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MB Sample ID: SA239103
Local Sample ID: | CTL_02_LIP329 |
Subject ID: | SU002489 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
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Subject:
Subject ID: | SU002489 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
CTL_02_LIP329 | SA239103 | FL029798 | CTL | Genotype |
Collection:
Collection ID: | CO002482 |
Collection Summary: | ASC were isolated from surgical samples of subcutaneous abdominal adipose tissue from a 25-year-old healthy woman with a normal body mass index (BMI). Adipose tissue was enzymatically digested with collagenase B (0.2%). |
Sample Type: | Adipose tissue |
Treatment:
Treatment ID: | TR002501 |
Treatment Summary: | After centrifugation, stromal vascular fraction was filtered, rinsed, plated and cultured in α-MEM with 10% Fetal Calf Serum (FCS), 1% GlutaMAX (#35050061, Thermo Fisher Scientific), 1% Penicillin/streptomycin (PS - 10,000 UI/mL), 1% HEPES and Fibroblast Growth Factor-2 (FGF-2 -145 nmol/L). After 24 h, only ASC adhered to plastic surfaces, while other cells were removed after culture medium replacement. ASC were maintained in an undifferentiated state in α-MEM supplemented with 10 % newborn calf serum (#CA-1151500; Biosera, MI, USA), 1% GlutaMAX, HEPES and P/S, and FGF-2 (145 nmol/L). Adipocyte differentiation was induced by treating 2-day post-confluent cultures with high-glucose (25 mmol/L) DMEM supplemented with 10 % FCS, 1 % PS, 1 µmol/L dexamethasone (#D4902; Sigma-Aldrich, MI, USA), 1 µM rosiglitazone (#D4902; Sigma-Aldrich), 250 µM 3-isobutyl-1-methyl xanthine (IBMX) (#I7018; Sigma-Aldrich) and 0.17 µmol/L insulin (#I0516; Sigma-Aldrich) for ten days. The medium was then replaced with high-glucose DMEM supplemented with 10% FCS, 1 % PS, 1 µmol/L rosiglitazone and 0.17 µM insulin, and changed to fresh medium every 2 days until the 20th day. |
Sample Preparation:
Sampleprep ID: | SP002495 |
Sampleprep Summary: | Lipid extraction. Lipids were extracted from ASC cells according to a modified Folch method. Cell pellets were resuspended in 100µl methanol and supplemented with deuterated internal standards. Lipids were extracted with 1.5 mL chloroform and 650 µL methanol, sonicated for 15 min. Phase separation was triggered by addition of 450 µL of ammonium carbonate (250 mM). Lower organic phase was dried and resuspended in 200 µL of liquid chromatography – mass spectrometry (LC-MS) solvent. |
Combined analysis:
Analysis ID | AN003907 | AN003908 | AN003909 | AN003910 | AN003911 |
---|---|---|---|---|---|
Analysis type | MS | MS | MS | MS | MS |
Chromatography type | HILIC | HILIC | HILIC | Reversed phase | Reversed phase |
Chromatography system | Shimadzu 20AD | Shimadzu 20AD | Shimadzu 20AD | Shimadzu 20AD | Shimadzu 20AD |
Column | Phenomenex Kinetex HILIC (150 x 3mm,2.6um) | Phenomenex Kinetex HILIC (150 x 3mm,2.6um) | Phenomenex Kinetex HILIC (150 x 3mm,2.6um) | Merck Supelco Ascentis Express C18 (150 x 2.1mm,2.7um) | Merck Supelco Ascentis Express C18 (150 x 2.1mm,2.7um) |
MS Type | ESI | ESI | ESI | ESI | ESI |
MS instrument type | Triple quadrupole | Triple quadrupole | Triple quadrupole | Triple quadrupole | Triple quadrupole |
MS instrument name | ABI Sciex 4000 QTrap | ABI Sciex 4000 QTrap | ABI Sciex 4000 QTrap | ABI Sciex 4000 QTrap | ABI Sciex 4000 QTrap |
Ion Mode | POSITIVE | POSITIVE | POSITIVE | POSITIVE | POSITIVE |
Units | mol% of total lipids | mol% of total lipids | mol% of total lipids | mol% of total lipids | mol% of total lipids |
Chromatography:
Chromatography ID: | CH002893 |
Instrument Name: | Shimadzu 20AD |
Column Name: | Phenomenex Kinetex HILIC (150 x 3mm,2.6um) |
Column Temperature: | 45 |
Flow Gradient: | - |
Flow Rate: | 300ul/min |
Solvent A: | 100% water; 0.2% acetic acid; 30 mM ammonium acetate |
Solvent B: | 100% acetonitrile; 0.2% acetic acid |
Chromatography Type: | HILIC |
Chromatography ID: | CH002894 |
Instrument Name: | Shimadzu 20AD |
Column Name: | Merck Supelco Ascentis Express C18 (150 x 2.1mm,2.7um) |
Column Temperature: | 43 |
Flow Gradient: | - |
Flow Rate: | 300ul/min |
Solvent A: | 60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate |
Solvent B: | 10% acetonitrile/90% isopropanol; 0.1% formic acid; 10 mM ammonium formate |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS003646 |
Analysis ID: | AN003907 |
Instrument Name: | ABI Sciex 4000 QTrap |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | MRM acquisition of broad chromatographic peaks of low abundant phospho- and sphingolipid classes: PS, PA, LPC, LPE and some ceramides this acquisition is called "long_1x" |
Ion Mode: | POSITIVE |
MS ID: | MS003647 |
Analysis ID: | AN003908 |
Instrument Name: | ABI Sciex 4000 QTrap |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | MRM acquisition of narrow chromatographic peaks of low abundant phospho- and sphingolipid classes: cer, PG, PI, PE, PE-P This acquisition is referred to as "short_1x" |
Ion Mode: | POSITIVE |
MS ID: | MS003648 |
Analysis ID: | AN003909 |
Instrument Name: | ABI Sciex 4000 QTrap |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | MRM acquisition of narrow chromatographic peaks of abundant phospho- and sphingolipid classes: PC and SM following 20-fold dilution This acquisition is referred to as "short_20x" |
Ion Mode: | POSITIVE |
MS ID: | MS003649 |
Analysis ID: | AN003910 |
Instrument Name: | ABI Sciex 4000 QTrap |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | MRM acquisition of low abundant neutral lipids: DG This acquisition is referred to as "C18_1x" |
Ion Mode: | POSITIVE |
MS ID: | MS003650 |
Analysis ID: | AN003911 |
Instrument Name: | ABI Sciex 4000 QTrap |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | MRM acquisition of abundant neutral lipids: CE and TG after 10 fold dilution This acquisition is referred to as "C18_10x" |
Ion Mode: | POSITIVE |