3 isobutyl 1 methylxanthine ibmx

Freshly egressed tachyzoites (RH TIR1-3FLAG and RH PDE-mAID-3HA lines) were harvested, counted on a hemocytometer, and inoculated (200 parasites/well) onto confluent HFF monolayers growing in 6-well plates containing D10 medium. To determine the effect of human phosphodiesterase inhibitors on Toxoplasma fitness, wells were treated with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (MP Biomedicals) prepared in 100% DMSO (Sigma-Aldrich), 0.5 mM zaprinast (Tocris) prepared in 100% DMSO, or vehicle (0.1% DMSO). To determine the effect of conditional knockdown of a TgPDE on Toxoplasma fitness, wells were treated with 0.5 mM IAA or 0.0789% (wt/vol) ethanol (vehicle). In both experiments, plates were left undisturbed for 8 days in a 37°C, 5% CO₂ incubator. Plaque formation was assessed by counting zones of clearance on EtOH-fixed, crystal violet-stained HFF monolayers. Each stained plate was scanned with high-definition digital scanner (Epson) to obtain representative images and for plaque area analysis. For plaque area measurements, the 10 centermost plaques from the representative wells shown were analyzed using ImageJ to define the plaque area in square pixels (pix²).