Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA116257

Local Sample ID:	M_20m_4a
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.

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.