Cultivation of Promastigote Leishmania and Trypanosoma

Promastigote form L. mexicana (WHO strain MNYC/BZ/62/M379), L. major Friedlin and their genetically modified derivatives were grown at 28°C in M199 medium (Life Technologies) supplemented with 2.2 g l⁻¹ NaHCO₃, 0.005% haemin, 40 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid pH 7.4 and 10% FCS. Relevant selection drugs (Melford Laboratories Ltd.) were added to the medium at the following concentrations: 32 µg ml⁻¹ Hygromycin B, 20 µg ml⁻¹ Puromycin Dihydrochloride, 5 µg ml⁻¹ Blasticidin S Hydrochloride, 40 µg ml⁻¹ G-418 Disulfate, 50 µg ml⁻¹ Nourseothricin Sulfate and 25 µg ml⁻¹ Phleomycin. Trypanosoma brucei SmOx B4 [49 (link)] was grown in HMI-9 [54 (link)] at 37°C in 5% CO₂, T. brucei SmOx P9 in SDM-79 [55 (link)] at 28°C. Relevant selection drugs (Melford Laboratories Ltd.) were added to the medium at the following concentrations: 0.2 µg ml⁻¹ Puromycin Dihydrochloride, 5 µg ml⁻¹ Blasticidin S Hydrochloride, 5 µg ml⁻¹ G-418 Disulfate, 5 µg/ml Hygromycin B. Cell culture densities were measured using a CASY model TT cell counter (Roche Diagnostics) with a 60 µm capillary and exclusion of particles with a pseudo diameter below 2.0 µm.