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دانلود رایکان مقاله انگلیسی ISI با موضوع حذف گزینشی لپتوموناز در هم کشتی آزمایشگاهی با لشمانیا

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حذف گزینشی لپتوموناز در هم کشتی آزمایشگاهی با لشمانیا

عنوان انگلیسی مقاله:

Selective elimination of Leptomonas from the in vitro co-culture with Leishmania

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2. Materials and methods

Leishmania donovani 1S (a cloned line from strain 1S, WHO designation: MHOM/SD/62/1S), L. donovani DD8 (ATCC #50212), L. donovani AG83 [14], L. donovani HP+/− (Kavita et al., unpublished) and L. seymouri (ATCC #30220) were used in all experiments. Among the Leishmania species, strain 1S was used for most experiments unless otherwise mentioned. Promastigote forms of all the L. donovani strains were grown in vitro in T25 cm2 culture flasks (Corning) at 26 °C in medium 199 (Sigma) [15] (pH 6.8) with 8 μM 6-Biotin, 25 mM Hepes (N-[2- hydroxyethyl]piperazine-N0-[2-ethanesulfonic acid; Sigma], 0.1 mM adenine (Sigma; in 25 mM Hepes), 8 μM hemin (4 mM stock made in 50% triethanolamine), 100 U/ml each of penicillin G and streptomycin (Gibco) and 10% (v/v) heat-inactivated fetal bovine serum (Gibco). Axenic amastigotes were grown in T25 cm2 culture flasks at 37 °C with 5% CO2 in RPMI-based medium [16] (pH 5.6) containing 15 mM KCl, 114.6 mM KH2PO4, 10.38 mM K2HPO4 · 3H2O, 0.5 mM MgSO4 · 7H2O and 24 mM NaHCO3, 1× liquid RPMI-1640 vitamin mix (Sigma); 1× liquid RPMI-1640 amino acid mix (Sigma), 4 mM Lglutamine (Gibco), 25 mM adenosine (Sigma), 23 μM folic acid (23 mM stock made in 1 N KOH Sigma), 100 U/ml each of penicillin G and streptomycin (Gibco), 1× liquid phenol-red (Gibco), 22 mM Dglucose (Sigma), 25 mM 2-(N-morpholino)ethanesulfonic acid (MES, Sigma) and 20% heat-inactivated fetal bovine serum (Gibco). Growth of both promastigotes and amastigotes were measured as described previously [17].