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MB Sample ID: SA314487
| Local Sample ID: | Ala-40-2-1 |
| Subject ID: | SU002990 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002990 |
| 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 |
|---|---|---|---|---|
| Ala-40-2-1 | SA314487 | FL037450 | Ala | factor |
Collection:
| Collection ID: | CO002983 |
| Collection Summary: | Cells were counted, washed with cold PBS and then flash-frozen in liquid N2 |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR002999 |
| Treatment Summary: | To trace L-Alanine metabolism, RAW264.7 cell were grown in DMEM (Hyclone) supplemented with 10% (v/v) cosmic calf (Hyclone), then transferred into L-Alanine-free medium and deprived of serum overnight. Subsequently, cells were incubated with 5 mM L-Alanine and 5mM [U-13C]-L-Alanine in serum-starved medium (DMEM/0.5% serum). Additionally, fresh media containing L-Alanine and [U-13C]-L-Alanine were exchanged 2 h before metabolite extraction for metabolic analysis. |
Sample Preparation:
| Sampleprep ID: | SP002996 |
| Sampleprep Summary: | Cells were homogenized with the first solvent (the mixture of chloroform, methanol and water (1:2:1, v/v/v)) for 30 s at 4 °C and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected and deposit was re-homogenized with the second solvent (methanol alone) before a second centrifugation. The 2 supernatants were mixed, and aliquot of sample was transferred to a GC sampling vial containing 5 μL 0.1 mg/mL ribitol (Sigma) as an analytical internal standard and then dried in a vacuum centrifuge concentrator before the subsequent derivatization. A total of 2 technical replicates were prepared for each sample. |
Combined analysis:
| Analysis ID | AN004714 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Thermo Scientific Trace GC Ultra with DSQ II GC/MS |
| Column | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| MS Type | EI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Thermo Scientific Trace GC Ultra with DSQ II GC/MS |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH003550 |
| Instrument Name: | Thermo Scientific Trace GC Ultra with DSQ II GC/MS |
| Column Name: | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| Column Temperature: | 270 °C |
| Flow Gradient: | none |
| Flow Rate: | 1.0 mL/min |
| Solvent A: | none |
| Solvent B: | none |
| Chromatography Type: | GC |
MS:
| MS ID: | MS004460 |
| Analysis ID: | AN004714 |
| Instrument Name: | Thermo Scientific Trace GC Ultra with DSQ II GC/MS |
| Instrument Type: | Triple quadrupole |
| MS Type: | EI |
| MS Comments: | samples was derivatized and then used to firstly protect carbonyl moieties through methoximation, through a 90 min 37 ℃ reaction with 40 μL of 20 mg/mL methoxyamine hydrochloride (Sigma-Aldrich) in pyridine, followed by derivatization of acidic protons through a 30 min 37 0C reaction with the addition of 80 μL N-methyl-N-trimethylsilyltrifluoroace-tamide (MSTFA, Sigma-Aldrich). The derivatized sample of 1 μL was injected into a 30m × 250 μm i.d. × 0.25 μm DBS-MS column using splitless injection and analysis was carried out by Trace DSQ II (Thermo Scientific). The initial temperature of the GC oven was held at 85 0C for 5 min followed by an increase to 330 0C at a rate of 15 0C min-1 then held for 5 min. Helium was used as carrier gas and flow was kept constant at 1 mL min-1. The MS was operated in a range of 50-600 m/z. |
| Ion Mode: | POSITIVE |
