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MB Sample ID: SA092007

Local Sample ID:MeOH-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 IDMB Sample IDFactor Level IDLevel ValueFactor Name
MeOH-H2OSA092007FL013197Methanol/WaterExctraction

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
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