Targeted Metabolomic Analysis of Immune Cells

Sample preparation, process and analysis was followed by previous report with modification (24 (link)). 5 x 10⁵ ABCs or FO B cells were sorted by cell sorter. Isolated cells were washed with ice-cold HBSS three times and snap-frozen until following steps. Cells were lysed by incubation with extraction solvents (50:50 v/v methanol:acetonitrile) for 1 hour at -20°C. Lysed cell extracts were centrifuged and upper layer (containing metabolites) were collected and further processed by LC-tandem mass spectrometry (MS/MS). Chromatrography was performed with a Shimadzu Nexera system (Shimadzu, Columbia, MD) coupled with a high-resolution hybrid quadrupole time-of-flight mass spectrometer (TripleTOF 5600, Framingham, MA).
Targeted metabolomic data were processed using Peak View 2.1 and MultiQuanta software version 3.0.2 (SCIEX). Chromatographic peaks of targeted metabolites were annotated, and each identified metabolite was quantified by integrating peak area using MultiQuant software. The quantitative analysis was based on the total peak areas of extracted ion chromatograms of feature ions. Principal component analysis plot and heat map were generated with MetaboAnalyst (v4.1).