Culturing Monomorphic Procyclic T. brucei

Cells used in all experiments were monomorphic procyclic form T. brucei Lister strain 427 or a tetracycline-inducible strain 427-based strain generated by introducing the Single Marker Oxford plasmid [30 (link)]. 427-based cell lines were grown at 27°C in Beck’s Medium (Hyclone, Logan, Utah) supplemented with 500 μg/mL penicillin-streptomycin-glutamine (Hyclone), 10% fetal bovine serum (Gemini Bioproducts, West Sacramento, CA), and 10 μg/mL gentamycin (ThermoFisher Scientific, Waltham, MA). SmOx-based cell lines were cultured at 27°C in SmOx medium comprised of Beck’s Medium supplemented with 500 μg/mL penicillin-streptomycin-glutamine, 1 μg/mL puromycin, 10% tetracycline-free fetal bovine serum (RnD Systems, Minneapolis, MN), and 10 μg/mL gentamycin. Cell counts were determined using a Z2 Coulter Counter particle counter (Beckman Coulter, Brea, CA).