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MB Sample ID: SA186819

Local Sample ID:	1 +AC 8h
Subject ID:	SU002080
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002080
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
1 +AC 8h	SA186819	FL023119	engulfing	Category

Collection:

Collection ID:	CO002073
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 7 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:	TR002092
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 7 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.

Treatment ID:

TR002092

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 7 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:	SP002086
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.

Sampleprep ID:

SP002086

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	AN003261	AN003262
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:	CH002405
Chromatography Summary:	Isocratic flow at 250 uL/min using 95% phase A (0.1% formic acid in water) and 5% phase 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 at 250 uL/min using 95% phase A (0.1% formic acid in water) and 5% phase 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:	CH002406
Chromatography Summary:	Isocratic flow at 250 uL/min using 100% phase 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:	MS003033
Analysis ID:	AN003261
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:	MS003034
Analysis ID:	AN003262
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