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MB Sample ID: SA218460

Local Sample ID:KO3
Subject ID:SU002366
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

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Subject ID:SU002366
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090


Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name


Collection ID:CO002359
Collection Summary:Mouse colonies were bred at the CNIC under specific pathogen-free conditions and on C57BL/6 background. Tfamf/f (Larsson et al., 1998) mice were kindly provided by Nils-Göran Larsson (Max Planck Institute for Biology of Ageing, Cologne, Germany). All floxed mouse lines were crossed with CD11cCre mice (Caton et al., 2007). Mice were group-housed, have not been used in previous procedures and were fed standard chow. Littermates of the same sex were randomly assigned to experimental groups. Male and female mice were used for all experiments. Mice with 6–10-weeks (adult) were used for the experiment. Bronchoalveolar lavage (BAL) was performed by inserting a venal catheter (BD) into the trachea and 3-10 washes with 0.3-1 ml FACS buffer to harvest BAL cells.
Collection Protocol Filename:Protocol_AM.pdf
Sample Type:Bronchoalveolar lavage
Collection Method:Bronchoalveolar lavage (BAL) was performed by inserting a venal catheter (BD) into the trachea and 3-10 washes with 0.3-1 ml FACS buffer to harvest BAL cells.
Storage Conditions:-80℃


Treatment ID:TR002378
Treatment Summary:Mice were group-housed and were fed standard chow.

Sample Preparation:

Sampleprep ID:SP002372
Sampleprep Summary:One million CD45+ F4/80+ CD11c+ FACS-sorted alveolar macrophages (AMs) from the bronchoalveolar lavage (BAL) of adult Tfamf/f and CD11c∆Tfam mice were collected. Each sample was generated by merging FACS-sorted AMs from 13 to 30 mice. Quality Control (QC) samples (n=4) were prepared by pooling equal volumes of cell extracts from each sample by following the same protocols used for the subject samples. Samples were subjected to two freeze–thaw cycles for metabolism quenching and complete metabolite extraction, specifically by placing the samples at -80ºC for 15 min and thawing them on ice for 10 min with brief vortex-mixing. The samples were then centrifuged at 20,000 xg at 4°C for 10 min and the supernatant collected. The supernatant was evaporated to dryness (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA) and derivatized with 10 μl O-methoxyamine hydrochloride (15mg/mL) in pyridine and 10 μl N,O-bis(trimethylsilyl)trifluoroacetamide in 1% trimethylchlorosilane. Finally, 100 μl of heptane containing 10 ppm of 4-nitrobenzoic acid (IS) was used as internal standard to monitor sample injection
Sampleprep Protocol Filename:Protocol_AM.pdf

Combined analysis:

Analysis ID AN003724
Analysis type MS
Chromatography type GC
Chromatography system Agilent 7890B
Column Agilent DB5-MS (30m x 0.25mm, 0.25um)
MS Type EI
MS instrument type QTOF
MS instrument name Agilent 7250 GC/Q-TOF
Units relative abundance


Chromatography ID:CH002758
Chromatography Summary:Derivative samples (2 μL) were injected into a GC column DB5–MS (30 m length, 0.250 mm i.d., 0.25 μm film 95% dimethyl/5% diphenylpolysiloxane) with a pre–column (10 m J&W integrated with Agilent 122–5532G). The temperature gradient was programmed at 60 °C (held for 1 min), with a ramping increase rate of 10 °C/min up to 325°C (held for 10 min). The total analysis time was 37.5 min. Two analytical replicates for each sample were injected.
Methods Filename:Protocol_AM.pdf
Instrument Name:Agilent 7890B
Column Name:Agilent DB5-MS (30m x 0.25mm, 0.25um)
Chromatography Type:GC


MS ID:MS003472
Analysis ID:AN003724
Instrument Name:Agilent 7250 GC/Q-TOF
Instrument Type:QTOF
MS Type:EI
MS Comments:The EI source was operated at 70 eV whereas the mass spectrometer operated in the scan mode over a mass range of m/z 50–600. Metabolite deconvolution and identification were carried out using Agilent MassHunter Unknowns Analysis version B.07.00, then, data was aligned in Agilent Mass Profiler Professional version B.12.1 and exported to Agilent MassHunter Quantitative Analysis version B.07.00. Metabolites were identified by comparing their retention time, retention index and mass fragmentation patterns with those available in an in-house library including both the NIST mass spectral database (version 2017) and Fiehn RTL library (version 2008). The different derivatives that were generated from the silylated compounds were unified by summing the abundance of all derivatives from the same metabolite. Finally, the median relative area of the two analytical replicates of the same sample was computed and used for subsequent statistical analysis.
Analysis Protocol File:Protocol_AM.pdf