Return to study ST001651 main page
MB Sample ID: SA151725
Local Sample ID: | Inulin_Cell_3 |
Subject ID: | SU001728 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Gender: | Female |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001728 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Gender: | Female |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Inulin_Cell_3 | SA151725 | FL017810 | 13C-Inulin | Treatment |
Collection:
Collection ID: | CO001721 |
Collection Summary: | Fresh fecal pellets were collected from each mouse (23 weeks old) and immediately placed in a sterile microcentrifuge tube. Samples were quickly transferred to an anaerobic chamber for immediate experimental setup to ensure microbial viability. |
Sample Type: | Feces |
Storage Conditions: | Described in summary |
Treatment:
Treatment ID: | TR001741 |
Treatment Summary: | Fresh fecal samples (ca. 110 mg) from each mouse were weighed and dispensed in 3 mL of prepared medium separately in an anaerobic chamber. The samples were pestled to suspend the microorganisms and particles. The suspensions were subjected to low-speed centrifugation and the supernatants were then collected and centrifuged to pellet microbes. The microbial cells were then suspended in 4 mL of culture medium, and divided equally into two Hungate tubes. To each paired tube was amended with either 13C-Inulin or 13C-cellulose aseptically to achieve a final concentration of 4 g/L fibers. The sealed Hungate tubes were incubated in a water bath (37°C) for 24 h. After incubation, the samples were centrifuged (3,000 g, 5 min) to collect supernatant (culture medium). Each pellet was washed with 1 mL of fresh culture medium and centrifuged to collect the microbial cells. All procedures were performed under anaerobic conditions. |
Sample Preparation:
Sampleprep ID: | SP001734 |
Sampleprep Summary: | The collected medium was weighed, and equal amounts of medium (50% of total medium weight) were accurately aliquoted from each sample and lyophilized. The microbial cells were quenched using 450 μL cold methanol right after collection. After brief agitation by vortex, the samples were transferred into glass tubes. Then 5 mL of methyl tert-butyl ether was added to each tube and the samples were mixed on a multi-tube vortexer (600 rpm, 30 min). Phase separation was then induced by adding 1.25 mL of Millipore deionized water. Samples were then vortexed again for 1 min, incubated at 4 °C for 10 min to allow phase separation and centrifuged (3,000 g, 10 min, 4 °C). Polar fractions were collected into clean tubes and lyophilized. |
Combined analysis:
Analysis ID | AN002697 | AN002698 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | HILIC |
Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
Column | SeQuant ZIC-HILIC (150 x 2.1mm,5um) | SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
Ion Mode | POSITIVE | NEGATIVE |
Units | peak area | peak area |
Chromatography:
Chromatography ID: | CH001990 |
Instrument Name: | Thermo Dionex Ultimate 3000 |
Column Name: | SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
Column Temperature: | 40 |
Flow Gradient: | 0–20 min, linear gradient from 80% to 20% B; 20–21 min, hold at 20% B min; 21-22 min, linear gradient to 80% B; 22-28 min, re-equilibrate at 80% B |
Flow Rate: | 0.150 ml/min |
Solvent A: | 100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide |
Solvent B: | 100% acetonitrile |
Chromatography Type: | HILIC |
MS:
MS ID: | MS002495 |
Analysis ID: | AN002697 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The mass spectrometer was operated in positive and negative ionization modes. Initial metabolite identification was performed using Compound Discoverer 3.1 (Thermo Fisher Scientific).Metabolites were further confirmed by comparison of the ion features in the samples with an in-house reference library of authentic chemical standards. Peak areas of metabolites and isotopologues were integrated and exported to Excel via the TraceFinder 5.0 software package (Thermo Fisher Scientific). |
Ion Mode: | POSITIVE |
MS ID: | MS002496 |
Analysis ID: | AN002698 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The mass spectrometer was operated in positive and negative ionization modes. Initial metabolite identification was performed using Compound Discoverer 3.1 (Thermo Fisher Scientific).Metabolites were further confirmed by comparison of the ion features in the samples with an in-house reference library of authentic chemical standards. Peak areas of metabolites and isotopologues were integrated and exported to Excel via the TraceFinder 5.0 software package (Thermo Fisher Scientific). |
Ion Mode: | NEGATIVE |