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MB Sample ID: SA092005
Local Sample ID: | ACN-H2O |
Subject ID: | SU001333 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Female |
Cell Biosource Or Supplier: | ATCC |
Cell Strain Details: | MCF-7 |
Cell Passage Number: | 30 |
Species Group: | Homo Sapien |
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Subject:
Subject ID: | SU001333 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Female |
Cell Biosource Or Supplier: | ATCC |
Cell Strain Details: | MCF-7 |
Cell Passage Number: | 30 |
Species Group: | Homo Sapien |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
ACN-H2O | SA092005 | FL013195 | Acetonitrile/Water | Exctraction |
Collection:
Collection ID: | CO001327 |
Collection Summary: | The breast adenocarcinoma cells (MCF-7) were cultured in DMEM medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin solution and incubated at 37oC in an atmosphere of 5% CO2. In preparation for an experiment, 3 × 106 cells were cultured in each of 15 tissue culture flasks (175 cm2) and cells were incubated for three days. When the cells reach 80% confluency, they were collected by trypsinization, counted in a cell counter and each 3 ×106 cells were suspended in 1 ml phosphate-buffered saline in an Eppendorf tube. |
Sample Type: | Breast cancer cells |
Collection Method: | trypsinization protocol |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR001348 |
Treatment Summary: | The aim of this study was to investigate the solvent extraction efficiency, so there was no cell treatment |
Sample Preparation:
Sampleprep ID: | SP001341 |
Sampleprep Summary: | A total of 5 triplicated MCF-7 cell culture samples were provided in Eppendorf vials dissolved in PBS, and stored at 4 °C for preservation purposes. Samples were centrifuged at 13000 rpm for 10 minutes at -4 °C. Supernatant was discarded, and cell pellets, each containing 3 million cells, were subjected to metabolomics analysis. In order to evaluate the influence of the solvents on the extraction rate, samples were divided in five different extraction groups: A, 0.2 % formic acid in water; B, 0.2 % ammonium hydroxide in water; C, ethyl acetate; D, methanol/water (1:1, v/v); and E, acetonitrile/water (1:1, v/v). Briefly, 300 µL of the extraction solvent was added to 3 million cell pellets, then vortexed for 2 minutes. All samples have been stored in ice for 1 hour, during which samples have been vortexed every 15 minutes. After this, cell insoluble matrix was centrifuged (13000 rpm, 10 minutes, -4 °C). Supernatant was collected then dried using EZ-2 Plus (GeneVac, Ipswich, UK) at 37 ± 1 °C. To detect all amino acids and metabolites, all samples were derivatized with methoxyamine hydrochloride and MSTFA + 1% TMCS prior to injection to GC-MS. |
Processing Storage Conditions: | Described in summary |
Extraction Method: | Direct extraction |
Extract Enrichment: | described in Summary |
Extract Storage: | Described in summary |
Sample Derivatization: | methoxyamine hydrochloride and MSTFA + 1% TMCS |
Combined analysis:
Analysis ID | AN002102 |
---|---|
Analysis type | MS |
Chromatography type | GC |
Chromatography system | Shimadzu GCMS-QP2010 Ultra |
Column | Restek Rtx-5Sil (30m x 0.25mm,0.25um) |
MS Type | EI |
MS instrument type | Single quadrupole |
MS instrument name | Shimadzu QP2010 Ultra |
Ion Mode | POSITIVE |
Units | Peak Area |
Chromatography:
Chromatography ID: | CH001534 |
Chromatography Summary: | A GC/MS-QP 2010 Ultra System (Shimadzu, Kyoto, Japan) was employed for the metabolomic analysis, along with LabSolutions GC-MS software (ver). A Restek Rtx®-5ms column (30.0 m × 0.25 mm, 0.25 µm) was utilized for separation of the metabolites. Helium (99.9%) was used as the carrier gas at the flow rate of 1.0 mL/min. The initial oven temperature was set at 60 °C and was held at this temperature for 2 minutes, then raised to 310 °C by 50 °C/min and held at this temperature during the analysis. Both the auxiliary temperature at the interface and the ionization temperature were 250 °C. Metabolites were analysed in full scan mode within the range of 50 – 650 amu. Total volume of 10 µL was injected in splitless mode, by employing AOC-20i Auto Injector (Shimadzu, Kyoto, Japan). GC total ion chromatograms (TIC) and fragmentation patterns of the metabolites identified using NIST/EPA/NIH Mass Spectral Library (NIST 14). Run time for each sample was 43.67 min. |
Methods Filename: | PAH scan |
Instrument Name: | Shimadzu GCMS-QP2010 Ultra |
Column Name: | Restek Rtx-5Sil (30m x 0.25mm,0.25um) |
Column Pressure: | 57.4 |
Column Temperature: | 60 |
Flow Rate: | 1 |
Injection Temperature: | 250 |
Retention Time: | many retention time |
Sample Injection: | 1 |
Solvent A: | Pyridine |
Analytical Time: | 43.67 |
Oven Temperature: | 60 |
Time Program: | 43.67 |
Transferline Temperature: | 250 |
Strong Wash Solvent Name: | Acetone |
Strong Wash Volume: | 10 uL |
Sample Syringe Size: | 10 uL |
Chromatography Type: | GC |
MS:
MS ID: | MS001953 |
Analysis ID: | AN002102 |
Instrument Name: | Shimadzu QP2010 Ultra |
Instrument Type: | Single quadrupole |
MS Type: | EI |
MS Comments: | A GC/MS-QP 2010 Ultra System (Shimadzu, Kyoto, Japan) was employed for the metabolomic analysis, along with LabSolutions GC-MS software (ver). A Restek Rtx®-5ms column (30.0 m × 0.25 mm, 0.25 µm) was utilized for separation of the metabolites. Helium (99.9%) was used as the carrier gas at the flow rate of 1.0 mL/min. The initial oven temperature was set at 60 °C and was held at this temperature for 2 minutes, then raised to 310 °C by 50 °C/min and held at this temperature during the analysis. Both the auxiliary temperature at the interface and the ionization temperature were 250 °C. Metabolites were analysed in full scan mode within the range of 50 – 650 amu. Total volume of 10 µL was injected in splitless mode, by employing AOC-20i Auto Injector (Shimadzu, Kyoto, Japan). GC total ion chromatograms (TIC) and fragmentation patterns of the metabolites identified using NIST/EPA/NIH Mass Spectral Library (NIST 14). Run time for each sample was 43.67 min. |
Ion Mode: | POSITIVE |
Gas Pressure: | 57.4 kPa |
Helium Flow: | 1 ml/min |
Ion Source Temperature: | 250 |
Ionization Energy: | 70 |
Source Temperature: | 250 |
Scanning Range: | 50-650 |