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MB Sample ID: SA017166
| Local Sample ID: | 10Glucose1hrReplicate3_0083.d |
| Subject ID: | SU000401 |
| Subject Type: | Cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Human |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000401 |
| Subject Type: | Cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Human |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 10Glucose1hrReplicate3_0083.d | SA017166 | FL006538 | 10.5mM Glu | Treatment |
| 10Glucose1hrReplicate3_0083.d | SA017166 | FL006538 | 1hr | Time |
Collection:
| Collection ID: | CO000395 |
| Collection Summary: | HepG2 cells (ATCC HB-8065) were cultured in MEM containing 10 % (v:v) FBS, 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA), 1 x MEM non-essential amino acids, and 5.5 mM glucose at 37 °C in a 5 % CO2 environment. Cells were grown for four to six passages in 10 cm tissue culture dishes with 14 mL MEM, and transferred to MULTIWELL™ 12 well culture dishes for incubation in 2 mL of the treatment medium. Upon reaching 80 % confluency, the cell culture medium was changed to media representative of experimental condition: 5.5 mM glucose MEM, 5.5 mM glucose MEM + 5 mM glucose, or 5.5 mM glucose MEM + 5 mM fructose. Media was replenished after 24 and 48 h. Cell material was collected at 10 min, 1, 6, and 24 h time points following the 48 h media replacement. Culture dishes were placed on ice and each well was washed twice with 1 mL ice-cold PBS. 4 mL ice-cold 3:1 methanol/H2O extraction solvent was added to each well. Cell material was manually scraped from each well, and the extraction solvent cell material suspension was transferred to collection tubes and frozen at −80 °C prior to further processing. |
| Collection Protocol Filename: | metabolomic_responses_of_cultured_HepG2_liver_cells.pdf |
| Sample Type: | Cell |
| Collection Location: | Invitrogen, Carlsbad, CA |
| Collection Frequency: | Cell material was collected at 10 min, 1, 6, and 24 h time points following the 48 h media replacement. |
| Tissue Cell Identification: | HepG2 |
Treatment:
| Treatment ID: | TR000415 |
| Treatment Summary: | 3 Treatments: A) Control group of HepG2 cells incubated in media with 5.5 mM glucose (Glc5) B) HepG2 cells incubated in media containing 5.5 mM glucose + 5.0 mM fructose (Glc5Fru5) C) HepG2 cells incubated in media containing 10.5 mM glucose (Glc10) |
| Treatment Protocol Filename: | metabolomic_responses_of_cultured_HepG2_liver_cells.pdf |
| Treatment Protocol Comments: | HepG2 cells incubated in media containing 10.5 mM glucose (Glc10) was included to enable differentiation of metabolic effects caused by fructose from metabolic effects caused by increased hexose resources |
| Cell Media: | Cells were acclimated to their respective media condition for 48 h prior to collection of sample material to obtain a metabolite profile representative of continued exposure instead of response to a sudden change in carbohydrate resources. Media was replenished after 24 and 48 h |
Sample Preparation:
| Sampleprep ID: | SP000408 |
| Sampleprep Summary: | Sample material was thawed on ice, vortexed for 20 s, sonicated for 5 min with a VWR 50HT Ultrasonic Bath (VWR International Inc., Bridgeport, NJ), and separated into 500 μL aliquots. Each aliquot was centrifuged for 5 min @ 14,000 rcf, and supernatant was collected and lyophilized to dryness. Samples was kept on ice and removed only for sonication, centrifugation, and lyophilization steps. Lyophilized material was used for HILIC-QTOF metabolite profiling without additional clean-up steps. Lyophilized material for GC-TOF analysis was redissolved in 1:1 acetonitrile/H2O, vortexed for 10 s, and centrifuged for 5 min @ 14,000 rcf. Supernatant was collected and lyophilized to dryness. |
| Sampleprep Protocol Filename: | metabolomic_responses_of_cultured_HepG2_liver_cells.pdf |
| Processing Method: | Lysophilization |
Combined analysis:
| Analysis ID | AN000614 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Agilent 6530 |
| Column | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Agilent 6530 QTOF |
| Ion Mode | POSITIVE |
| Units | counts |
Chromatography:
| Chromatography ID: | CH000440 |
| Methods Filename: | Data_Dictionary_Fiehn_laboratory_HILIC_QTOF_MS.pdf |
| Instrument Name: | Agilent 6530 |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| Column Pressure: | 200-700 bar |
| Column Temperature: | 45 C |
| Flow Gradient: | 100% B to 30% B |
| Flow Rate: | 0.4 mL/min |
| Injection Temperature: | 4 C |
| Internal Standard: | See data dictionary |
| Retention Time: | See data dictionary |
| Sample Injection: | 3uL |
| Solvent A: | 100% water; 0.125% formic acid; 10 mM ammonium formate, pH 3 |
| Solvent B: | 100% water; 0.125% formic acid; 10 mM ammonium formate, pH 3 |
| Analytical Time: | 14 min |
| Capillary Voltage: | 4500 V |
| Time Program: | 16.75 min |
| Weak Wash Solvent Name: | 1:1 ACN:H2O |
| Strong Wash Solvent Name: | 1:1 ACN:H2O |
| Target Sample Temperature: | Autosampler temp 4 C |
| Randomization Order: | Excel generated |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS000547 |
| Analysis ID: | AN000614 |
| Instrument Name: | Agilent 6530 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
| Capillary Voltage: | 3500 |
| Collision Gas: | Nitrogen |
| Dry Gas Flow: | 8 L/min |
| Dry Gas Temp: | 325 C |
| Fragment Voltage: | 120 V |
| Fragmentation Method: | Auto MS/MS |
| Ion Source Temperature: | 325 |
| Ion Spray Voltage: | 1000 |
| Ionization: | Pos |
| Precursor Type: | Intact Molecule |
| Reagent Gas: | Nitrogen |
| Source Temperature: | 325 C |
| Dataformat: | .d |
| Desolvation Gas Flow: | 11 L/min |
| Desolvation Temperature: | 350 C |
| Nebulizer: | 35 psig |
| Octpole Voltage: | 750 |
| Resolution Setting: | extended dynamic range |
| Scan Range Moverz: | 60-1700 Da |
| Scanning Cycle: | 2 Hz |
| Scanning Range: | 60-1700 Da |
| Skimmer Voltage: | 1850 V |
