Hemozoin

Fresh tonsil mononuclear cells (MNC) were isolated as described above and re-suspended at ∼1×10⁶ cells/ml in pre-warmed PBSA. Two microliters of 5 mM CFSE (carboxyfluorescien diacetate, succinimidyl ester) in DMSO (Invitrogen, Life Technologies, Grand Island, USA) was added per ml of cells (final concentration 10 µM CFSE). The cells were then incubated for 10 minutes at 37°C. Cells were spun down and re-suspended in 2 volumes of ice-cold culture medium and incubated on ice for 5 minutes. Cells were re-pelleted and re-suspended in 2 volumes ice cold culture medium (2 times). Washing was done one more time in 2 volumes of pre-warmed medium and cells were finally re-suspended in fresh pre-warmed cell culture medium (described above). A final concentration of 3 µM of CpG-2006 (Hycult Biotech, Plymouth Meeting, USA), 0.25 µg/ml CD40 ligand (eBioscience, San Diego, USA), 5 ng/ml IL-4 (eBioscience, San Diego, USA), 2.5 or 5 µg/ml Anti-human IgG+IgM, (Jackson ImmunoResearch, West Grove, USA) 10 µg/ml of crude parasite extract, and hemozoin (InvivoGen, San Diego, USA) 50 µg/ml were used in the tonsil mononuclear cell stimulation. Cells were harvested on days 3, 5, 7 and 10, stained with CD19 and then B cells were sorted with the MoFlo.