Culturing Trypanosome Bloodstream Forms

Lister 427 (WT) bloodstream form (BSF) cells were grown in HMI-9 (Gibco^®) (Hirumi and Hirumi, 1989), supplemented with 20% (v/v) heat inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin-streptomycin solution (stock at 10 000 U/ml) (Gibco^®). In the case of the 2T1 cell line, the cells were grown in HMI-11 thymidine-free media, consisting of Iscove's modified Dulbecco's medium (IMDM) (Gibco^®), 10% (v/v) of FBS (Gibco^®,tetracycline free), 1% of penicillin-streptomycin solution (10 000 U/ml) (Gibco^®). For the RNAi cell lines used in EdU replication assays, the cells were grown in HMI-11 thymidine free media, consisting of Iscove's modified Dulbecco's medium (IMDM) (Gibco^®), 10% (v/v) of FBS (Gibco^®, tetracycline free), 1% of penicillin–streptomycin solution (10 000 U/ml; Gibco^®), 4% (v/v) of HMI-9 mix (0.05 mM of bathocuproine disulphonic acid, 1 mM of sodium pyruvate, and 1.5 mM of l-cysteine, 1 mM of hypoxanthine and 0.0014% of 2-mercaptoethanol (Sigma Aldrich)). For Lister 427 (WT), no drugs were added to the media. The selective drugs used for 2T1 cells were puromycin (0.2 μg/ml) and phleomycin (2.5 μg/ml), for RNAi cell lines phleomycin (2.5 μg/ml) and hygromycin (5 μg/ml) and for N50 (41 (link)) neomycin (2.5 μg/ml). The tagged cells were grown in medium with 10 μg/ml blasticidin.