Bradyzoite differentiation assay.

Bradyzoite differentiation was measured by staining the cyst wall. Briefly, purified parasites were inoculated onto HFF cells in a 24-well plate at a concentration of 1,000 parasites/well. After a 2-h incubation, the medium was changed to fresh medium and incubated under a 5% CO₂ condition (normal) or was changed to induction medium (DMEM supplemented with 1% FBS and 50 mM HEPES, adjusted to pH 8.2 with NaOH) and incubated under an 0.5% CO₂ condition (pH 8.2 and CO₂ depletion). For the bradyzoite induction with the RH strain, ambient air with a humid box was used; thus, CO₂ concentration was around 0.02%. Infected host cells were extensively incubated for 48 h, fixed with 4% paraformaldehyde in PBS, and stained as described for the immunofluorescence assay. Salmon E monoclonal antibody (1:500) for cyst wall protein 1 (CST1) (31) and 1:4,000-diluted rabbit anti-Toxoplasma serum were used as primary antibodies to detect cyst wall and total parasites, respectively. Alexa 488- and 594-conjugated anti-mouse or -rabbit IgG antibody diluted 1:1,000 was used as a secondary antibody. Green fluorescent signal at the parasitophorous vacuole membrane was identified as the cyst wall. At least 100 vacuoles were scored to determine the number of parasitophorous vacuoles with or without cyst walls.