A culture containing uRBCs and 5–10% late-stage (trophozoite/schizont) iRBCs was washed twice in RPMI and three times in PBS-G. RBCs were incubated in 3% BSA (in PBS-G) for 45 minutes with a 1/40 dilution of 1 mg/mL recombinantly synthesised cholesterol-binding probe (θ-D4) conjugated to the fluorophore mCherry [76], a gift from Donatienne Tyteca, or with buffer alone. RBCs were washed three times in PBS-G, and incubated with 5 μg/mL Hoechst 33342 for 20 minutes. Events were measured on a LSRFortessa Flow Cytometer. mCherry fluorescence was detected at 561 nm ex/ 610 nm em. Hoechst fluorescence was detected at 350 nm ex/ 530 nm em), and data were processed as described above. Data were normalised between experiments (uRBCs set to 100%) and analysed using the Mann-Whitney Test. Images were collected at 37°C on a Deltavision Deconvolution microscope, and processed as specified.

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