Gs500

Individual colonies of methylcellulose cultures were picked, washed in PBS and immobilized on microscope slides by cytospin (4 min, 800 rpm, Shandon Cytospin 3). Cells were fixed with methanol for 1 min, air-dried, Giemsa-stained for 3 min (Sigma, GS500) and analysed by microscopy.

For the colony formation assay, 600 cells were seeded into the 60 mm dishes and treated with 100 μM scrambled C2ORF40 mimic peptide or 100 μM C2ORF40 mimic peptide. Cells were incubated in a homogeneous atmosphere with 5% CO₂ at 37°C for two weeks. Then methal alcohol was used to fix the clones for 20 minutes and Giemsa (Sigma, GS-500) was used to stain the clones for 30 minutes. Lastly we counted the clones under a microscope.

EVs were obtained from 25 ml of alanine-synchronized infected RBCs (iRBCs) in late schizont stage or uninfected ones (uRBCs). Cultures with different parasitaemia (ranging from 2% to 25%) were used, depending on the conditions for each experiment. Parasitaemia was evaluated by optical light microscopy using Giemsa staining (GS500, Sigma-Aldrich). The isolation of EVs was based on previous reports^{24 (link),45 (link)}. Briefly, the cultures were collected and centrifuged at 2000 × g for 15 min. The 2000 × g supernatants were then centrifuged at 15,000 × g at 4 °C for 30 min to remove cell debris. Next, these supernatants were filtered through 0.2 μm low-binding protein filters (Acrodisc, Pall Life Science, Port Washington, USA) and the filtered supernatants were ultracentrifuged at 110, 000 × g 4 °C for 70 min to pellet small vesicles. The pellet was washed once by resuspending it in sterile double-filtered (0.2 μm) PBS 1X and further ultracentrifuged at 110,000 × g for an additional 70 min. The pellet was finally resuspended in 100 μl of double-filtered PBS 1× for analysis.

Cells were stained on slides with May-Grünwald–Giemsa and diaminobenzidine hydrochloride reagents (Sigma-Aldrich GS-500 and D-9015) for morphological analyses. Hemoglobin content was measured at 540 nm wavelength light after incubating one million cells with Drabkin’s reagent (RICCA Chemical Company, 2660-16). Human hemoglobin (Sigma-Aldrich H7379) was used for the standard curve to calculate hemoglobin amounts from the O.D. 450 nm value.