Fluorescence Microscopy of Malaria Parasites

The transgenic P. berghei parasite line pv5-tag-GFP^PV (27 (link)) was imaged live using an AxioImager Z2 epifluorescence microscope equipped with an AxioCam MR3 camera (Zeiss). For mechanical parasite expansion, 1 to 2 µL of infected blood was incubated under a 22 × 40-mm coverslip for several minutes until erythrocyte lysis became apparent. P. falciparum parasites were imaged on an Eclipse Ni light microscope (Nikon) fitted with a C11440 digital camera (Hamamatsu). Immunofluorescence analysis was performed with P. falciparum pv5-3xHA:loxP parasites that were fixed in 4% formaldehyde using rat anti-HA (1:500; Sigma Aldrich) and rabbit anti-SERA5 (1:500) (55 (link)) primary antibodies in combination with appropriate fluorophore-coupled secondary antibodies (1:1,000; Thermo Fisher Scientific). Staining with Lysosensor blue DND-167 (Thermo Fisher Scientific), BODIPY 581/591 C11 (Image-iT Lipid Peroxidation Kit, Thermo Fisher Scientific), and CellROX Green (Thermo Fisher Scientific) was performed according to the manufacturer’s instructions.