Return to study ST002489 main page
MB Sample ID: SA248866
| Local Sample ID: | AG-23-TC-14 |
| Subject ID: | SU002579 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002579 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| AG-23-TC-14 | SA248866 | FL031391 | aCD25 | factor |
Collection:
| Collection ID: | CO002572 |
| Collection Summary: | C57BL/6 mice were orthotopically implanted with PK5L1940 cells. 8 Gy RT was administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per week beginning day 7 post-implantation. Spleens and lymph nodes of C57BL/6 mice were harvested 17 days post implantation and homogenized to a single cell suspension by mashing through a 70 uM cells strainer. For spleen processing, and the erythrocytes were lysed with ACK (ammonium-chloride-potassium) lysis buffer for 5 min at RT. CD8 T cells were sorted with a CD8-negative selection Stemcell isolation kit following manufacturer instructions. |
| Sample Type: | T-cells |
Treatment:
| Treatment ID: | TR002591 |
| Treatment Summary: | CD8+ T cells were cultured for 6h in the presence of 25 mM U-13C-glucose in glucose-free RPMI. Cells were washed in PBS at 1200RPM, 4degC for 10 minutes and pellets were flash frozen. |
Sample Preparation:
| Sampleprep ID: | SP002585 |
| Sampleprep Summary: | Metabolites from frozen T-cell pellets were extracted with cold 5:3:2 MeOH:acetonitrile:water at a concentration of 4e6 cells/ml. Samples were vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10 min, 10,000 g, 4 degrees C) and transferred to autosampler vials. |
| Processing Storage Conditions: | 4℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN004063 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 2.1 x 150 mm, 1.7 um |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH003009 |
| Chromatography Summary: | Negative C18 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 2.1 x 150 mm, 1.7 um |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B |
| Flow Rate: | 450 uL/min |
| Sample Injection: | 10 uL |
| Solvent A: | 95% water 5% acetonitrile 1 mM ammonium acetate |
| Solvent B: | 95% acetonitrile 5% water 1 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003810 |
| Analysis ID: | AN004063 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database. |
| Ion Mode: | NEGATIVE |
