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MB Sample ID: SA009132
Local Sample ID: | 10 |
Subject ID: | SU000186 |
Subject Type: | Animal |
Subject Species: | Rattus norvegicus |
Taxonomy ID: | 10116 |
Genotype Strain: | SpragueDawley rats |
Weight Or Weight Range: | 300400 g |
Species Group: | Mammal |
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Subject:
Subject ID: | SU000186 |
Subject Type: | Animal |
Subject Species: | Rattus norvegicus |
Taxonomy ID: | 10116 |
Genotype Strain: | SpragueDawley rats |
Weight Or Weight Range: | 300400 g |
Species Group: | Mammal |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
10 | SA009132 | FL002158 | SFM+Def cold 24-120h | Treatment |
Collection:
Collection ID: | CO000172 |
Collection Summary: | Hepatocytes were isolated from male SpragueDawley rats (300400 g; Harlan, Indianapolis, IN, USA) by a two-step perfusion method as previously described (33). All harvests yielded hepatocytes with viability exceeding 95% by trypan blue dye exclusion. Freshly isolated hepatocytes were suspended in SFM, composed of Williams E supplemented with 0.2 U/ml insulin, 6 ?g/ml transferrin, 100 U/ml penicillin G, 100 mg/ml streptomycin, 3 g/L human albumin, 2.2 g/L sodium bicarbonate, 1 g/L L-carnitine, 2.0 mM L-glutamine, 100 nM dexamethasone, 40 ng/ml glucagon, 20 ng/ml Gly-His-Lys, 1,000 U/L heparin, 1 mg/L warfarin, and 5 ng/ml of mouse epidermal growth factor (EGF) (3). The cells, suspended in SFM, were placed in a spheroid box (33 × 28 × 6 cm) custom-made of polycarbonate by Mayo Division of Engineering and siliconized with Sigmacote for 30 min (20) and gently rocked continuously at a frequency of 10 cycles per minute (0.17 Hz) to induce spheroid formation and to maintain spheroids in suspension. Final hepatocyte concentration was 5 × 105 cells/ml per spheroid box. All culture conditions were maintained in a 5% CO2, 37°C incubator as previously described (20). Spheroids were centrifuged and resuspended in fresh culture media every 24 h. |
Sample Type: | Liver |
Treatment:
Treatment ID: | TR000192 |
Treatment Summary: | UW alone|UW + 1 mM Def|SFM alone|SFM + 1 mM Def|SFM + 1 mM Def + 1 µM CsA|SFM + 1 µM CsA |
Treatment Protocol Filename: | PMID-23507348-Liu-Nydberg-CellTrans-2014.pdf |
Treatment Protocol Comments: | Treatment annotation Cold 24 Indicates 24 hours cold storate prior to treatment Cold 48 Indicates 48 hours cold storate prior to treatment -96h Indicates treated for 96 hours after cold storage -120h Indicates treated for 120 hours after cold storage |
Treatment Compound: | University of Wisconsin (UW) solution|UW + deferoxamine (Def)| serum-free medium; Sigma-Aldrich (St. Louis, MO, USA)|SFM + Def|SFM + Def + cyclosporin A (CsA)|SFM + CsA |
Sample Preparation:
Sampleprep ID: | SP000186 |
Sampleprep Summary: | After 48 h of continuous rocking, the spheroids were washed with SFM at room temperature. The culture medium was changed to the cold storage medium (UW alone, UW + 1 mM Def; SFM alone; SFM + 1 mM Def; SFM + 1 mM Def + 1 ?M CsA; SFM + 1 ?M CsA) at room temperature. Spheroids were left in rocked culture without cold storage as control. Spheroids intended for cold storage were rocked in glass dishes (10 × 8 × 2 cm) custom-made by Mayo Division of Engineering and siliconized with Sigmacote for 30 min and then transferred into 50-ml conical tubes (BD Falcon; 1 × 106cells/ml, 20 ml in total) and put on ice for 1 h. Of note, spheroid box and glass dishes differ in size (i.e., volume capacity) but possess similar properties of spheroid formation and culture. Finally, tubes of spheroids were placed in a refrigerator to be cold stored at 4°C. After 24 or 48 h of cold storage treatment, the spheroids were centrifuged at 50 × g for 5 min, and the supernatant fluid was removed. Warm SFM (20 ml) at 37°C was added to each tube. The tubes were mixed gently prior to adding 20 ml of cell suspension to each glass culture dish and continued to rocking culture. Cultures were maintained in a humidified incubator at 37°C with a 5% CO2 atmosphere. |
Combined analysis:
Analysis ID | AN000261 |
---|---|
Analysis type | MS |
Chromatography type | |
Chromatography system | |
Column | |
MS Type | ESI |
MS instrument type | Triple quadrupole |
MS instrument name | - |
Ion Mode | POSITIVE |
Units | ng/ml |
Chromatography:
Chromatography ID: | CH000184 |
Chromatography Summary: | Concentrations of diazepam and its two major metabolites (nordiazepam and temazepam) were determined by high-performance liquid chromatography (HPLC) with mass spectrometry detection in the CTSA Metabolomics Core lab at Mayo Clinic. |
MS:
MS ID: | MS000210 |
Analysis ID: | AN000261 |
Instrument Name: | - |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
Ion Mode: | POSITIVE |