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MB Sample ID: SA002830
| Local Sample ID: | MR2_GSK_213 |
| Subject ID: | SU000074 |
| Subject Type: | Animal cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | FLT3-ITD AML (MR2) |
| Cell Biosource Or Supplier: | Dr. Tim Pardee (Wake Forest Univ.) |
| Cell Primary Immortalized: | Primary |
| Species Group: | Mammal |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000074 |
| Subject Type: | Animal cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | FLT3-ITD AML (MR2) |
| Cell Biosource Or Supplier: | Dr. Tim Pardee (Wake Forest Univ.) |
| Cell Primary Immortalized: | Primary |
| Species Group: | Mammal |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| MR2_GSK_213 | SA002830 | FL000738 | GSK inhibitor | Treatment |
| MR2_GSK_213 | SA002830 | FL000738 | 0 | Collection Time (h) |
Collection:
| Collection ID: | CO000057 |
| Collection Summary: | Conditioned Medias:Cellular Fraction_LC-MS:Cellular Fraction_WB |
| Collection Protocol Comments: | Processed simultaneously/time point:Processed simultaneously/time point:Processed simultaneously/time point |
| Sample Type: | conditioned treatment media |
| Collection Method: | Total contents pipette-transferred to tubes & centrifuged at 1800 rpm for 5 minutes. Medias transferred into aliquot tubes.:Cellular fractions pelleted & resuspended in 1X PBS. Aliqotted into collection vials based on cell counts. Re-centrifuged at 8,000 rpm for 3 min at 4 °C. PBS was aspirated off pellets for storage.:Cellular fractions pelleted & resuspended in 1X PBS. Aliqotted into collection vial based on cell counts. Re-centrifuged at 8,000 rpm for 3 min at 4 °C. PBS was aspirated off pellets for storage. |
| Collection Location: | RTI RCMRC Tissue culture suite:RTI RCMRC Wet lab:RTI RCMRC Wet lab |
| Collection Frequency: | 0h, 4h, 24h and 48h:0h, 4h, 24h and 48h:0h, 4h, 24h and 48h |
| Collection Duration: | 10 minutes:10 minutes:5 minutes |
| Collection Time: | 0h, 4h, 24h and 48h:0h, 4h, 24h and 48h:0h, 4h, 24h and 48h |
| Volumeoramount Collected: | 1 mL x 5 aliquots:2x10^7 cells:2x10^6 cells |
| Storage Conditions: | -80C:-80C:-80C |
| Collection Vials: | 15 mL conical tubes:15 mL conical tubes:15 mL conical tubes |
| Storage Vials: | 1.5 mL Eppendorf Protein Lo-Bind tubes:1.5 mL Eppendorf Protein Lo-Bind tubes:1.5 mL Eppendorf Protein Lo-Bind tubes |
| Collection Tube Temp: | chilled in ice:chilled in ice:chilled in ice |
Treatment:
| Treatment ID: | TR000075 |
| Treatment Summary: | MEK kinase inhibitors |
| Treatment Compound: | GSK | AZD |
| Treatment Route: | added to media |
| Treatment Dose: | 10nM | 10nM |
| Treatment Vehicle: | dilutions made in 1X sterile PBS (orginally dissolved in DMSO) |
| Cell Storage: | liquid nitrogen, live maintenance in 5% CO2, humidified incubator |
| Cell Growth Container: | T-75 culture flask |
| Cell Growth Config: | suspension |
| Cell Growth Rate: | double ~every 20 hours |
| Cell Media: | 50:50 DMEM:IMDM + 10% FBS, 1% P/S |
| Cell Harvesting: | Transfer 1 mL to new flask containing 10 mL of fresh culture media |
| Cell Pct Confluence: | maintained at ~ 2 x10^6/mL |
Sample Preparation:
| Sampleprep ID: | SP000070 |
| Sampleprep Summary: | Samples were extracted using 90:10 methanol:chloroform. For reversed phase, samples were dried down and reconstituted in 95:5 water:methanol. For HILIC, samples were directly transferred to autosampler vials. |
| Sampleprep Protocol Filename: | RTI_GRAVES_Metabolomics_Procedure.docx |
| Extraction Method: | 90:10 methanol:chloroform |
| Extract Storage: | -80 C |
| Sample Resuspension: | 95:5 Water:Methanol |
| Sample Spiking: | L-Tryptophan-d5 |
| Cell Type: | FLT3-ITD AML (myeloid) |
Combined analysis:
| Analysis ID | AN000093 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity |
| Column | Waters Acquity HSS T3 (100 x 2.1mm,1.8um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Waters Synapt G2 QTOF |
| Ion Mode | POSITIVE |
| Units | Normalized abundance |
Chromatography:
| Chromatography ID: | CH000061 |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity HSS T3 (100 x 2.1mm,1.8um) |
| Column Pressure: | 6000-10000 |
| Column Temperature: | 50 C |
| Flow Gradient: | Time(min) Flow Rate %A %B Curve ; 1. Initial 0.400 100.0 0.0 ; 2. 1.00 0.400 100.0 0.0 6 ; 3. 16.00 0.400 0.0 100.0 6 ; 4. 20.00 0.400 0.0 100.0 6 ; 5. 22.00 0.400 100.0 0.0 6 |
| Flow Rate: | 0.4 mL/min |
| Injection Temperature: | 8 C |
| Internal Standard: | L-Tryptophan-d5 |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% methanol; 0.1% formic acid |
| Analytical Time: | 22 min |
| Weak Wash Solvent Name: | 5%MeOH |
| Weak Wash Volume: | 1000 uL |
| Strong Wash Solvent Name: | 80%MeOH |
| Strong Wash Volume: | 1000 uL |
| Target Sample Temperature: | 8 C |
| Sample Loop Size: | 10 uL |
| Sample Syringe Size: | 100 uL |
| Randomization Order: | Yes |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS000070 |
| Analysis ID: | AN000093 |
| Instrument Name: | Waters Synapt G2 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 110 C |
| Capillary Voltage: | 3.2 kV |
| Collision Energy: | 4 |
| Helium Flow: | 180 |
| Ionization: | ES+ |
| Mass Accuracy: | 10 ppm |
| Source Temperature: | 110 C |
| Dataformat: | Continuum |
| Desolvation Gas Flow: | 400 L/Hr |
| Desolvation Temperature: | 400 C |
| Resolution Setting: | 18000 |
| Scan Range Moverz: | 50-1000 m/s |
| Scanning Cycle: | 1 s |
| Tube Lens Voltage: | 75 |
