Setting of a colorimetric method to determine the viability of Trypanosoma cruzi epimastigotes

Abstract

The method most commonly used in screening of drugs for the treatment of Chagas' disease, microscopic counting of viable trypanosomes, is time-consuming, labor-intensive, and dependent on the observer. Although the tetrazolium dye [MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is comparatively quick and accurate, it requires careful attention in design as well as in interpretation of the results. Therefore, we examined under various conditions the sensitivity and specificity of the MTT assay versus microscopic counting for determination of the viability of Trypanosoma cruzi for drug-screening purposes. We tested different concentrations of MTT in phenazine methosulfate (PMS) against T. cruzi epimastigotes of the Y strain in different stages of logarithmic growth. In our model, in tests of benznidazole and nifurtimox the optimal concentration of MTT was 2.5 mg/ml of PMS and the optimal incubation period was 75 min. This method detected parasite concentrations of approx. 500,000 epimastigotes/ml (P<0.01), and the linear correlation between absorbance values and numbers of epimastigotes per milliliter was very strong (approx. R = 0.99). The present MTT assay results in faster determination of the activity of compounds, is more objective, and enables testing of several drugs simultaneously.

Biological activity evaluation of dibenzilbutirolactones lignans derivatives against Leishmania braziliensis

“…Preparation of dibenzylbutyrolactone derivatives Synthesis of compounds (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17): The acid 4-(3',4'-methylenedioxifenil)-3-methoxicarbonyl-3-butenoic (3), acid 4-(3',4'-methylenedioxifenil)-3-methoxicarbonil-3-butanoic (4) preparation was previously described by Landais et al, 1999. Lactone (5), the raceme methylpluviatolide (9) and its intermediates compounds (7) and (8) are displayed in Scheme 1.…”

“…The compounds were added in different concentrations 8, 32 and 128 µM, in microtiter plate (96 wells) and it was incubated for 24 hours at 22°C along with the parasites and culture medium. The determination of the biological activity was quantitatively obtained by colorimetric MTT method 9 .…”

Actividad contra Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) de extractos metanólicos de plantas de uso medicinal en México

“…Evaluación de la actividad tripanosomicida de los extractos: Se realizó una solución de cada extracto metanólico de 15 mg disueltos en dimetil sulfoxido 1 % (DMSO) e incubados por 24 h. Los bioensayos se realizaron por duplicado; para cada extracto se tomó como unidad experimental una microplaca de 96 pozos, donde se depositaron 200 μL de la suspensión del medio LIT con 1.5 x 10 6 parásitos/mL, en cada pozo se agregó cada una de las cinco diferentes concentraciones del extracto con tres réplicas de cada uno de los tratamientos (1 000 μg/mL, 750 μg/mL, 500 μg/mL, 250 μg/mL y 100 μg/mL); simultáneamente, se colocaron el control positivo y negativo, se utilizó nifurtimox (Sigma-Aldrich) a 10 µg/mL y medio de cultivo con parásitos respectivamente, con un volumen igual al de los extractos de DMSO 1 % (Muelas-Serrano, Nogal-Ruiz, & Gómez-Barrio, 2000). Las concentraciones expresadas en μg/mL de los extractos se prepararon en una balanza analítica con capacidad de 80 g a 0.0001 g (Nimbus 84i, Adam Equipment) y mediante diluciones con micropipetas (Eppendorf) con capacidad de 1 mL a 0.5 mL.…”

Oleanonic acid from Lippia lupulina (Verbenaceae) shows strong in vitro antileishmanial and antitrypanosomal activity

“…All the assays were performed in triplicate. Test samples concentrations corresponding to 50% of parasite growth inhibition (IC 50 ) were determined from non-linear regression (Muelas-Serrano et al 2000;Santos et al 2012).…”