were isolated from adult male C57BL/6 mice (Charles River Laboratories, Wilmington, MA). All procedures involving mice were approved by the University of North Carolina Institutional Animal and Use Committee. Mice were sacrificed by cervical dislocation and pancreas was infused in situwith collagenase P (1 mg/ml, Roche Diagnostics Corp., Indianapolis, IN) via the common bile duct. Pancreas was then removed and digested in the collagenase solution for 12 min at 37 °C. The digestate was washed and islets were purified by centrifugation in a gradient of Ficoll PM 400 (GE Healthcare, Uppsala, Sweden) (Szot et al., 2007).The isolated islets were cultivated overnight at 37 °C with 5% CO2 in RPMI 1640 medium (Mediatech, Manassas, VA) with 10% fetal bovine serum, 10 mM Hepes, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Sigma-Aldrich, St. Louis, MO). The same medium was used in experiments in which the islets were exposed to iAsIII (sodium arsenite; Sigma-Aldrich), MAsIII(methylarsine oxide) or DMAsIII (iododimethylarsine). |
| Sample Type: | Pancreas |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001403 |
| Treatment Summary: | The isolated islets were incubated overnight at 37°C with 5% CO2 at 6-well plates in RPMI 1640 medium with 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Gibco). The islets were then exposed to 2 μM iAsIII, 0.25 μM MAsIII, or 0.5 μM DMAsIII for 48 hours. Control islets were incubated in the culture medium without arsenicals. |
| Cell Storage: | -80 °C |
Sample Preparation:
| Sampleprep ID: | SP001396 |
| Sampleprep Summary: | With a randomized order, islet samples were put on dry ice before sample preparation. A volume of 400 uL ice cold methanol-water (80:20) was added, and then vortex for 5 min at 5000 rpm. All contents in the tube were transferred into pre-labeled MagNaLyzer tubes (with 10-15 beads inside). The tubes were put on bead homogenizer using quick run setting for bacterial cells (2 ml) with speed at 6.30 m/s for 45 sec in 1 cycle. All samples were centrifuged at 16,000 rcf for 20 min at 4°C. A volume of 300 µl supernatant were transferred into pre-labeled 1.5 ml Low-bind Eppendorf tube. For quality control purpose, 32 µl of the supernatant from individual sample was transferred and mixed in new 2-ml tube to make a sample pool (SP). A volume of 300 µl SP was transferred into a pre-labeled 2.0 ml Low-bind Eppendorf tubes. All samples, including study samples and pools, were dried under speed-vac. For immediate analysis, 100 µL of Water-Methanol (95:5) containing 500 ng/mL L-tryphtophan-d5 was added to the residue, and then vortexed for 10 mins at 5000 rpm. After centrifuge at 4°C, 16000 rcf for 4 min, the supernatant from individual sample was transferred to pre-labeled auto-sampler vial for LC-MS analysis. |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN002188 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH001603 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um) |
| Column Pressure: | 6000-10000 psi |
| Column Temperature: | 50 |
| Flow Rate: | 0.4 ml/min |
| Injection Temperature: | 8 |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% methanol; 0.1% formic acid |
| Analytical Time: | 22 min |
| Capillary Voltage: | 3.75 KV |
| Weak Wash Solvent Name: | 10:90 Methanol:Water with 0.1% FA solution |
| Strong Wash Solvent Name: | 75:25 2-Propanol: Water with 0.1% FA solution |
| Randomization Order: | Yes |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002035 |
| Analysis ID: | AN002188 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | We used DDA mode to acquire the MS and MS/MS data. Progenesis QI was used for peak picking, alignment, and normalization. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 275 °C |
| Capillary Voltage: | 3.75 KV |
| Collision Energy: | 10-35, ramp |
| Collision Gas: | N2 |
| Dry Gas Flow: | 45 |
| Dry Gas Temp: | 325°C |
| Fragmentation Method: | CID |
| Ionization: | ES+ |
| Mass Accuracy: | 5ppm |
| Dataformat: | Profile |
| Desolvation Gas Flow: | 45 |
| Desolvation Temperature: | 325°C |
| Resolution Setting: | 120000 |
| Scan Range Moverz: | 70-1050 m/z |
| Scanning Range: | 70-1050 m/z |
