Parasite strains cultivation and differentiation

Type I strain RH⁷⁹, RH-Δku80Δhxgprt (RHΔku80)⁸⁰, GT1⁸¹, Type II ME49⁸², NTE⁸², Pru-Δku80Δhxgprt(BSG-4) (Pru-GFP)⁴², Pru-Δhxgprt tdTomato (Pru-tdTomato)⁸³ and Type III NED⁸⁴, VEG (⁸⁵, provided by G. Schares), were maintained in vitro in HFF monolayers grown in DMEM supplemented with 25 mM glucose, 4 mM l-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin and 1% heat-inactivated FBS (tachyzoite medium). Freshly egressed parasites were passaged by transfer to new HFF monolayers.

Differentiation of tachyzoites into tissue cysts was facilitated by CO₂ depletion at pH 7.4 if not otherwise indicated. To avoid acidification, the medium was changed to low glucose (5 mM), 50 mM HEPES (Sigma-Aldrich) buffered RPMI 1640 medium (Gibco) supplemented with 4 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 2% HOS, and 10 µg/ml human insulin, 5 µg/ml human holo-transferrin and 1.7 ng/µl sodium selenite (bradyzoite medium). The cells were incubated at 37 °C and ambient CO₂ levels. Medium was changed every two days and cells were washed once per week with PBS. The day after infection, infected monolayers were washed with prewarmed PBS to remove non-invaded parasites. Medium was changed every second day and bradyzoite cultures were washed with PBS once a week. For assays involving plate reader-based fluorescence measurements, phenol red was omitted from the medium.