Microscopy

Imaging of live cells was performed by staining infected erythrocytes with 20 nM MitoTracker® (Thermo Fisher Scientific) which stains the mitochondrion and 0.5 μg/mL of DAPI to visualise the nucleus. Briefly, 500 μL of parasite culture was pelleted, resuspended in MitoTracker®, and washed twice with PBS (1x), before final staining with DAPI. For visualisation of ACP-DsRed/CS-eYFP 3D7 P. falciparum, nuclear staining was carried out prior to fluorescence microscopy.

Immunofluorescence assay (IFA) in solution was carried out to analyse protein subcellular localisation in intraerythrocytic parasites. Infected erythrocytes (8–10% parasitemia) were fixed in PBS (1x) containing 4% (v/v) paraformaldehyde and 0.0075% (v/v) glutaraldehyde for 30 min, permeabilised with 0.1% (v/v) Triton X-100 for 10 min, and blocked with 3% (w/v) BSA for 30 min. Cells were pelleted and incubated for one hr in blocking solution with mouse anti-GFP and rabbit anti-ACP as primary antibodies. Subsequent incubation was carried out in Alexa Fluor® 488 and 594-conjugated anti-mouse and anti-rabbit as secondary antibodies. Cells were washed with 500 mg/mL of DAPI before final resuspension in DABCO and PBS (1x).

Fluorescence microscopy was performed using the Zeiss Axioplan 2 imaging and Leica SP5 Confocal imaging platforms. Images were processed using ImageJ⁵³.