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MB Sample ID: SA186818

Local Sample ID:unexp1
Subject ID:SU002079
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

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Subject:

Subject ID:SU002079
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
unexp1SA186818FL023118unexposed macrophagescategory

Collection:

Collection ID:CO002072
Collection Summary:LPS-primed murine peritoneal macrophages were fed fluorescently-tagged apoptotic Jurkat cells for 1h. Unengulfed targets were washed off and macrophages were left to degrade targets for a further 3 hours. Macrophages were then collected and sorted into non-engulfing or engulfing populations, alongside control unexposed macrophages. Sorted cells were pelleted at 400xg and dry pellets were snap frozen and stored at -80C until metabolite extraction.
Sample Type:Macrophages

Treatment:

Treatment ID:TR002091
Treatment Summary:LPS-primed murine peritoneal macrophages were fed fluorescently-tagged apoptotic Jurkat cells for 1h. Unengulfed targets were washed off and macrophages were left to degrade targets for a further 3 hours. Macrophages were then collected and sorted into non-engulfing or engulfing populations, alongside control unexposed macrophages. Sorted cells were pelleted at 400xg and dry pellets were snap frozen and stored at -80C until metabolite extraction.

Sample Preparation:

Sampleprep ID:SP002085
Sampleprep Summary:To process cells for assessment of intracellular metabolites, cells were pelleted at 400xg in tubes coated with 0.06% BSA. Supernatant was aspirated and discarded; residual liquid was carefully wicked away from the pellet with a kimwipe. Dry pellets were immediately snap frozen and stored at -80C until processing. To process culture supernatants for assessment of metabolites, supernatant was centrifuged at 400xg to pellet any cells. Cell-free supernatant was then transferred to a fresh tube, snap frozen, and stored at -80C until processing. Ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) was performed by the University of Colorado School of Medicine Metabolomics Core. Metabolites from frozen cell pellets were extracted at 2e6 cells/mL in ice cold 5:3:2 MeOH:acetonitrile:water (v/v/v). Media was thawed on ice and a 10 L aliquot treated with 240 L of the same extraction solution. Extractions were carried out using vigorous vortexing for 30 min at 4C. Supernatants were clarified by centrifugation (10 min, 18,000 g, 4C) and analyzed using a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive mass spectrometer.

Combined analysis:

Analysis ID AN003259 AN003260
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
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:CH002403
Chromatography Summary:Isocratic flow of 250 uL/min of 95% A (0.1% formic acid in water) and 5% B (0.1% formic acid in acetonitrile).
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:25
Flow Gradient:Isocratic flow of 250 uL/min of 95% A (0.1% formic acid in water) and 5% B (0.1% formic acid in acetonitrile).
Flow Rate:250 ul/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH002404
Chromatography Summary:Isocratic flow of 250 uL/min of 100% A (95% water, 5% acetonitrile, 10 mM ammonium acetate).
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:25
Flow Gradient:isocratic
Flow Rate:250 ul/min
Solvent A:95% water/5% acetonitrile; 10 mM ammonium acetate
Solvent B:N/A
Chromatography Type:Reversed phase

MS:

MS ID:MS003031
Analysis ID:AN003259
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Global metabolomics analyses were performed using a 3 min isocratic run in positive and negative ion modes (separate runs) as described previously (Nemkov et al., 2015, Nemkov et al., 2017); stable isotope tracing samples were analyzed using a 5 min C18 gradient in positive and negative ion modes (separate runs) as described (Nemkov et al., 2019, Gehrke et al., 2019). For all analyses, the MS scanned in MS1 mode across the m/z range of 65 to 950. Peaks were annotated (in conjunction with the KEGG database), integrated, and quality control performed using Maven (Princeton University) as described. Stable isotope tracing results were isotopically corrected for the natural abundance of 13C1, 13C2, 15N1, and 15N2 (Nemkov et al., 2017).
Ion Mode:POSITIVE
  
MS ID:MS003032
Analysis ID:AN003260
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Global metabolomics analyses were performed using a 3 min isocratic run in positive and negative ion modes (separate runs) as described previously (Nemkov et al., 2015, Nemkov et al., 2017); stable isotope tracing samples were analyzed using a 5 min C18 gradient in positive and negative ion modes (separate runs) as described (Nemkov et al., 2019, Gehrke et al., 2019). For all analyses, the MS scanned in MS1 mode across the m/z range of 65 to 950. Peaks were annotated (in conjunction with the KEGG database), integrated, and quality control performed using Maven (Princeton University) as described. Stable isotope tracing results were isotopically corrected for the natural abundance of 13C1, 13C2, 15N1, and 15N2 (Nemkov et al., 2017).
Ion Mode:NEGATIVE
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