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8544264849
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5 parasites/mL, were cultured in biphasic medium containing 1.5% nutrient agar with the addition of 0.2% rabbit defibrinated blood and Brain Heart Infusion medium (Difco Michigan, USA) at 28°C. Drug concentrations ranging from 0 to 200 μM were added. The diluent, ethanol, remained below 0.125%. Parasite concentration was microscopically determined in samples obtained at 48, 72, 96, and 144 h of incubation using a Neubauer chamber
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16
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8544271137
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note
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Effect of CsA and CsA derivatives on trypomastigote lysis: the assay was performed in 96-well microplate with 50.000 bloodstream trypomastigotes per well incubated with different drug concentrations ranging from 0 to 400 μM. Cultures were incubated for 24 h at 4°C at a final volume of 0.1 mL. Drug lysis was evaluated by counting remaining parasites in an haemocytometer. The final diluent concentration ethanol remained below 0.125%, including in drug nontreated control cultures
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17
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8544280231
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note
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2 and then, thoroughly washed with serum-free medium and incubated with complete medium without drugs for 36 h. Infected and drug nontreated cell cultures were used as controls with 0.3% ethanol. Cell cultures were set up in triplicate for each drug concentration. Covers with cells were removed from the culture plates after 36 h of treatment, gently rinsed with phosphate buffer saline, air dried, fixed in ethanol, and stained with Giemsa. Statistical analysis: experiments were performed in duplicate or in triplicate, as indicated, in two to four independent experiments. The dose response curves were obtained by linear regression analysis using computer programs. Statistical analysis of the in vitro parasite experiments and enzyme kinetics was performed using Student's t test. p < 0.05 was regarded as statistically significant
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18
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21,22 The effect of CsA and derivatives on PPIase activity was analyzed by incubating 20 nM of purified TcCyP19 during 5 min at 5°C with different concentrations of drugs ranging from 0 to 1 μM
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