Quantifying Parasite Ingestion in Host Cells

Parasite ingestion was determined using modifications of a previously reported assay (16 (link)). In brief, mCherry was expressed in inducible Chinese hamster ovary (iCHO) cells with the addition of 2 μg/ml of doxycycline (DOX). Parasites from infected iCHO cells were harvested, purified, treated with pronase and saponin, and imaged on Cell-Tak (Fisher Scientific)-coated slides using a Zeiss Axiovert Observer Z1 inverted fluorescence microscope. For each biological replicate, more than 97 parasites of each genotype or treatment were enumerated for host-derived mCherry accumulation within parasites. Samples were coded during the time of harvesting to blind the experimenter during imaging and quantification.
In this study, we refer to the in vitro conditions that promote tachyzoite conversion to bradyzoite cysts as “bradyzoite-inducing conditions.” This includes the use of alkaline (conversion) media and growth without CO₂. For all experiments, 2 μg/ml DOX was used. Detailed changes for each ingestion assay are described below.