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MB Sample ID: SA239098

Local Sample ID:ADH1B_KO_03_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 IDMB Sample IDFactor Level IDLevel ValueFactor Name
ADH1B_KO_03_LIP329SA239098FL029797ADH1B_KOGenotype

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 QTRAP QTRAP QTRAP QTRAP QTRAP
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:QTRAP
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:QTRAP
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:QTRAP
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:QTRAP
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:QTRAP
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
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