β-Galactosidase Assay to Evaluate Drug-Induced Cytotoxicity in Parasites

Add benznidazole solution to epimastigotes, Vero cells with amastigotes, or trypomastigotes, as described in the text manuscript. Then, incubate the epimastigotes at 28 degrees Celsius for 72 hours, and the trypomastigotes or infected Vero cells with amastigotes for 24 hours at 37 degrees Celsius and 5% carbon dioxide.

For the colorimetric assay, set up the blank wells by adding 100 microliters of PBS or corresponding medium. Next, add 40 microliters of CPRG substrate solution, and 10 microliters of the detergent solution to each well for obtaining a final concentration of 200 micromolar CPRG and 0.1% detergent in a final volume of 250 microliters in each well.

After incubation at 37 degrees Celsius for 2 hours, measure the absorbance at 595 nanometers using a microplate spectrophotometer.