Inducing Toxoplasma gondii Bradyzoites

Toxoplasma gondii parasites were maintained in continuous passage in Hs27 human foreskin fibroblast (HFF) cell monolayers (ECACC number 94041901). Parasites were grown in DMEM supplemented with 10% foetal bovine serum (Invitrogen, Paisley, UK) and passaged between 4 and 15 times prior to bradyzoite induction. Bradyzoites were induced in culture using the high (pH 8.1) pH shock method (49). The number of parasitized cells, vacuoles and the number of parasites per vacuole were scored every 24 hours to monitor differentiation. Four parasitized monolayers were harvested every 24 hours by trypsinization and parasites purified by passage through a 21 gauge needle and washing in phosphate-buffered saline (PBS). Parasites were enumerated and pellets were resuspended in Qiagen RNeasy kit RTL buffer prior to RNA extraction. For Western blotting, parasites were harvested as in Roberts et al. (50). Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane and probed with anti-rat-tyrosine hydroxylase monoclonal antibody.