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MB Sample ID: SA029941
| Local Sample ID: | 5_C3 |
| Subject ID: | SU000592 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6 |
| Species Group: | Mammal |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000592 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6 |
| Species Group: | Mammal |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 5_C3 | SA029941 | FL007099 | none (GF) | colonized bacteria |
Collection:
| Collection ID: | CO000586 |
| Collection Summary: | Feces of GF or GF mice reconstituted with Esherichia coli (EC), Bacteroides acidifaciens (Bac), or Clostridia consortium (CL) |
| Sample Type: | Feces |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR000606 |
| Treatment Summary: | Animals Specific-pathogen-free (SPF) C57BL/6 mice were originally purchased from Jackson Laboratories. Wild-type mice on GF background were bred and maintained at the Germ-free Animal Core Facility of the University of Michigan. GF mice were maintained in flexible film isolators and were checked weekly for germ-free status by aerobic and anaerobic culture. The absence of microbiota was verified by microscopic analysis of stained cecal contents to detect unculturable contamination. All animal studies were performed according to approved protocols by the University of Michigan Committee on the Use and Care of Animals. Reconstitution of GF mice with cecal contents and defined bacteria Cecal contents from single neonatal and 7-week old SPF mice were diluted into 50 ml (per one adult mouse) or 5 ml (per one 4, 12, 16-day old mouse) of PBS, and passed through a 70 μm cell strainer to eliminate clumps and debris under sterile conditions) under an atmosphere of 5% H2, 5% CO2 and 90% N2 in an anaerobic growth chamber (Coy Manufacturing, Grass Lake, MI). Then 200 μl of the suspension was processed by intragastric gavage into each of the recipient GF mice. Bacteroides species were anaerobically grown at 37°C overnight in pre-reduced chopped-meat broth (Anaerobe systems, Morgan Hill, CA). E. coli were grown aerobically overnight in LB broth with shaking. |
| Treatment Protocol Filename: | Treatment_Protocol.pdf |
| Treatment: | Biotic |
| Treatment Compound: | Suspension of diluted cecal contents |
| Treatment Route: | Intragastric gavage |
| Treatment Dose: | 200 μl |
Sample Preparation:
| Sampleprep ID: | SP000599 |
| Sampleprep Summary: | CE-TOFMS-based metabolome analysis(sample preparation) Fecal samples were freeze dried, and disrupted by vigorous shaking at 1,500 rpm for 10 min with four 3 mm zirconia beads by Shake Master NEO (Bio Medical Science Inc.). 10 mg (±0.5 mg) fecal samples were homogenized with 500 μl MeOH containing internal standards (20 μM each of methionine sulfone, and D-camphor-10-sulfonic acid (CSA)) and 100 mg of 0.1 mm and four of 3 mm zirconia/silica beads (BioSpec Products). After vigorous shaking (1,500 rpm for 5 min) by Shake Master NEO (Bio Medical Science Inc.), 200 μl of Milli-Q water and 500 μl of chloroform was added and then shaking in a same manner as before. After centrifugation at 4,600 × g for 15 min at 4°C, the supernatant was transferred to a 5kDa cutoff centrifugal filter tube. The filtrate was centrifugally concentrated at 40°C and reconstituted with 40 μl of Milli-Q water. |
| Sampleprep Protocol Filename: | Sample_Prep_Protocol.pdf |
| Processing Method: | Homogenized with 500 μl MeOH containing internal standards and 100 mg of 0.1 mm and four of 3 mm zirconia/silica beads |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Chloroform |
| Extract Cleanup: | 5kDa cutoff centrifugal filter tube |
| Sample Resuspension: | 40 μl of Milli-Q water |
Combined analysis:
| Analysis ID | AN000877 | AN000878 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | CE | CE |
| Chromatography system | Agilent CE | Agilent CE |
| Column | COSMO(+),50um(anion),Fused-silica,50um(cation) | COSMO(+),50um(anion),Fused-silica,50um(cation) |
| MS Type | ESI | ESI |
| MS instrument type | TOF | TOF |
| MS instrument name | Agilent CE-TOFMS | Agilent CE-TOFMS |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | nmol/g | nmol/g |
Chromatography:
| Chromatography ID: | CH000623 |
| Instrument Name: | Agilent CE |
| Column Name: | COSMO(+),50um(anion),Fused-silica,50um(cation) |
| Internal Standard: | 20 μM each of methionine sulfone, and D-camphor-10-sulfonic acid (CSA) |
| Chromatography Type: | CE |
MS:
| MS ID: | MS000778 |
| Analysis ID: | AN000877 |
| Instrument Name: | Agilent CE-TOFMS |
| Instrument Type: | TOF |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
| MS ID: | MS000779 |
| Analysis ID: | AN000878 |
| Instrument Name: | Agilent CE-TOFMS |
| Instrument Type: | TOF |
| MS Type: | ESI |
| Ion Mode: | NEGATIVE |
