Development of resazurin microtiter assay for drug sensibility testing of Trypanosoma cruzi epimastigotes.
A quantitative colorimetric assay using the oxidation-reduction indicator resazurin was developed to measure cytotoxicity of compounds against the protozoan parasite Trypanosoma cruzi. This method is based on the detection of colorimetric changes caused by the oxidation (blue) and reduction (pink) capabilities of resazurin dye, an indicator for metabolic cell function. To validate the assay, the experimental conditions were adjusted, such as number of parasites, dye concentration, and time of incubation, with respect to linearity and lower limit of detection. We found that absorbances increased linearly, with the plating density of parasites as low as 5-100 x 10(4)/well (r=0.99; p<0.001) when they were incubated for 5 h at 28 degrees C in the presence of 10% resazurin solution (3 mM). When the cytotoxicity of the reference drugs nifurtimox and benznidazole was measured with this assay and compared to the microscopic counting method, the same range was obtained, demonstrating that the resazurin microtiter assay is valid for the screening of new trypanocidal compounds. This test is very simple, fast, sensitive, and cheap.
Rolón
Miriam
M
Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain.
Vega
Celeste
C
Escario
José A
JA
Gómez-Barrio
Alicia
A
D000818:Animals; D003124:Colorimetry; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D009547:Nifurtimox / Q000494:pharmacology*; D009593:Nitroimidazoles / Q000494:pharmacology*; D010078:Oxazines / Q000378:metabolism*; D010084:Oxidation-Reduction; D021261:Parasitic Sensitivity Tests / Q000379:methods; D012680:Sensitivity and Specificity; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014349:Trypanosoma cruzi / Q000187:drug effects*; D014966:Xanthenes / Q000378:metabolism*
D007202:Indicators and Reagents; D009593:Nitroimidazoles; D010078:Oxazines; D014344:Trypanocidal Agents; D014966:Xanthenes; C005843:resazurin; D009547:Nifurtimox; C009999:benzonidazole