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MB Sample ID: SA245021
| Local Sample ID: | 3_3 |
| Subject ID: | SU002539 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | Primary microglia isolated from 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 |
| Species Group: | Mammals |
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Subject:
| Subject ID: | SU002539 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | Primary microglia isolated from 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 |
| Species Group: | Mammals |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 3_3 | SA245021 | FL030704 | E3 | Genotype |
Collection:
| Collection ID: | CO002532 |
| Collection Summary: | Primary microglia were plated at 7x106 cells/well in 6-well plates (VWR #10062-894) and incubated at 5% CO2 37°C. Upon reaching confluence, cells were removed from the incubator 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, and the resulting pellet was briefly dried at 10-3 mbar using a CentriVap vacuum concentrator (LabConco) to evaporate remaining methanol, followed by determination of protein content via BCA assay (ThermoFisher #23225) to normalize metabolite concentrations to total protein amount of each sample. |
| Sample Type: | Cultured cells |
| Collection Method: | Ice-cold methanol was added to primary microglia cultures in 6-well plates after which cells were harvested using a cell scraper |
| Collection Location: | University of Kentucky |
| Volumeoramount Collected: | 1ml |
| Storage Conditions: | -80℃ |
| Collection Tube Temp: | -80 |
Treatment:
| Treatment ID: | TR002551 |
| Treatment Summary: | Cells were not treated, as we were just comparing APOE3/3 vs. APOE4/4 genotypes |
| Cell Growth Container: | 6-well plate |
| Cell Media: | DMEM/F12 with 10% FBS, 10% LCCM supplement (see full methods in associated publication for preparation details), and 1% Penicillin/Streptomycin |
| Cell Envir Cond: | 37C, 5% CO2 |
| Cell Harvesting: | Cell scraper |
| Cell Pct Confluence: | 100 |
| Cell Media Lastchanged: | 2-4 days prior to harvest |
Sample Preparation:
| Sampleprep ID: | SP002545 |
| 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% methanol |
| Extract Storage: | -80℃ |
| Sample Derivatization: | MSTFA |
Combined analysis:
| Analysis ID | AN003998 |
|---|---|
| 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 5977 |
| Ion Mode | POSITIVE |
| Units | Relative Abundance |
Chromatography:
| Chromatography ID: | CH002952 |
| Instrument Name: | Agilent 8890 |
| Column Name: | Agilent HP5-MS (30m x 0.25mm, 0.25 um) |
| Chromatography Type: | GC |
MS:
| MS ID: | MS003746 |
| Analysis ID: | AN003998 |
| Instrument Name: | Agilent 5977 |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | Metabolites were identified and quantified using DExSI v1.11 (https://github.com/DExSI/DExSI) |
| Ion Mode: | POSITIVE |
