Procyclic Differentiation Assays for Trypanosomes

Procyclic differentiation assays refer to the previous literature with modifications as described (McDonald et al., 2018 (link)). Pleomorphic T. brucei (Tbp1) parasites, laboratory-adapted monomorphic passages (includingTbp40, Tbp80, Tbp120) and monomorphic T. evansi and T. equiperdum parasites, were purified from whole blood and resuspended at 2 × 10⁶ cell ml^–1 in SDM-79 + 10% FBS (v/v) media supplemented with 6 mM cis-aconitate (Sigma) to induce procyclic differentiation. These were then incubated at 27°C in 5% CO₂. After 1, 6, 12, 24, or 48 h cultivation (depending on the strains), 1 ml of the cell culture was concentrated at 1000 g, and then air-dried smears were prepared from the parasite pellets and fixed in 1% paraformaldehyde for stage-specific procyclin coat expression analysis by immunofluorescence. Meanwhile, CA was removed from the medium after 6- or 24-h treatments and the parasites were used for subculture in vitro.