Sdm 79 medium

PCF cells of T. brucei Lister 427 29-13 (TetR T7RNAP), which co-express the tetracycline (Tet) repressor and T7 RNA polymerase, were maintained in SDM-79 medium (Invitrogen) containing 10% fetal bovine serum, 50 μg hygromycin ml⁻¹ and 2.5 μg G-418 ml⁻¹, at 25°C with 5% CO₂ in a water-saturated incubator. Transgenic lines were generated by the limiting dilution method (Krazy & Michels, 2006 (link)) and selection with 2.5 μg phleomycin ml⁻¹. For RNAi studies, PCF cells were grown in glucose-free medium (Wickstead et al, 2002 (link)).
BSF cells of T. brucei Lister 427 VSG 221 (TetR T7RNAP) were maintained in HMI-9 medium containing 10% fetal bovine serum, 10% Serum Plus (Sigma-Aldrich), and 2.5 μg G-418 ml⁻¹. The cultures were maintained at 37°C with 5% CO₂ in a water-saturated incubator. Transgenic lines were obtained by the limiting dilution method and selection using 2.5 μg phleomycin ml⁻¹.

Tbb were cultivated in HMI9 medium containing 10% heat-inactivated foetal bovine serum (FBS) (Sigma-Aldrich), 150 mM L-cysteine and 20 mM beta-mercaptoethanol at 37 °C (CO₂ 5%). Lmm (MHOM/BZ/84/BEL46) were cultivated in SDM-79 medium (Gibco) supplemented with 15% heat-inactivated FBS (Sigma-Aldrich) and 5 mg/L hemin at 28 °C (CO₂ 5%). Antiparasitic bioassays were performed as described by Bero et al. [17 (link)]. Suramin (SUR) and pentamidine (PEN) were used as positive controls respectively. The hops compounds (xanthohumol, desmethylxanthohumol, humulone, lupulone) concentration that inhibits 50% of the cell viability (IC₅₀) was determined using GraphPad Prism, version 5.01 (GraphPad Software, San Diego, CA, USA).

T. b. brucei Lister 427 bloodstream form cells were cultured in the standard HMI-11 medium (Gibco), supplemented with 10% fetal bovine serum (Sigma-Aldrich) at 37°C, 5% CO₂ [32 (link)]. Phleomycin (Invivogen) was used at 1 μg/ml, hygromycin (Sigma-Aldrich) at 1 μg/ml for bloodstream and 50 μg/ml for insect-stage, puromycin (Sigma-Aldrich) at 1 μg/ml, and blasticidin (Melford) at 5 μg/ml, as appropriate. RNAi was induced using tetracycline (Sigma-Aldrich) at 1 μg/ml. Differentiation into insect-stage cells was performed as described [59 (link)]. Briefly, 2 x 10⁷ cells were washed in DTM medium, and resuspended in 5 ml of DTM supplemented with 15% heat-inactivated fetal bovine serum, 3 mM cis-aconitate (Sigma-Aldrich) and 3 mM sodium isocitrate (Sigma-Aldrich). Cells were cultivated for at least 7 days at 27°C prior to analysis. Established insect-stage cells were cultured in SDM-79 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), GlutaMAX (Gibco) and 2 mg/l hemin (Sigma-Aldrich) as described [60 (link)]. Genetic manipulation was performed by electroporation in cytomix using an Amaxa nucleofector (Lonza) for BSF cells, and a Gene Pulser (BioRad) for insect-stage cells.

Trypanosoma brucei brucei procyclic SmOxP927 cells [50 (link)] were cultured at 28°C in SDM-79 medium (Life Technologies) supplemented with 10% fetal bovine serum (Gibco, 10270106) [51 (link)]. Transgenic cell lines with flagellar proteins KIN-E (Tb927.5.2410 [42 (link)]) and RSP4/6 (Tb927.11.4480 [52 (link)]) tagged with a 3 × HA tag were generated using the pPOT tagging approach [49 (link)]. Bloodstream Trypanosoma brucei brucei cells of the 427 cell line and promastigote Leishmania major cells were provided by the laboratory of Martin Zoltner, Charles University, Prague, Czech Republic.