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MB Sample ID: SA317311
Local Sample ID: | Ctrl_2 |
Subject ID: | SU003035 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU003035 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Ctrl_2 | SA317311 | FL037792 | Control | Treatment |
Collection:
Collection ID: | CO003028 |
Collection Summary: | The SGBS cells were obtained from Prof. Martin Wabitsch laboratory at the University Clinic Ulm. SGBS preadipocytes were differentiated according to the standard protocol described previously (Wabitsch et al., 2001). |
Sample Type: | Adipose tissue |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR003044 |
Treatment Summary: | SGSB preadipocytes were maintained at 37°C and 5% CO2 in 95% humidity. To investigate the effects of the plasticizer DINCH and its primary metabolite MINCH on adipocyte differentiation, SGBS preadipocytes were treated for 12 days with differentiation media without the PPARG agonist rosiglitazone supplemented with DINCH or MINCH (10 nM, 100 nM, 1 µM, and 10 µM). To obtain an adipogenesis reference, SGBS cells were differentiated in the presence of rosiglitazone; to obtain an untreated control, they were differentiated in the absence of rosiglitazone. A final concentration of 0.01% (v/v) MeOH and 0.02% (v/v) DMSO was added to all conditioned differentiation media. Continuous exposure was mimicked by replacing the cell culture medium every second day. Each treatment was performed in four biological replicates (n=4). |
Sample Preparation:
Sampleprep ID: | SP003041 |
Sampleprep Summary: | Extraction of intracellular and extracellular metabolites was performed by a 1:1:1 methanol:water:chloroform extraction protocol. For the extraction of intracellular metabolites, the culture medium was removed and the cells were rinsed twice with 1 ml of 0.9% ice-cold NaCl. The rinsing solution was removed, and the metabolism of the cells was stopped by adding 400 µL of MeOH (-20 °C) followed by 400 µL of ice-cold H2O containing 10 µM d6-glutarate. Cells were collected using a cell lifter and 400 µL of chloroform was added. After shaking at 1,400 rpm and 4 °C for 20 min, the extraction mixture was centrifuged at 18,000 g and 4 °C for 5 min. Subsequently, 300 µL volume of the polar upper phase was collected and evaporated to complete dryness. For the extraction of extracellular metabolites, 300 µL of the supernatant was extracted by adding 400 µL MeOH (-20 °C) containing 100 nM MEHP, 100 µL ice-cold H2O containing 40 µM d6-glutarate, and 400 µL chloroform (-20 °C). Subsequent sample preparation was identical to the extraction of intracellular metabolites. Note: After measurement of the samples by LC-MS, the raw AUC values uploaded here were normalized to the internal standard (d6-glutarate, if applicable) and DNA content per well (measured by DAPI fluorescence). After normalization, log2 fold changes were calculated by dividing the normalized peak area from each replicate of each treatment by the normalized peak area from each control. Insulin data were not normalized to DAPI because fold changes were calculated by dividing the intensities of the insulin-stimulated cells by the noninsulin-stimulated cells from each treatment. |
Combined analysis:
Analysis ID | AN004792 |
---|---|
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+ QTrap |
Ion Mode | NEGATIVE |
Units | Peak AUC |
Chromatography:
Chromatography ID: | CH003623 |
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: | MS004538 |
Analysis ID: | AN004792 |
Instrument Name: | ABI Sciex 6500+ QTrap |
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 |