Maintenance of Sand Fly Colonies and Leishmania Cultures

Laboratory colonies of four sand fly species, Phlebotomus orientalis (from Ethiopia), P. argentipes (India), Sergentomyia schwetzi (Ethiopia) and P. duboscqi (Senegal), were maintained in the insectary of the Charles University in Prague under standard conditions (at 26°C, fed on 50% sucrose,) with a 14 h light/10 h dark photoperiod as described previously [16 (link)]. The latter two sand fly species were included as non-permissive vector controls.
Leishmania donovani Parent 1 (P1) (MHOM/ET/2010/GR347) expressing enhanced green fluorescence protein (eGFP) and L. donovani Parent 2 (P2) (MHOM/ET/2010/AM459) expressing red fluorescent protein (dsRFP), both strains originating from Ethiopia, were cultured in M199 medium (Sigma) containing 10% heat-inactivated foetal calf serum (Gibson) supplemented by 1% BME vitamins (Sigma), 2% sterile human urine, 250 μg/ml amikacin (Amikin, Bristol-Myers Squibb), and 150 μg/ml selective antibiotic G 418 (Sigma) and Hygromycin B (Sigma), respectively [4 (link),7 (link)]. To obtain amastigote stages, mouse macrophage line J774 was exposed to stationary-phase parasites at a parasite to macrophage ratio 8:1. Both infected and uninfected macrophages were cultured in RPMI medium containing 10% FBS, 100 U/ml of penicillin, 100 μg/ml of streptomycin, 2mM L-glutamine, and 0.05 mM β-mercaptoethanol (all from Sigma) at 37°C with 5% CO₂.