Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA207626

Local Sample ID:	cont_3
Subject ID:	SU002253
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002253
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
cont_3	SA207626	FL025462	Control	Culture conditions
cont_3	SA207626	FL025462	No treatment	Treatment

Collection:

Collection ID:	CO002246
Collection Summary:	4T1 cells were cultured in 10 cm dishes for 48 hours and the culture supernatant was collected. The supernatant was stored as the 4T1-conditioned media at 4°C until use. AML cells per a well were cultured in a 6 well plate for 24 hours, and then the media was switched to the 4T1-conditioned media. After 24 hours, the treated AML12 cells were collected.
Sample Type:	AML cells

Treatment:

Treatment ID:	TR002265
Treatment Summary:	AML cells per well were cultured in a 24 well plate for 24 hours. The media was then switched to the bovine-serum free media, and TNF alpha was added at the concentration of 20 ng/ml or 200 ng/ml (Roche). After 24 hours, the treated AML12 cells were collected.

Sample Preparation:

Sampleprep ID:	SP002259
Sampleprep Summary:	Metabolites were extracted from AML12 cells (less than 6 × 10E5 cells/well (6 well plate)) using the Bligh and Dyer’s method with some modifications. Briefly, each sample was mixed with 1 mL of cold methanol containing 10-camphorsulfonic acid (1.5 nmol) and piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES, 1.5 nmol) as internal standards for mass spectrometry-based metabolomic analysis. The samples were vigorously mixed by vortexing for 1 min followed by 5 min of sonication. The extracts were then centrifuged at 16,000 × g for 5 min at 4 °C, and the resultant supernatant (400 uL) was collected. After mixing 400 uL of supernatant with 400 uL of chloroform and 320 uL of water, the aqueous and organic layers were separated by vortexing and subsequent centrifugation at 16,000 × g and 4 °C for 5 min. The aqueous (upper) layer (500 uL) was transferred into a clean tube. After the aqueous layer extracts were evaporated under vacuum, the dried extracts were stored at −80 °C until the analysis of hydrophilic metabolites. Prior to analysis, the dried aqueous layer was reconstituted in 50 uL of water.

Sampleprep ID:

SP002259

Sampleprep Summary:

Metabolites were extracted from AML12 cells (less than 6 × 10E5 cells/well (6 well plate)) using the Bligh and Dyer’s method with some modifications. Briefly, each sample was mixed with 1 mL of cold methanol containing 10-camphorsulfonic acid (1.5 nmol) and piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES, 1.5 nmol) as internal standards for mass spectrometry-based metabolomic analysis. The samples were vigorously mixed by vortexing for 1 min followed by 5 min of sonication. The extracts were then centrifuged at 16,000 × g for 5 min at 4 °C, and the resultant supernatant (400 uL) was collected. After mixing 400 uL of supernatant with 400 uL of chloroform and 320 uL of water, the aqueous and organic layers were separated by vortexing and subsequent centrifugation at 16,000 × g and 4 °C for 5 min. The aqueous (upper) layer (500 uL) was transferred into a clean tube. After the aqueous layer extracts were evaporated under vacuum, the dried extracts were stored at −80 °C until the analysis of hydrophilic metabolites. Prior to analysis, the dried aqueous layer was reconstituted in 50 uL of water.

Combined analysis:

Analysis ID	AN003550	AN003551
Analysis type	MS	MS
Chromatography type	Ion exchange	Reversed phase
Chromatography system	Thermo Dionex ICS-5000+	Shimadzu Nexera X2
Column	Dionex IonPac AS11-HC (250 x 2mm,4um)	Discovery HS (150 x 2.1mm,3um)
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:	CH002622
Chromatography Summary:	Anionic polar metabolites (i.e., organic acids, sugar phosphates, nucleotides, etc.) were analyzed via IC/HRMS/MS.
Instrument Name:	Thermo Dionex ICS-5000+
Column Name:	Dionex IonPac AS11-HC (250 x 2mm,4um)
Chromatography Type:	Ion exchange

Chromatography ID:	CH002623
Chromatography Summary:	Cationic polar metabolites (i.e., amino acids, bases, nucleosides, NAM, SAM, MNAM, SAH, me2PY, me4PY, etc) were analyzed via PFPP-LC/HRMS/MS.
Instrument Name:	Shimadzu Nexera X2
Column Name:	Discovery HS (150 x 2.1mm,3um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003307
Analysis ID:	AN003550
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	-
Ion Mode:	NEGATIVE

MS ID:	MS003308
Analysis ID:	AN003551
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	-
Ion Mode:	POSITIVE