Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA316419

Local Sample ID:	SGBS_ctrl_d9_1
Subject ID:	SU003027
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU003027
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
SGBS_ctrl_d9_1	SA316419	FL037676	d9	Treatment
SGBS_ctrl_d9_1	SA316419	FL037676	1	Replicate
SGBS_ctrl_d9_1	SA316419	FL037676	Homo sapiens	Species

Collection:

Collection ID:	CO003020
Collection Summary:	Human SGBS cells were provided by the laboratory of Prof. Dr. Wabitsch at the University Clinic Ulm. Cells were differentiated according to the standard protocol described previously (Wabitsch et al., 2001). 3T3-L1 mouse embryonic fibroblasts were purchased from the American Type Culture Collection (ATCC, CL-173, USA).
Sample Type:	Adipose tissue
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR003036
Treatment Summary:	Both cell lines were maintained under 5% CO2 at 37°C and 95% humidity. Cell culture experiments for metabolomic (SGBS and 3T3L1) data analysis were performed in 6 replicates. Cells were cultivated until day0,day6 and day12 for the SGBS cell line, and until day0, day5 and day10 for the 3T3-L1 cells.

Sample Preparation:

Sampleprep ID:	SP003033
Sampleprep Summary:	Intracellular metabolites were extracted using a 1:1:1 methanol:water:chloroform extraction protocol. Briefly, culture media was removed and cells were rinsed with icecold 0.9 % sodium chloride solution. The rinsing solution was removed and the cells metabolism was quenched by adding equal volumes of methanol, followed by icecold deionized water. Cells were scraped off the culture plate and combined chloroform. Samples were vortexed and kept on a shaker at 4°C for 20 min at 1400 rpm. Cell debris was removed by centrifugation. A fixed volume of the polar upper phase was transferred to new tubes and dried to completeness.

Sampleprep ID:

SP003033

Sampleprep Summary:

Intracellular metabolites were extracted using a 1:1:1 methanol:water:chloroform extraction protocol. Briefly, culture media was removed and cells were rinsed with icecold 0.9 % sodium chloride solution. The rinsing solution was removed and the cells metabolism was quenched by adding equal volumes of methanol, followed by icecold deionized water. Cells were scraped off the culture plate and combined chloroform. Samples were vortexed and kept on a shaker at 4°C for 20 min at 1400 rpm. Cell debris was removed by centrifugation. A fixed volume of the polar upper phase was transferred to new tubes and dried to completeness.

Combined analysis:

Analysis ID	AN004784
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1290 Infinity II
Column	Waters XSelect XP HSS T3 (150 x 2.1mm, 2.5um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex 6500+
Ion Mode	NEGATIVE
Units	AUC

Chromatography:

Chromatography ID:	CH003615
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Waters XSelect XP HSS T3 (150 x 2.1mm, 2.5um)
Column Temperature:	40
Flow Gradient:	0-5 min 0% B, 5-9 min 0%- 2% B, 9-9.5 min 2-6% B, 9.5-11.5 min 6% B, 11.5-12 min 6-11% B, 12-13.5 min 11% B, 13.5-15.5 min 11-28% B, 15.5-16.5 min 28-53% B, 16.5-22.5 53% B, 22.5-23 min 53-0% B, 23-33 min 0% B
Flow Rate:	0-15.5 min 0.4 mL/min, 15.5-16.5 min 0.4-0.15 mL/min, 16.5-23 min 0.15 mL/min, 23-27 min 0.15-0.4 mL/min, 27-33 min 0.4 mL/min
Solvent A:	10mM TBA, 10mM acetic acid, 5% MeOH, 2% IPA in water
Solvent B:	100% IPA
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004530
Analysis ID:	AN004784
Instrument Name:	ABI Sciex 6500+
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	For identification and quantitation, a scheduled MRM method was used, with specific transitions for every metabolite. Data acquisition and peak integration were performed in Analyst® software (Version 1.7.1).
Ion Mode:	NEGATIVE