To generate and purify schizonts and establish synchronous infections, a previously published protocol for schizont transfection was followed (38). In summary, two naïve Balb/c mice were injected with 200 μl of a P. berghei stabilate. Upon reaching a parasitemia of up to 5% (usually at days 4 to 5 after infection, verified by Wright staining), blood was collected by intracardiac puncture. Complete RPMI1640 culture medium (125 ml) was prepared by adding 25 ml of freshly thawed fetal bovine serum to 100 ml of RPMI1640 culture medium and 200 μl of gentamicin stock solution. Between 15:00 and 18:00, a total of 1.5 to 2 ml of infected blood was collected from each of the two mice by cardiac puncture following euthanasia by CO2 inhalation. The blood was immediately added to a 50-ml Falcon tube containing 5 ml of complete media supplemented with 350 μl of heparin stock solution. Infected erythrocytes were then harvested by centrifugation for 8 min at 450g, and the supernatant was carefully discarded. The iRBCs were resuspended in 50 ml of complete media and mixed with the 120 ml of RPMI1640 complete media. The total volume was divided equally between two tubes/flasks, each containing around 85 ml. The tubes/flasks were gassed for 90 s at 1.5 to 2 bar pressure with the gas mixture of 5% CO2, 5% O2, and 90% N2. Tubes were closed tightly following gassing and placed immediately at a 37°C shaking/rotating incubator. The parasites were incubated overnight for 16 hours, and viable schizonts were collected between 9:00 and 12:00 of the following morning as follows. First, 500 μl of the schizonts in media was collected and centrifuged for 5 min at 16,000g. The supernatant was discarded, and a blood smear was prepared and stained with Wright stain. The smear was verified by light microscopy for contamination and schizont enrichment. Before schizont isolation, a 55% Nycodenz–1× phosphate-buffered saline (PBS) solution (v/v) was prepared in a 50-ml Falcon tube by adding 27.5 ml of Nycodenz solution [138 g of Nycodenz powder dissolved in 500 ml of 5 mM tris/HCl, 3 mM KCl, and 0.3 mM CaNa2-EDTA in deionized water (dH2O) at pH 7.5] to 22.5 ml of 1× PBS. Schizont cultures (170 ml) were divided into four separate 50-ml Falcon tubes (each containing 25 to 30 ml). Using a 20-ml pipette, 10 ml of Nycodenz-PBS solution was underplayed under the culture suspension in each tube. All tubes were centrifuged for 20 min at 450g in a swing-out rotor at room temperature, without brake or initial acceleration. After 20 min, using a Pasteur pipette, the brown-grayish interface of schizonts between the two suspensions was collected from each tube. The isolate (10 μl) was Hoechst-stained, and schizont purity and number per volume were quantified. Aliquots of 6 × 106 schizont were separated into Eppendorf tubes, centrifuged for 5 min at 450g, resuspended in 200 μl of 1× PBS, and injected intravenously into naïve UBC-GFP C57BL/6 or WT C57BL/6 mice. Depending on the time when schizont purification was initiated, injection into mice occurred between 10:00 and 13:00. At all consecutive times, mice were kept in a strict 12-hour light cycle to keep parasite synchronicity. Schizont localization in the recipient mice’s bodies (by bioluminescence imaging) or in the BMs (by IVM) was performed at 22 to 24 hours after infection. For isolation of merozoites, rings, and trophozoites, the mice infected with schizonts were regarded as intermediate donor mice and were bled by intracardiac puncture at 4 hours after infection for isolation of rings and at 11 hours after infection for isolation of trophozoites. Upon obtaining 1.5 to 2 ml of synchronously infected blood, a Percoll density gradient was used to separate the corresponding parasite stages. Pure rings or pure trophozoites were resuspended in 200 μl of 1× PBS and intravenously injected into naïve UBC-GFP C57BL/6 or WT C57BL/6 mice. Mice were imaged 2 hours after infection. For merozoite injection, isolated schizonts were mechanically ruptured, and merozoites were briefly incubated in 100 μl of heparin, followed by intravenous injection into naïve UBC-GFP C57BL/6 or WT C57BL/6 mice. Mice were imaged by IVM or bioluminescence immediately following injections.

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