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MB Sample ID: SA339215
| Local Sample ID: | Conv2 |
| Subject ID: | SU003248 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57Bl/6J |
| Age Or Age Range: | 8-10 weeks |
| Weight Or Weight Range: | 20-29g |
| Height Or Height Range: | - |
| Gender: | Male |
| Animal Animal Supplier: | Taconic |
| Animal Housing: | Gnotobiotic isolators/conventional housing |
| Animal Light Cycle: | 12/12 |
| Animal Feed: | RM1A (P) and RM3A (P) from Special Diet Services |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003248 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57Bl/6J |
| Age Or Age Range: | 8-10 weeks |
| Weight Or Weight Range: | 20-29g |
| Height Or Height Range: | - |
| Gender: | Male |
| Animal Animal Supplier: | Taconic |
| Animal Housing: | Gnotobiotic isolators/conventional housing |
| Animal Light Cycle: | 12/12 |
| Animal Feed: | RM1A (P) and RM3A (P) from Special Diet Services |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Conv2 | SA339215 | FL040170 | Caecal Contents | Sample source |
| Conv2 | SA339215 | FL040170 | Conv_Pre | Factor |
Collection:
| Collection ID: | CO003241 |
| Collection Summary: | The acute restraint stress procedure was performed using a clean perforated polypropylene screw-cap 50 mL conical tubes. Cages were randomly assigned to either non-stress or stress groups. Each mouse that underwent stress was placed into the 50 mL tube and restrained for 15 minutes. After 15 minutes of restraint stress, mice were removed from the restrainer and either transported immediately to the cull room or returned to their home cage and left undisturbed for 45 minutes. To control for the variable of transport stress, mice from the non-stress control group were placed into new cages containing fresh bedding and transported the same distance before entering the cull room. Upon entering the cull room, mice were immediately decapitated, trunk blood collected, and tissues harvested. Caecal contents and intestinal samples were manually dissected and stored in PCR-grade microfuge tubes at -80°C until analyses. |
| Sample Type: | Cecum |
| Storage Conditions: | -80? |
Treatment:
| Treatment ID: | TR003257 |
| Treatment Summary: | All animal work carried was approved by the Animal Experimentation Ethics Committee of University College Cork and Health Products Regulatory Authority (HPRA) before beginning this study. All experimentation was carried out in accordance with European Directive 2010/63/EU and was approved by both the Animal Experimentation Ethics Committee of University College Cork (Project Authorization AE19130/P160) and United States Air Force Surgeon General's Office of Research Oversight and Compliance. C57/BL6 mice breeding pairs were acquired from Taconic Biosciences, and F1-generation male and female offspring were used in all experiments. Germ-free, ex-germ-free, and conventional mice were housed 2-4 mice/cage under a 12-hour light/dark cycle and maintained on ad libitum autoclaved water and autoclaved, pelleted diet (Special Diet Services). Housing conditions for germ-free, conventional, and colonized germ-free adhered to the same environmental conditions of temperature (21 ± 1°C) and humidity (55%-60%). Germ-free mice were housed in gnotobiotic flexible-film isolators. Colonized germ-free mice were born and maintained as germ-free mice in gnotobiotic flexible-film isolators until postnatal day 21 when they were removed from the isolators and, for the remaining duration of this study, re-located to the standard animal facility and housed in wire-top cages that contained used-bedding from age- and sex-matched conventional mice. The acute restraint stress procedure was performed using a clean perforated polypropylene screw-cap 50 mL conical tubes. Cages were randomly assigned to either non-stress or stress groups. Each mouse that underwent stress was placed into the 50 mL tube and restrained for 15 minutes. |
Sample Preparation:
| Sampleprep ID: | SP003255 |
| Sampleprep Summary: | Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis. |
Combined analysis:
| Analysis ID | AN005135 | AN005136 | AN005137 | AN005138 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | Reversed phase | HILIC |
| Chromatography system | Waters Acquity | Waters Acquity | Waters Acquity | Waters Acquity |
| Column | Waters Acquity BEH C18 (100 x 2.1mm, 1.7um) | Waters Acquity BEH C18 (100 x 2.1mm, 1.7um) | Waters Acquity BEH C18 (100 x 2.1mm, 1.7um) | Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) |
| MS Type | Other | Other | Other | Other |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | POSITIVE | NEGATIVE | NEGATIVE |
| Units | Raw Peak Area | Raw Peak Area | Raw Peak Area | Raw Peak Area |
Chromatography:
| Chromatography ID: | CH003885 |
| Chromatography Summary: | Low pH polar (LC/MS Pos early) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity BEH C18 (100 x 2.1mm, 1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 5% B to 80% B over 3.35 minutes |
| Flow Rate: | 0.35 mL/min |
| Solvent A: | 100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Solvent B: | 100% methanol; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003886 |
| Chromatography Summary: | Low pH Lipophilic (LC/MS Pos late) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity BEH C18 (100 x 2.1mm, 1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 40% B to 99.5% B over 1.0 minute, hold 99.5% B for 2.4 minutes. |
| Flow Rate: | 0.60 mL/min |
| Solvent A: | 100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Solvent B: | 50% methanol/50% acetonitrile; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003887 |
| Chromatography Summary: | High pH (LC/MS Neg) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity BEH C18 (100 x 2.1mm, 1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 0.5 to 70% B over 4.0 minutes, then rapid gradient to 99% B in 0.5 minutes. |
| Flow Rate: | 0.35 mL/min |
| Solvent A: | 100% water; 6.5 mM ammonium bicarbonate, pH 8 |
| Solvent B: | 95% methanol/5% water; 6.5 mM ammonium bicarbonate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003888 |
| Chromatography Summary: | HILIC (LC/MS Polar Neg) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 5% B to 50% B in 3.5 minutes, then linear gradient from 50% B to 95% B in 2 minutes. |
| Flow Rate: | 0.50 mL/min |
| Solvent A: | 15% water/5% methanol/80% acetonitrile; 10 mM ammonium formate, (effective pH 10.16 with NH4OH) |
| Solvent B: | 50% water/50% acetonitrile; 10 mM ammonium formate, (effective pH 10.60 with NH4OH) |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004871 |
| Analysis ID: | AN005135 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Pos early) |
| Ion Mode: | POSITIVE |
| MS ID: | MS004872 |
| Analysis ID: | AN005136 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Pos late) |
| Ion Mode: | POSITIVE |
| MS ID: | MS004873 |
| Analysis ID: | AN005137 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Neg) |
| Ion Mode: | NEGATIVE |
| MS ID: | MS004874 |
| Analysis ID: | AN005138 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Polar)#_1 MS_METABOLITE_DATA |
| Ion Mode: | NEGATIVE |
