Circular micro cover glass until confluent

Tachyzoites were differentiated in vitro into bradyzoites within cysts essentially as previously and elegantly described by Knoll and colleagues (57 (link)). Differentiation medium contained Roswell Park Memorial Institute medium (RPMI) without bicarbonate supplemented with 2.05 mM l-glutamine (HyClone), 20 mM HEPES-free acid (IBI Scientific), 1% XL-glutamine (a long-lasting stable form of glutamine; VWR), 1% FBS, and 1% penicillin-streptomycin. The pH of differentiation medium was adjusted to 8.1 with sodium hydroxide and filter sterilized. HFF cells were cultured on circular micro cover glass until confluent (Electron Microscopy Sciences), and confluent monolayers were infected with type II Pru parasites at a multiplicity of infection (MOI) of ∼0.5. Infected cells were washed 3 h after infection once in Dulbecco’s phosphate-buffered saline (DPBS) supplemented with Ca²⁺ and Mg²⁺ and incubated in differentiation medium for 6 h, 1 day, 2 days, 3 days, 7 days, or 10 days at 37°C in ambient air. Medium was changed on days 3 and 7.

Tachyzoites were differentiated in vitro into bradyzoites within cysts as previously described by Tobin and colleagues (44 (link)). The differentiation medium contained Roswell Park Memorial Institute medium (RPMI) without bicarbonate supplemented with 2.05 mM l-glutamine (HyClone), 20 mM HEPES (free acid) (IBI Scientific), 1% XL-glutamine (a long-lasting stable form of glutamine; VWR), 1% FBS, and 1% penicillin-streptomycin. The pH of differentiation medium was adjusted to 8.1 with sodium hydroxide and filter sterilized. HFF cells were cultured on circular micro cover glass until confluent (Electron Microscopy Sciences), and confluent monolayers were infected with type II Pru parasite at an MOI of ∼0.5. Infected HFF cells were washed 3 h after infection once in DPBS supplemented with Ca²⁺ and Mg²⁺ and incubated in differentiation medium for 6 h, 1 day, 2 days, 3 days, 7 days, or 10 days at 37°C in ambient air. The medium was changed on day 3 and day 7.

Tachyzoites were differentiated in vitro into bradyzoites within cysts essentially as previously and elegantly described by Tobin and colleagues (22 (link)). Differentiation medium contained Roswell Park Memorial Institute medium (RPMI) without bicarbonate supplemented with 2.05 mM l-glutamine (HyClone), 20 mM HEPES (free acid) (IBI Scientific), 1% XL-glutamine (a long-lasting stable form of glutamine) (VWR), 1% FBS, and 1% penicillin-streptomycin. The pH of the differentiation medium was adjusted to 8.1 with sodium hydroxide and filter sterilized. HFF cells were cultured on circular micro cover glass until confluent (Electron Microscopy Sciences), and confluent monolayers were infected with type II Pru parasites at a multiplicity of infection (MOI) of ∼0.5. Three hours after infection, infected cells were washed once in DPBS supplemented with Ca²⁺ and Mg²⁺ and incubated in differentiation medium for 6 h, 1 day, 2 days, 3 days, 7 days, or 10 days at 37°C in ambient air. The medium was replaced with fresh medium on day 3 and day 7.