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MB Sample ID: SA245036
| Local Sample ID: | 3_4 |
| 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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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 |
