Parasite growth assays

Growth over one 48h growth cycle was measured by Malaria SYBR Green I-based fluorescence (MSF) assay, essentially as previously described [60]. Trophozoite-stage cultures of all parasite lines being tested were seeded in triplicate into 96-well plates at 1% parasitaemia, 4% haematocrit. The outer wells of each plate were filled with medium to prevent evaporation. Plates were incubated for 48h in a gassed chamber at 37°C. Following this, 100 μL of sample from each well was transferred to plate wells containing 100 μL MSF lysis buffer (20 mM Tris pH 7.5, 5 mM EDTA, 0.008% saponin, 0.8% Triton X-100) supplemented with 0.2 μL mL^-1 of SYBR Green I (Sigma). After a 1h incubation in the dark at room temperature, SYBR Green I fluorescence was measured using the blue fluorescent module (excitation 490 nm: emission 510–570 nm) of a GloMax multidetection system (Promega). Percentage parasite growth was calculated as follows: 100x[μ_(s)—μ_(-)/μ₍₊₎—μ_(-)] where μ_(s), μ_(-) and μ₍₊₎ are the means of the fluorescent readouts from sample wells (μ_(s)), control wells with 100μM chloroquine (μ_(-), representing 0% growth), and control wells with wildtype 3D7 parasites (μ₍₊₎, 100% growth).

Growth over two 48h cycles was measured by seeding parasites at 0.1% parasitaemia and then counting the parasitaemia via blood smear microscopy (with a minimum of 100 parasites) at 48h intervals. All growth assays were conducted in triplicate.

Parasite DNA content was measured by two methods: merozoite-counting by microscopy, and flow cytometry. Flow cytometry was carried out on a Guava easyCyte system, using parasites isolated by Percoll and then held for 4h in the egress inhibitor E64 (10μM) before staining with SYBR Green 1, fixing with formaldehyde, and quantifying the fluorescence of 5000 parasites.