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MB Sample ID: SA165043
| Local Sample ID: | 2h TGF-β_4 |
| Subject ID: | SU001856 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | Human Lung epithelial A549 cancer cells |
| Cell Passage Number: | 10 |
| Cell Counts: | 5 million cells |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001856 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | Human Lung epithelial A549 cancer cells |
| Cell Passage Number: | 10 |
| Cell Counts: | 5 million cells |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 2h TGF-β_4 | SA165043 | FL019756 | 2h TGF-β | Treatment |
Collection:
| Collection ID: | CO001849 |
| Collection Summary: | A549 lung cancer cells were treated with control, ATP, TGF-beta and then collected according to sample preparation protocol. |
| Collection Protocol Filename: | Collection protocol |
| Sample Type: | Lung |
| Collection Method: | Ice cold methanol (80%) using cell scrappers |
| Collection Location: | 150mm dish |
| Collection Frequency: | Once |
| Collection Duration: | 1-2 sec |
| Volumeoramount Collected: | 1 ml |
| Storage Conditions: | -80℃ |
| Collection Vials: | Polypropylene 1.5 ml tubes |
| Storage Vials: | Polypropylene 1.5 ml |
| Collection Tube Temp: | 4C |
| Tissue Cell Quantity Taken: | 5 million |
Treatment:
| Treatment ID: | TR001869 |
| Treatment Summary: | 5 million A549 cells were treated with control (no treatment), 0.5 mM ATP or 10 ng/ml TGF-beta for 2, 6 and 12 hours. |
| Treatment: | In vitro treatment with small molecules |
| Treatment Compound: | 0.5 mM ATP and 10 ng/ml TGF-beta |
| Treatment Dosevolume: | 10 ml DMEM media containing appropriate compounds |
| Cell Storage: | 37C; 5% CO2 incubator |
| Cell Growth Container: | 150 mm Dish tissue culture treated |
| Cell Media: | DMEM (10% FBS; 1% Pen/Strep) |
| Cell Harvesting: | after 2, 6 and 12 hours |
| Cell Pct Confluence: | 80% |
| Cell Media Lastchanged: | NA |
Sample Preparation:
| Sampleprep ID: | SP001862 |
| Sampleprep Summary: | 5 × 106 A549 cells were treated with control (no treatment), 0.5 mM ATP or 10 ng/ml TGF-beta for 2, 6 and 12 hours. After treatment, cells were washed twice with deionized water and polar metabolites were then extracted with cryogenically cold 80% methanol/water mixture. LC-MS grade water, methanol, and acetonitrile (Fischer Scientific, PA, USA) were used. Methanol-extracted samples were then sonicated in cycles of sonication phase and rest phase for 10 minutes (5 second sonication phase and 10 seconds halt). The samples were then centrifuged at 13,000 rpm for 10 minutes and supernatant was then collected. Supernatants collected from in vitro and in vivo extraction were then lyophilized. Briefly, the supernatant was then lyophilized by using a speed vacuum evaporator. The samples were then dissolved into a mixture of acetonitrile/water (1:1; v/v). |
| Sampleprep Protocol Filename: | Sample preparation protocol |
| Processing Method: | CEll scrapping Quenching |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Quenching with Ice cold methanol |
| Extract Enrichment: | Speed vaccum evaporator |
| Extract Storage: | -80℃ |
| Sample Resuspension: | Acetonitrile/water (1:1) |
Combined analysis:
| Analysis ID | AN002888 | AN002889 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Agilent 1290 | Agilent 1290 |
| Column | Poroshell 120 SB-C18 (100 x 2.1mm,2.7um) | Poroshell 120 SB-C18 (100 x 2.1mm,2.7um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Agilent 6545 QTOF | Agilent 6545 QTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | normalized | normalized |
Chromatography:
| Chromatography ID: | CH002141 |
| Chromatography Summary: | The entire LC/MS-MS experiment was performed in the Campus Chemical Instrumentation Center’s Mass Spectrometry and Proteomics facility at The Ohio State University. Lyophilized samples were dissolved in equal amounts of LC-MS grade water and acetonitrile and run with LC/MS-MS analysis, using an untargeted metabolomics approach by utilizing Agilent Q-TOF 6545 mass spectrometer connected to an Agilent 1290 UHPLC system with a Poroshell 120 SB-C18 (2 x 100 mm, 2.7 µm particle size) column. The LC gradient consisted of solvent A, H2O with 0.1 % Formic acid, and solvent B, 100 % acetonitrile at a 200 µL/min flow rate with an initial 2 % solvent B with a linear ramp to 95 % B at 15 min, holding at 95% B for 1 minutes, and back to 2 % B from 16 min and equilibration of 2 % B until min 32. A 5 µL volume sample was injected for each run and the top 5 ions were selected for data-dependent analysis with a 15 second exclusion window. |
| Instrument Name: | Agilent 1290 |
| Column Name: | Poroshell 120 SB-C18 (100 x 2.1mm,2.7um) |
| Column Temperature: | 40 |
| Flow Gradient: | an initial 2 % solvent B with a linear ramp to 95 % B at 15 min, holding at 95% B for 1 minutes, and back to 2 % B from 16 min and equilibration of 2 % B until min 32. |
| Flow Rate: | 200 µL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002681 |
| Analysis ID: | AN002888 |
| Instrument Name: | Agilent 6545 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | Masshunter software was used to collect the raw data. Progenesis was used for peak intergration. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002682 |
| Analysis ID: | AN002889 |
| Instrument Name: | Agilent 6545 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | Masshunter software was used to collect the raw data. Progenesis was used for peak intergration. |
| Ion Mode: | NEGATIVE |
