Isolation of Plasmodium berghei Extracellular Vesicles

C57BL/6 mice with 8 weeks of age were intraperitoneally injected with 10⁶Plasmodium berghei ANKA-GFP (PbA)-iRBCs. Parasitemia was determined as the percentage of GFP⁺ RBCs, using flow cytometry analysis (FACScan Cell Analyzer; Becton Dickinson). At day 5–6 post-infection when parasitemia was 10–20%, blood was collected (between 13:00h and 15:00h) by cardiac puncture with a syringe containing 100 μl of heparin. The collected blood was suspended in 5 ml of RPMI1640 medium and centrifuged at 500g for 10 min. iRBCs were then synchronized in RPMI1640 medium supplemented with FBS, to a final concentration of 20%, and neomycin (100 μg/ml) in 250-ml culture flasks flushed with a gas mixture of 5% CO2, 5% O2, and 90% N2 using a 0.2-μm filter unit connected to the gas hose as previously described (Janse et al. 2006 (link)). After 16 h, RBCs and iRBCs were collected by centrifugation and the supernatant used to collect EPs. The pellet was resuspended in PBS containing 2% BSA and mature-stage iRBC were selected and concentrated using automatic magnetically activated cell sorting (MACS) as previously described (de Moraes et al. 2016 (link)). Supernatant from RBCs and iRBCs was further centrifuged at 1600g for 20 min to remove lysed cells. The resultant supernatant was further centrifuged at 20,000g for 20 min to obtain a pellet with EPs. Medium and PBS solutions used to prepare EPs were previously filtered through a 0.2-μm filter.