Non-targeted metabolome profiling in splenic monocyte-derived dendritic cells from Plasmodium chabaudi-infecetd mice

Spleens from mice infected with Plasmodium chabaudi were processed and stained for localization of monocyte-derived dendritic cells (MODCs) by flow cytometry. The markers utilized were: Live/Dead, F4/80, CD11b, DCSign, MHCII, CD11c and CD3. After sorted out, MODCs were frozen in liquid nitrogen, followed by metabolite extraction, as recommended by The Metabolomics Facility at MD Anderson. Metabolites were extracted using ice-cold 0.1% Ammonium hydroxide in 80/20 (v/v) methanol/water. Extracts were centrifuged at 17,000 g for 5 min at 4°C, and supernatants were transferred to clean tubes, followed by evaporation to dryness under nitrogen. Dried extracts were reconstituted in deionized water, and 5 μL was injected for analysis by ion chromatography (IC)-MS. IC mobile phase A (MPA; weak) was water, and mobile phase B (MPB; strong) was water containing 100 mM KOH. A Thermo Scientific Dionex ICS-5000+ system included a Thermo IonPac AS11 column (4 µm particle size, 250 x 2 mm) with column compartment kept at 30°C. The autosampler tray was chilled to 4°C. The mobile phase flow rate was 350 µL/min and gradient from 1mM to 100mM KOH was used. The total run time was 60 min. To assist the desolvation for better sensitivity, methanol was delivered by an external pump and combined with the eluent via a low dead volume mixing tee. Data were acquired using a Thermo Orbitrap Fusion Tribrid Mass Spectrometer under ESI negative ionization mode at a resolution of 240,000.