Return to study ST003265 main page
MB Sample ID: SA354279
| Local Sample ID: | W3 |
| Subject ID: | SU003385 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003385 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| W3 | SA354279 | FL041342 | wild-type | Genotype |
| W3 | SA354279 | FL041342 | CD8 T cell | Sample source |
Collection:
| Collection ID: | CO003378 |
| Collection Summary: | Mouse CD8+ T cells were isolated from the spleen and lymph nodes of ME2 wild-type and ME2 knockout mice (knockdown of ME2 in T cells). The mice were euthanized and soaked in 75% alcohol for 3 minutes. Separate mouse spleens using a dissector and grind them into single-cell suspensions using a grinder. Single-cell suspension was used for CD8+ T cell isolation using the Mouse Lymphocyte Isolation Kit (Biolegend, 480044, China). T cells were cultured in RMPI-1640 medium supplemented with 10% FBS, 10μg/ml β-mercaptoethanol, and 1% penicillin and streptomycin. For metabolite analysis, CD8+ T cells were stimulated with 4 μg/ml anti-CD3 and 1μg/ml anti-CD28 antibodies for 24 h. |
| Sample Type: | T-cells |
| Storage Conditions: | On ice |
Treatment:
| Treatment ID: | TR003394 |
| Treatment Summary: | Naive ME2+/+ and ME2-/- CD8+ T cells were isolated and activated with anti-CD3 (4μg/ml) and anti-CD28 (1μg/ml). |
Sample Preparation:
| Sampleprep ID: | SP003392 |
| Sampleprep Summary: | For metabolite analysis, naive ME2+/+ and ME2-/- CD8+ T cells were isolated and activated with anti-CD3 (4μg/ml) and anti-CD28 (1μg/ml). Cells were washed twice with ice-cold PBS and metabolites were extracted with ice-cold 80% methanol. The extracts were analyzed by LC-MS/MS. |
| Extraction Method: | Cells were washed twice with ice-cold PBS and metabolites were extracted with ice-cold 80% methanol. |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN005349 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Focus |
| Ion Mode | UNSPECIFIED |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH004051 |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| Column Temperature: | 35 |
| Flow Gradient: | 0-2 min, 100% phase A; 2-11 min, 0% to 100% B; 11-13 min, 100% B; 13-15 min, 0% to 100% A. |
| Flow Rate: | 0.4 ml/min |
| Solvent A: | 100% water; 0.1 % formic acid |
| Solvent B: | 100% methanol; 0.1 % formic acid |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS005079 |
| Analysis ID: | AN005349 |
| Instrument Name: | Thermo Q Exactive Focus |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The Q Exactive mass spectrometer was operated in full MS and polarity switching mode (acquisition from m/z 50 to 500) with a scan resolution set at 35,000. Five independent cell cultures were measured for each condition and samples were randomised in order to avoid bias in sample analyses due to machine drift. Q Exactive LC-MS/MS vendor raw data files were converted to the open source format mzML using the program ProteoWizard, and then the XCMS program was used for peak alignment, retention time correction and peak area extraction. Significance was determined using an unpaired Student's t-test. p-value < 0.05 was considered as statistically significant. The acquired spectra were analysed using XCalibur Qual Browser and XCalibur Quan Browser software (Thermo Scientific) by referencing to an internal library of compounds. |
| Ion Mode: | UNSPECIFIED |
