Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA392738

Local Sample ID:	1-89-5-
Subject ID:	SU003735
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Gender:	Not applicable

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Subject:

Subject ID:	SU003735
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Gender:	Not applicable

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
1-89-5-	SA392738	FL045477	6	time (h)
1-89-5-	SA392738	FL045477	live	E coli treatment
1-89-5-	SA392738	FL045477	WT	genotype
1-89-5-	SA392738	FL045477	murine bone marrow derived macrophages	Sample source

Collection:

Collection ID:	CO003728
Collection Summary:	BMDMs were seeded at 2.5E6 cell/well in a non-tissue treated 6-well plate (2 wells per condition) the day before the experiment. To harvest, BMDMs were washed with cold PBS and harvested with 5 mM EDTA in PBS, frozen as dry cell pellet, and stored at -80˚C until processing.
Sample Type:	Macrophages

Treatment:

Treatment ID:	TR003744
Treatment Summary:	Tg.hUbC-cre-8 ERT2+/T::Kras+/G12V mice were originally generated in the laboratory of M. Barbacid and provided by D. Santamaria. HrasG12S mice were originally generated at the Institut Clinique de la Souris (ICS) and provided by R. Rossignol. All transgenic mice were on a C57BL/6N background. We used 8- to 10-week-old male or female animals for all experiments and did not observe any gender bias. Preparation of macrophages: Murine bone marrow-derived macrophages (BMDMs) were generated as described previously, in RPMI 1640 supplemented with M-CSF (30% mycoplasma-free L929 cell supernatant, NCBI Biosample accession # SAMN00155972) and 10% FBS, plus 100 µg/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 1 nM sodium pyruvate and 50 µM 2-mercaptoethanol (all from Gibco). For Tg.hUbC-cre-ERT2+/T::Rragaf/f and Tg.hUbC-cre-ERT2+/T::Kras+/G12V mice, tamoxifen was added to the culture on day 2 or 3 to a final concentration of 1 µg/ml in order to induce CRE expression. BMDMs were used on day 5 to 7 after seeding. Period of differentiation of the cells, concentration of cells when replating and time-lapse between replating and stimulation with bacteria were important parameters to maintain metabolic backgrounds homogenous between experiments. Preparation of viable and killed U-[13C]Bacteria: ThyA- E. coli were grown overnight with shaking in LB supplemented with thymidine (500 µg/ml) and trimethoprim (50 µg/ml), diluted 1/40, and grown until log-phase [optical density at 600 nm (OD600) of 0.8-1.2]. Bacteria were washed with phosphate buffer saline (PBS) to remove LB salts before addition to cells. For labeling of bacteria, 10 µl of an overnight cultured of thyA- E. coli was added to 20 ml of a filtered M9 minimal medium salts (Life Technologies) supplemented with 1 mM thiamine, 1 mM MgSO4, 0.1 M CaCl2, 500 µg/ml thymidine, 50 µg/ml trimethoprim, and 0.5% U-[13C6] glucose (Campro Scientific). Bacteria were grown for 72h, washed with PBS and subjected to heat-killing by re-suspension in PBS and subsequently incubation at 60˚C for 60-90 min. For antibiotic killing, bacteria were incubated for 6h to 12h with Streptomycin or Gentamycin (50 µg/ml). Bacteria were kept at 4˚C until use. Efficient killing was confirmed by overnight plating on LB-agar plates. Treatment of macrophages: 2E6 BMDMs were plated 12-16h prior stimulation in non-tissue cultured treated 6-well plate (BD Falcons). Cells were then stimulated with live or killed labelled-E. coli at MOI 50, centrifuged at 2000 rpm for 5min. BMDMs were incubated for 5 min and washed with PBS to remove non-ingested bacteria and further incubated for 6h or 18h.

