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MB Sample ID: SA245067
| Local Sample ID: | 47_3_15 |
| Subject ID: | SU002540 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | APOE-targeted replacement mice homozygous for human E3 (B6.129P2-Apoe^tm2(APOE*3)Mae N8, Taconic #1548-F) or human E4 (B6.129P2- Apoe^tm3(APOE*4)Mae N8, Taconic #1549-F) alleles |
| Age Or Age Range: | P0-P3 |
| Gender: | Pooled |
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Subject:
| Subject ID: | SU002540 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | APOE-targeted replacement mice homozygous for human E3 (B6.129P2-Apoe^tm2(APOE*3)Mae N8, Taconic #1548-F) or human E4 (B6.129P2- Apoe^tm3(APOE*4)Mae N8, Taconic #1549-F) alleles |
| Age Or Age Range: | P0-P3 |
| Gender: | Pooled |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 47_3_15 | SA245067 | FL030707 | E3_proinflammatory | Group |
| 47_3_15 | SA245067 | FL030707 | E3/E3 | APOE genotype |
| 47_3_15 | SA245067 | FL030707 | Proinflammatory | Treatment |
Collection:
| Collection ID: | CO002533 |
| Collection Summary: | Primary microglia were plated at 7x10^6 cells/well in 6-well plates (VWR #10062-894) and incubated at 5% CO2 37°C. Upon reaching confluence, cells were washed with warm, sterile phosphate-buffered saline (PBS; Thomas #QZY-11666789001-4L) to remove traces of non-13C media and then incubated in glucose- and sodium pyruvate-free DMEM (Thermo #11966-025) containing 2mM GlutaMAX (Thermo #35050-061), 1% penicillin/streptomycin, and 10mM universally labelled 13C-glucose (Cambridge Isotope Laboratories # CLM-1396-PK) for two hours with either a pro-inflammatory cocktail of 20ng/ml interferon-γ (IFNγ, R&D Systems #485-MI-100) and 50ng/ml tumor necrosis factor α (TNFα, R&D Systems #410-MT-025) or vehicle control. Cells were then removed from the incubator and washed with warm 0.9% NaCl solution. Culture plates were placed on a bed of crushed dry ice and 1mL of ice cold 50% methanol (HPLC-grade, Sigma #A456-4) was added to quench cellular metabolic activity followed by a 10 minute incubation at -80°C to ensure cell lysis. After removing from the freezer, cells were detached with a cell scraper (VWR #10062-906) and the entire contents collected into a microcentrifuge tube, vortexed briefly, and placed on ice until all samples were collected. The tubes were then placed on a Disruptor Genie Cell Disruptor Homogenizer (Scientific Industries) for 5 min at 3,000 rpm. Tubes were then centrifuged at 20,000 x g for 10 min at 4 °C. The supernatant containing polar metabolites was isolated to a new tube and stored at -80C until use. |
| Sample Type: | Brain |
Treatment:
| Treatment ID: | TR002552 |
| Treatment Summary: | APOE3/3 and APOE4/4 primary microglia were treated for 2 hours with a pro-inflammatory cocktail of 20ng/ml interferon-γ (IFNγ, R&D Systems #485-MI-100) and 50ng/ml tumor necrosis factor α (TNFα, R&D Systems #410-MT-025) or vehicle control. |
Sample Preparation:
| Sampleprep ID: | SP002546 |
| Sampleprep Summary: | The supernatant fraction containing polar metabolites was thawed gently on ice and dried at 10-3 mbar using a CentriVap vacuum concentrator (LabConco) to evaporate methanol. The dried polar metabolite pellet was derivatized by a two-step methoxyamine protocol first by addition of 70µL methoxyamine HCl (Sigma-Aldrich #226904-5G) in pyridine (20 mg/mL; Sigma-Aldrich #TS25730) to each pellet followed by 90 min dry heat incubation at 30°C. Samples were then centrifuged at 20,000 x g for 10 minutes after which 50µL of each sample was transferred to an amber V-shaped glass chromatography vial (Agilent #5184-3554) containing 80µL N-methyl-trimethylsilyl-trifluoroacetamide (MSTFA; ThermoFisher #TS48915) and gently vortexed followed by 30 min dry heat incubation at 37°C. |
| Extraction Method: | 50% ice-cold methanol |
| Extract Storage: | -80℃ |
| Sample Derivatization: | MSTFA |
Combined analysis:
| Analysis ID | AN003999 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 8890 |
| Column | Agilent HP5-MS (30m x 0.25mm, 0.25 um) |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Agilent 5977B |
| Ion Mode | POSITIVE |
| Units | Fractional Enrichment (adjusted for natural abundance) |
Chromatography:
| Chromatography ID: | CH002953 |
| Instrument Name: | Agilent 8890 |
| Column Name: | Agilent HP5-MS (30m x 0.25mm, 0.25 um) |
| Column Temperature: | 60°C, rising at 10°C/min to 325°C, and held for 10 min |
| Flow Gradient: | N/A |
| Flow Rate: | 0.581 mL/min |
| Solvent A: | N/A |
| Solvent B: | N/A |
| Chromatography Type: | GC |
MS:
| MS ID: | MS003747 |
| Analysis ID: | AN003999 |
| Instrument Name: | Agilent 5977B |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | Metabolites were identified and quantified using DExSI v1.11 (https://github.com/DExSI/DExSI) - Dagley, M. J. and McConville, M. J. (2018) "DExSI: A new tool for the rapid quantitation of 13C-labelled metabolites detected by GC-MS", Bioinformatics, bty025, https://doi.org/10.1093/bioinformatics/bty025. |
| Ion Mode: | POSITIVE |
