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MB Sample ID: SA002841
Local Sample ID: | MR2_CTL_222 |
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_CTL_222 | SA002841 | FL000744 | Media alone | Treatment |
MR2_CTL_222 | SA002841 | FL000744 | 4 | 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 |