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MB Sample ID: SA254713
| Local Sample ID: | 20221116_QE2_JAS_Job2640_67 |
| Subject ID: | SU002631 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002631 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 20221116_QE2_JAS_Job2640_67 | SA254713 | FL032635 | RPMI | Media |
| 20221116_QE2_JAS_Job2640_67 | SA254713 | FL032635 | Day_1 | Collection_Time |
Collection:
| Collection ID: | CO002624 |
| Collection Summary: | 100,000-150,000 cells were seeded in 2mL of culture medium in six-well plates with 3 technical replicates per condition per time point and allowed to attach overnight. The following day (day 1), cells were washed twice with 2mL PBS. They were then given 2mL of media, either TIFM or RPMI. An unspent media sample was collected at this time as well and stored at -80 °C. Cell number on day 1 was measured using a Vi-CELL XR Cell Viability Analyzer (Beckman Coulter). 24h later (day 2), 1mL of spent media from cells was collected, centrifuged and stored at -80 °C. Cell number was counted again. Quantification of metabolite levels in unspent (day 1) and day 2 (conditioned media) cell culture media samples was performed. |
| Sample Type: | Cell culture media |
Treatment:
| Treatment ID: | TR002643 |
| Treatment Summary: | NA |
Sample Preparation:
| Sampleprep ID: | SP002637 |
| Sampleprep Summary: | We extracted metabolites from 5µL of cell culture media samples using 45µL of a 75:25:0.1 HPLC grade acetonitrile:methanol:formic acid extraction mix with labelled stable isotope internal standards. Samples in extraction mix were vortexed for 10 min at 4°C and centrifugated at 15,000x rpm for 10 min at 4°C to pellet insoluble material. 20µL of the soluble polar metabolite supernatant was moved to sample vials for analysis by LC-MS. |
Combined analysis:
| Analysis ID | AN004165 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | Peak area (m/z) |
Chromatography:
| Chromatography ID: | CH003084 |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 25 |
| Flow Gradient: | 0–20 min: linear gradient from 80% to 20% B; 20–20.5 min: linear gradient from 20% to 80% B; 20.5–28 min: hold at 80% B |
| Flow Rate: | 0.150 mL/min |
| Solvent A: | 20 mM ammonium carbonate, 0.1% ammonium hydroxide |
| Solvent B: | 100% Acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003912 |
| Analysis ID: | AN004165 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | XCalibur 965 2.2 software (Thermo 966 Fisher Scientific) was used identification and relative quantification for metabolites. |
| Ion Mode: | UNSPECIFIED |
