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MB Sample ID: SA239062
| Local Sample ID: | SCM4_Medium-1 |
| Subject ID: | SU002488 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Subject Comments: | OP9 cells were left as stromal cells or were differentiated into adipocytes. These cells were allowed to condition media, with the media being the analyte in this study. |
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Subject:
| Subject ID: | SU002488 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Subject Comments: | OP9 cells were left as stromal cells or were differentiated into adipocytes. These cells were allowed to condition media, with the media being the analyte in this study. |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| SCM4_Medium-1 | SA239062 | FL029795 | SCM | Media |
Collection:
| Collection ID: | CO002481 |
| Collection Summary: | OP-9 bone marrow stromal cells were maintained in α-MEM (cat#15-012-CV, Corning, Glendale, AZ) supplemented with 20% FBS. For adipocyte differentiation, 105 OP-9 cells were plated in 6-well plates in DMEM (cat#10-017-CV, Corning, Glendale, AZ) supplemented with 10% FBS as previously described [18, 26]. After 24 hours of culture, the media was removed and switched to differentiation media which is composed of α-MEM supplemented with 1.8mM oleate (cat#O7501, Sigma, St. Louis, MO) bound to BSA (cat#A6003, Sigma, St. Louis, MO) with molar ratio 5.5:1 along with 175nM insulin (cat#I6634, Sigma, St. Louis, MO) and 0.2% FBS. ACM was collected after 3 days of differentiation and used in the experiments describe in this study. For SCM, OP-9 cells were plated in DMEM supplemented with 10% FBS and conditioned media were collected on day 3 of culture. |
| Sample Type: | Cultured cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002500 |
| Treatment Summary: | Cells and media were not treated with anything |
Sample Preparation:
| Sampleprep ID: | SP002494 |
| Sampleprep Summary: | Media samples (50 µL) were mixed with acetonitrile (Sigma) (100 µL) and vortexed. All samples were incubated 30 min on ice and centrifuged at 20,627 g for 10 min at 4 ˚C. The supernatant was transferred to autosampler vials (ThermoFisher) held at 4 ˚C for LC-MS acquisition. |
| Processing Storage Conditions: | On ice |
Combined analysis:
| Analysis ID | AN003906 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH002892 |
| Chromatography Summary: | A VanquishTM Horizon Binary ultrahigh performance liquid chromatography system coupled to a Q Exactive High Field Hybrid Orbitrap mass spectrometer (ThermoFisher) was used for data collection. A SeQuant® ZIC-HILIC column (Millipore Sigma, 3.5 µm, 100 Å, 150 x 2.1 mm, PEEK) was pumped with a mixture of mobile phases of 0.1% formic acid in water (mobile phase A) or 0.1% formic acid in acetonitrile (mobile phase B) (all reagents from Millipore Sigma). The column was equilibrated with 90% B for 8.3 min at 0.25 mL/min, after which 2.5 mL of extracted sample was injected. After this, a gradient ran from 90% to 20% B over 17.5, then was held at 20% B at 0.35 mL/min for 7.5 min. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) |
| Flow Gradient: | The column was equilibrated with 90% B for 8.3 min at 0.25 mL/min, after which 2.5 mL of extracted sample was injected. After this, a gradient ran from 90% to 20% B over 17.5 min, then was held at 20% B at 0.35 mL/min for 7.5 min |
| Flow Rate: | 0.35 mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003645 |
| Analysis ID: | AN003906 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Compound Discoverer 3.3 (Thermo Scientific) was used for data analysis of metabolomic experiments, including retention time alignment across runs, cubic spline normalization of areas for each metabolite in quality control pooled samples, and gap-filling based on real peak detection. The minimum peak intensity was 5x105, and metabolites were excluded if they had >50% relative standard deviation after correction or a max sample area <5-fold that of the blank. Furthermore, to be considered a high-confidence metabolite for analysis, metabolites required a) an accurate mass (5 ppm) match and either (i) retention time (±5%) matching of reference standards, and/or (ii) matching to MS2 spectra in mzCloud (mzcloud.org) (including at least one matching product ion); b) an accurate mass and retention time match to a lipid identity determined by LipidSearch (ThermoFisher, all default parameters used) based on MS2 data; and/or c) an MS2 fragment ion matching to a particular class of compounds (184.1 for phosphatidylcholine). When a chromatographic peak matched to two standards of similar retention time that were indistinguishable by MS2, we prioritized the metabolite with higher abundance in human or retained both annotations. The naming convention for the metabolites reported herein is as follows – a) if the metabolite was an accurate mass and retention time match to our curated library or to our LipidSearch library, it is listed only as the metabolite name; b) if the metabolite had an MS2 library match but no match in the aforementioned libraries, it is listed as the MS2 match and the retention time; c) if the metabolite had an MS2 compound class match, it is listed as the compound class, the chemical formula (or the molecular weight if formula was not available), and the retention time. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 275 |
| Ionization Potential: | +3.5 kV |
| Automatic Gain Control: | 1E6 |
