Transfection of P. falciparum with pfhsp70-1-mCherry

pfhsp70-1-mCherry was subcloned into a P. falciparum expression vector, pfYC103 as previously reported (Wagner et al., 2013 (link)). The plasmid was purified using a Qiagen Maxi kit and resuspended in CytoMix (25 mM HEPES, pH 7.6, 2 mM EGTA (Sigma), 5 mM MgCl₂ (Fisher Scientific), 8.66 mM K₂HPO₄ (Fisher Scientific), 1.34 mM KH₂PO₄ (VWR International), 120 mM KCl (Fisher Scientific), 0.15 mM CaCl₂ (Sigma)) (Rug and Maier, 2013 (link); Crabb et al., 2004 (link)). For transfection of P. falciparum 3D7, 100 μL ring-stage parasites (14–18 hpi) at 5% parasitemia was resuspended in 300 μL CytoMix containing 75 μg plasmid DNA in a 0.2 cm electroporation cuvette (Bio-Rad). Electroporation was performed at 0.31 kV and 950 μF with maximal capacitance using a Gene Pulser II system (Bio-Rad). The parasites were then cultured in complete medium at 1% hematocrit at 37°C. Selection of transfectants with 200 nM pyrimethamine (Sigma) started at 3 days post-transfection. PfHsp70-1-mCherry expression in transfected parasites was verified by fluorescence microscopy.