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MB Sample ID: SA116256
Local Sample ID: | M_20m_3a |
Subject ID: | SU001489 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | C57BL/6J |
Age Or Age Range: | 4 months & 20 months |
Gender: | Male and female |
Animal Animal Supplier: | Jackson Laboratories |
Animal Housing: | SPF animal facility at USC |
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Sample Preparation:
Sampleprep ID: | SP001497 |
Sampleprep Summary: | Metabolites and lipids were extracted from neutrophil cell pellets and analyzed in a randomized order. Extraction was performed using a biphasic separation protocol with ice-cold methanol, methyl tert-butyl ether (MTBE) and water (Contrepois et al., 2018). Briefly, 300μL of methanol spiked-in with 54 deuterated internal standards provided with the Lipidyzer platform (SCIEX, cat #5040156, LPISTDKIT-101) was added to the cell pellet, samples were vigorously vortexed for 20 seconds and sonicated in a water bath 3 times for 30 seconds on ice. Lipids were solubilized by adding 1000μL of MTBE and incubated under agitation for 1h at 4°C. After addition of 250μL of ice-cold water, the samples were vortexed for 1 min and centrifuged at 14,000g for 5 min at 20°C. The upper phase containing the lipids was then collected and dried down under nitrogen. The dry lipid extracts were reconstituted with 300μL of 10 mM ammonium acetate in 9:1 methanol:toluene for analaysis. The lower phase containing metabolites was subjected to further protein precipitation by adding 4 times of ice-cold 1:1:1 isopropanol:acetonitrile:water spiked in with 17 labeled internal standards and incubating for 2 hours at -20°C. The supernatant was dried down to completion under nitrogen and re-suspended in 100μL of 1:1 MeOH:Water for analysis. |