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MB Sample ID: SA011159
| Local Sample ID: | PA2 |
| Subject ID: | SU000261 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | HepG2 |
| Subject Comments: | NA |
| Cell Primary Immortalized: | Immortalized |
| Cell Passage Number: | NA |
| Cell Counts: | NA |
| Species Group: | Human |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000261 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | HepG2 |
| Subject Comments: | NA |
| Cell Primary Immortalized: | Immortalized |
| Cell Passage Number: | NA |
| Cell Counts: | NA |
| Species Group: | Human |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| PA2 | SA011159 | FL002721 | 500 | Concentration (uM) |
| PA2 | SA011159 | FL002721 | Palmitic acid | Treatment_compound |
Collection:
| Collection ID: | CO000252 |
| Collection Summary: | - |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR000272 |
| Treatment Summary: | Cells treated 24hr with 500 µM Fa, analogue or BSA control. Harvested and lipids extracted and then resolved using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) s |
| Treatment Protocol ID: | CBC_treatment |
| Treatment Protocol Filename: | See_Comments |
| Treatment Protocol Comments: | Human hepatoma HepG2 cells (American Type Culture Collection; HB-8065) were grown in 75 ml tissue cell culture flasks in Eagle's minimal essential medium (EMEM) augmented with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2. For treatment with fatty acids or analogues, the cells were seeded at a density of 2 × 106 cells in a T25-cm2 flask for 24 h. Each FA or analogue was added to the culture medium as a fatty acid-bovine serum albumin (BSA) complex (2.5:1, FA:BSA) to give 500µM final concentration. The controls in these experiments were HepG2 cells with BSA alone. After 24 h treatment, the media was collected, cells were rinsed twice with PBS and cells were harvested for complex lipid analysis. |
| Treatment Compound: | Fatty acid/BSA |
| Treatment Dose: | 500 µM (fatty acid) |
| Treatment Doseduration: | 24 hr |
| Treatment Vehicle: | PBS |
| Cell Growth Container: | 75 ml tissue cell culture flasks |
| Cell Media: | Eagle's minimal essential medium (EMEM) augmented with 10% fetal bovine serum |
| Cell Envir Cond: | 37°C in a humidified atmosphere with 5% CO2 |
| Cell Harvesting: | Typsinize and scrape |
| Cell Media Lastchanged: | 24 hr |
Sample Preparation:
| Sampleprep ID: | SP000266 |
| Sampleprep Summary: | - |
| Sampleprep Protocol Comments: | For metabolomics analysis, the lipid extracts were resuspended in chloroform/methanol, 2:1 (v/v).All analyses were carried out using an Agilent 1200 Series HPLC, ACE 5 C8-300 column (2.1 x 100 mm) and linear gradient elution at a flow rate of 0.1 mL/min. Mobile phase A and B consisted of 0.1% formic acid; 10 mM ammonium acetate in H2O and 0.1% formic acid; 10 mM ammonium acetate in ACN/isopropanol (50/50; v/v), respectively. The injection volume was 4 µL. Separation of metabolites was achieved at the following gradient: T=0 min: 30% B; T=1 min: 30% B; T=25 min: 100% B; T=45 min: 100% B; T=47 min: 30% B; and T=60 min: 30% B (re-equilibration). The HPLC system was directly coupled to a Bruker Soalrix 70 Hybrid FTMS instrument equipped with electrospray ionization source (ESI) (Bruker Daltonics). The system was controlled by HyStar v.3.4.8.0 software. MS data was collected with resolving power of 78,000 (at m/z 400) in positive or negative mode under following conditions: a capillary voltage of (+/-) 4,500 V and an end plate offset of -500 V. The dry temperature was set at 180°C. Dry gas flow was maintained 4 L/min. Acquisition range was 244-1,800 m/z with 0.2 s ion accumulation time. LC-MS data was converted into mzXML format using CompassXport v. 3.0.6 and processed by mzMine v.2.10 [25] or XCMS data analysis software. Data processing involved mass detection, chromatographic peak detection and deconvolution, isotopic peaks grouping, normalization and peak alignment. |
| Extraction Method: | Folche Lipid Extraction: Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J biol Chem. 1957;226:497-509. |
| Sample Spiking: | 25 µg C15:0 PE, C17:0 PC and C71:1 TAG added to 2 X 10e6 cells prior to lipid extraction |
| Cell Type: | HepG2 |
Combined analysis:
| Analysis ID | AN000373 | AN000374 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Agilent 1200 | Agilent 1200 |
| Column | ACE 5 C8-300 (100 x 2.1mm) | ACE 5 C8-300 (100 x 2.1mm) |
| MS Type | ESI | ESI |
| MS instrument type | FT-ICR-MS | FT-ICR-MS |
| MS instrument name | Bruker SolariX FT-ICR-MS | Bruker SolariX FT-ICR-MS |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak area | Peak area |
Chromatography:
| Chromatography ID: | CH000270 |
| Methods Filename: | jac8lipidneg.m |
| Chromatography Comments: | jalcms1lipneg.m |
| Instrument Name: | Agilent 1200 |
| Column Name: | ACE 5 C8-300 (100 x 2.1mm) |
| Flow Rate: | 0.1 mL/min |
| Internal Standard: | 25 g C15:0 PE, C17:0 PC and C71:1 TAG |
| Solvent A: | 100% water; 0.1% acetic acid; 2 mM ammonium acetate |
| Solvent B: | 50% acetonitrile/50% isopropanol; 0.1% formic acid; 10 mM ammonium acetate |
| Analytical Time: | 60 min |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS000319 |
| Analysis ID: | AN000373 |
| Instrument Name: | Bruker SolariX FT-ICR-MS |
| Instrument Type: | FT-ICR-MS |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
| Dataformat: | *.d |
| MS ID: | MS000320 |
| Analysis ID: | AN000374 |
| Instrument Name: | Bruker SolariX FT-ICR-MS |
| Instrument Type: | FT-ICR-MS |
| MS Type: | ESI |
| Ion Mode: | NEGATIVE |
| Dataformat: | *.d |
