Motility Analysis of Trypanosomes

For motility analysis in T. brucei, a sedimentation assay was conducted as previously described by Ralston et al. (2006) (link). 5×10⁶ uninduced and 6 days induced trypanosomes were incubated at 27°C in 1 ml medium in spectrophotometric cuvettes, and optical density was measured every 2 h compared to that in controls in which cells were resuspended. In L. mexicana, swimming behaviours are analysed for cells in the exponential growth phase in normal culture medium essentially as previously described (Wheeler, 2017 (link)). For cell swimming analysis, a 25.6 s video at five frames/s under darkfield illumination was captured from 5 μl of cell culture in a 250 μm deep chamber using a Zeiss Axioimager.Z2 microscope with a 10×/0.3 NA objective and a Hamamatsu ORCA-Flash4.0 camera. Particle tracks were traced automatically, and mean cell speed, mean cell velocity and cell directionality (the ratio of velocity to speed) were calculated as previously described (Beneke et al., 2019 (link)).