Treatment ID:

TR003744

Treatment Summary:

Tg.hUbC-cre-8 ERT2+/T::Kras+/G12V mice were originally generated in the laboratory of M. Barbacid and provided by D. Santamaria. HrasG12S mice were originally generated at the Institut Clinique de la Souris (ICS) and provided by R. Rossignol. All transgenic mice were on a C57BL/6N background. We used 8- to 10-week-old male or female animals for all experiments and did not observe any gender bias. Preparation of macrophages: Murine bone marrow-derived macrophages (BMDMs) were generated as described previously, in RPMI 1640 supplemented with M-CSF (30% mycoplasma-free L929 cell supernatant, NCBI Biosample accession # SAMN00155972) and 10% FBS, plus 100 µg/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 1 nM sodium pyruvate and 50 µM 2-mercaptoethanol (all from Gibco). For Tg.hUbC-cre-ERT2+/T::Rragaf/f and Tg.hUbC-cre-ERT2+/T::Kras+/G12V mice, tamoxifen was added to the culture on day 2 or 3 to a final concentration of 1 µg/ml in order to induce CRE expression. BMDMs were used on day 5 to 7 after seeding. Period of differentiation of the cells, concentration of cells when replating and time-lapse between replating and stimulation with bacteria were important parameters to maintain metabolic backgrounds homogenous between experiments. Preparation of viable and killed U-[13C]Bacteria: ThyA- E. coli were grown overnight with shaking in LB supplemented with thymidine (500 µg/ml) and trimethoprim (50 µg/ml), diluted 1/40, and grown until log-phase [optical density at 600 nm (OD600) of 0.8-1.2]. Bacteria were washed with phosphate buffer saline (PBS) to remove LB salts before addition to cells. For labeling of bacteria, 10 µl of an overnight cultured of thyA- E. coli was added to 20 ml of a filtered M9 minimal medium salts (Life Technologies) supplemented with 1 mM thiamine, 1 mM MgSO4, 0.1 M CaCl2, 500 µg/ml thymidine, 50 µg/ml trimethoprim, and 0.5% U-[13C6] glucose (Campro Scientific). Bacteria were grown for 72h, washed with PBS and subjected to heat-killing by re-suspension in PBS and subsequently incubation at 60˚C for 60-90 min. For antibiotic killing, bacteria were incubated for 6h to 12h with Streptomycin or Gentamycin (50 µg/ml). Bacteria were kept at 4˚C until use. Efficient killing was confirmed by overnight plating on LB-agar plates. Treatment of macrophages: 2E6 BMDMs were plated 12-16h prior stimulation in non-tissue cultured treated 6-well plate (BD Falcons). Cells were then stimulated with live or killed labelled-E. coli at MOI 50, centrifuged at 2000 rpm for 5min. BMDMs were incubated for 5 min and washed with PBS to remove non-ingested bacteria and further incubated for 6h or 18h.

Sample Preparation:

Sampleprep ID:	SP003742
Sampleprep Summary:	Metabolites from frozen pellets were extracted at 4e6 cells per mL using ice cold 5:3:2 methanol:acetonitrile:water (v/v/v) with vigorous vortexing at 4 degrees C followed by centrifugation as described for 10 min at 18,000 g. Supernatants were maintained at 4°C until analysis that same day.
Processing Storage Conditions:	4℃
Extract Storage:	-80℃

Combined analysis:

Analysis ID	AN005925	AN005926
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	NEGATIVE	POSITIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH004500
Chromatography Summary:	Negative C18
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B
Flow Rate:	450 uL/min
Sample Injection:	6 uL
Solvent A:	95% water/5% acetonitrile; 1 mM ammonium acetate
Solvent B:	95% acetonitrile/5% water; 1 mM ammonium acetate
Chromatography Type:	Reversed phase

Chromatography ID:	CH004501
Chromatography Summary:	Positive C18
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B
Flow Rate:	450 uL/min
Sample Injection:	6 uL
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005642
Analysis ID:	AN005925
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:	NEGATIVE

MS ID:	MS005643
Analysis ID:	AN005926
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:	POSITIVE