Protocol to enhance pre-sexual and sexual differentiation of Toxoplasma gondii using retinal cells and intestinal organoid-derived monolayers

Toxoplasma gondii undergoes pre-sexual and sexual differentiation primarily in feline hosts, limiting experimental study. Here, we present a protocol for enhancing T. gondii stage differentiation using non-feline in vitro systems. We describe culturing human retinal pigment epithelial cells and murine intestinal organoid-derived monolayers in felid-environment-like with linoleic acid excess (FELIX) medium to mimic feline intestinal lipid biochemistry, combined with conditional MORC depletion to promote stage conversion. We detail procedures for host cell preparation, parasite culture, infection, and assessment of stage-specific gene and protein expression. For complete details on the use and execution of this protocol, please refer to Cancela et al.¹.