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MB Sample ID: SA163676

Local Sample ID:scfa_Exp2_7_Hu+PBS_week2
Subject ID:SU001825
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:GF 129SvEv background IL-10-deficient mice (Il10-/-); Il10-eGFP-reporter (Il10+/eGFP) mice (on a C57BL/6J background)
Age Or Age Range:8-12 week old
Animal Animal Supplier:8-12 week-age GF 129SvEv background IL-10-deficient mice (Il10-/-) were obtained from University of North Carolina National Gnotobiotic Rodent Resource Center; Il10-eGFP-reporter (Il10+/eGFP) mice on a C57BL/6J background mice were originally provided by Dr. C. L. Karp (Global Health, Bill & Melinda Gates Foundation, USA)
Animal Housing:Germ-free and gnotobiotic conditions at National Gnotobiotic Rodent Resource Center, University of North Carolina

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Subject:

Subject ID:SU001825
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:GF 129SvEv background IL-10-deficient mice (Il10-/-); Il10-eGFP-reporter (Il10+/eGFP) mice (on a C57BL/6J background)
Age Or Age Range:8-12 week old
Animal Animal Supplier:8-12 week-age GF 129SvEv background IL-10-deficient mice (Il10-/-) were obtained from University of North Carolina National Gnotobiotic Rodent Resource Center; Il10-eGFP-reporter (Il10+/eGFP) mice on a C57BL/6J background mice were originally provided by Dr. C. L. Karp (Global Health, Bill & Melinda Gates Foundation, USA)
Animal Housing:Germ-free and gnotobiotic conditions at National Gnotobiotic Rodent Resource Center, University of North Carolina

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
scfa_Exp2_7_Hu+PBS_week2SA163676FL019465Before GUT108/PBS therapy (baseline)Treatment

Collection:

Collection ID:CO001818
Collection Summary:For samples for targeted metabolomics analysis, mouse cecal content and/or feces were collected.
Collection Protocol ID:University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee (18-266.0-B)
Sample Type:Feces
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001838
Treatment Summary:For treatment 200 µl of diluted human donor stool was applied by oral gavage on day 1 to Il10-/- mice. The stool was derived from a single healthy donor, Donor-Y, and was previously found to induce moderate to severe colitis in Il10-/- mice. One gram stool was diluted 100- fold with anaerobic PBS, and vigorously mixed for 5 minutes under anaerobic conditions. For application of GUT-103 and GUT-108, 300µl resuspended strain mixture in anaerobic PBS was applied per mouse by oral gavage. GUT-103 and GUT-108 strains were grown individually, subsequently mixed to equal concentrations (cfu/ml), and provided at a dose of 2.0x10+7 cfu/strain in a total volume of 300 µl. The strain mixture was provided four times via oral gavage on days 15, 17, 22 and 25 (Il10-/- mice) or on days 1, 3, 8 and 11 (Il10+/eGFP mice).

Sample Preparation:

Sampleprep ID:SP001831
Sampleprep Summary:Bile acids, SCFAs, and tryptophan metabolites were extracted (with careful cleanup or derivatization procedures whenever necessary) and analyzed using mass spectrometry-based platforms.
Sampleprep Protocol Filename:Protocols.pdf
Processing Storage Conditions:On ice
Extraction Method:Bile acid: SPE and LLE; SCFA: alkaline aqueous extraction; tryptophan metabolites: 1:1 MeOH:Water LLE
Extract Cleanup:For Bile acids, C18 solid-phase extraction cleanup and concentration was used.
Extract Storage:-80℃
Sample Derivatization:For SCFAs, propyl chloroformate derivatization was performed.

Combined analysis:

Analysis ID AN002845 AN002846 AN002847
Analysis type MS MS MS
Chromatography type GC Reversed phase Reversed phase
Chromatography system Agilent 7820A Thermo Vanquish Thermo Vanquish
Column Agilent DB5-MS (30m x 0.25mm, 0.25um) Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um) Waters Acquity BEH C18 (150 x 2mm,1.7um)
MS Type EI ESI ESI
MS instrument type Single quadrupole Orbitrap Triple quadrupole
MS instrument name Agilent 5977B Thermo Q Exactive Orbitrap Thermo Quantis QQQ
Ion Mode POSITIVE POSITIVE NEGATIVE
Units μmol/kg ng/g mg/g

Chromatography:

Chromatography ID:CH002106
Chromatography Summary:Generic DB-5ms GC capillary column for analysis of SCFA derivatives.
Instrument Name:Agilent 7820A
Column Name:Agilent DB5-MS (30m x 0.25mm, 0.25um)
Chromatography Type:GC
  
Chromatography ID:CH002107
Chromatography Summary:C18 LC (HSS-T3) for quantitation of tryptophan metabolites
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Chromatography Type:Reversed phase
  
Chromatography ID:CH002108
Chromatography Summary:C18 LC (BEH C18) and (HSS T3) for quantitation of bile acids
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity BEH C18 (150 x 2mm,1.7um)
Chromatography Type:Reversed phase

MS:

MS ID:MS002638
Analysis ID:AN002845
Instrument Name:Agilent 5977B
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:fullscan data with scan range of m/z 30-600; Peak integration was performed in MassHunter Qual.
Ion Mode:POSITIVE
  
MS ID:MS002639
Analysis ID:AN002846
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Optimized PRM method scanning characteristic MS2 fragment ion for absolute quantitation.
Ion Mode:POSITIVE
  
MS ID:MS002640
Analysis ID:AN002847
Instrument Name:Thermo Quantis QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Optimized SRM method scanning characteristic MS2 fragment ion for absolute quantitation.
Ion Mode:NEGATIVE
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