Fractionation of a sex-inducing substance from flatworms using open-column chromatography and reverse-phase high-performance liquid chromatography

A substance that sexualizes planarians, an ancestral group of parasitic flatworms, is widely present in planarians and parasitic flatworms. Here, we present a protocol for extracting and purifying the active fraction with sex-inducing activity. We describe steps for homogenization of flatworms, sample concentration, open-column chromatography, and reverse-phase high-performance liquid chromatography. We then detail a feeding bioassay to confirm sex-inducing activity. The obtained active fraction may positively affect parasitic flatworm sexual maturation and can be tested by adding it into the culture media. For complete details on the use and execution of this protocol, please refer to Sekii et al. (2023).(1)