Return to study ST002365 main page
MB Sample ID: SA237055
Local Sample ID: | Got1_KO_01 |
Subject ID: | SU002454 |
Subject Type: | Cultured cells |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57BL/6J |
Age Or Age Range: | 6-8 weeks |
Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002454 |
Subject Type: | Cultured cells |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57BL/6J |
Age Or Age Range: | 6-8 weeks |
Gender: | Male and female |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Got1_KO_01 | SA237055 | FL029357 | GOT1_KO | Treatment |
Collection:
Collection ID: | CO002447 |
Collection Summary: | Cells were spun down and washed once with pre-warmed PBS and metabolites were immediately extracted or stored at -80℃ until further extraction. |
Sample Type: | Cultured cells |
Treatment:
Treatment ID: | TR002466 |
Treatment Summary: | CD8+ T cells were isolated from spleens and lymph nodes from WT, T cell conditional GOT1 or GLUD1 knockout mice. Cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours. CD8+ T cells were counted and resuspended in full media containing 4 mM [U-13C]glutamine at 2 E6 mL-1. Normal FBS was substituted with dialyzed FBS. Cells were collected for LC-MS analysis 4-6 hrs post incubation. |
Sample Preparation:
Sampleprep ID: | SP002460 |
Sampleprep Summary: | Cells were spun down and washed once with pre-warmed PBS and metabolites were immediately extracted by adding methanol:water (80:20, v/v) extraction solution, sonicated and stored at -80 °C for at least 2 hrs to precipitate the proteins. Supernatant after centrifugation at 14,000xg for 10 minutes was dried under nitrogen gas. Metabolites were then reconstituted using ACN:water (50:50, v/v) overnight at 4 °C. Soluble metabolites after centrifugation at 14,000xg for 10 minutes were subjected to analysis by liquid chromatography mass spectrometry (LC-MS). |
Combined analysis:
Analysis ID | AN003860 |
---|---|
Analysis type | MS |
Chromatography type | Ion pair |
Chromatography system | Agilent 1290 Infinity |
Column | Agilent Zorbax Extend C18 (150 x 2.1mm,1.8 um) |
MS Type | ESI |
MS instrument type | QTOF |
MS instrument name | Agilent 6520 QTOF |
Ion Mode | NEGATIVE |
Units | AUC |
Chromatography:
Chromatography ID: | CH002858 |
Instrument Name: | Agilent 1290 Infinity |
Column Name: | Agilent Zorbax Extend C18 (150 x 2.1mm,1.8 um) |
Chromatography Type: | Ion pair |
MS:
MS ID: | MS003601 |
Analysis ID: | AN003860 |
Instrument Name: | Agilent 6520 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | The optimized ESI Q-TOF parameters for MS experiments were: ion polarity, negative; gas temperature, 325 °C; drying gas, 10 L min-1; nebulizer pressure, 45 psig; capillary voltage, 4,000 V; fragmentor, 140 V; skimmer, 65 V; mass range, 50-1100 m/z; acquisition rate, 1.5 spectra s-1; instrument state, extended dynamic range (1700 m/z, 2 GHz). Spectra were internally mass calibrated in real time by continuous infusion of a reference mass solution using an isocratic pump connected to a dual sprayer feeding into an electrospray ionization source. Data were acquired with MassHunter Acquisition software. A metabolite database with retention times based on the ion-pairing method was developed using Agilent MassHunter PCDL manager software. The isotopologue peak extractions were achieved by Agilent MassHunter Profinder software. |
Ion Mode: | NEGATIVE |