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

1. β-galactosidase assay

NOTE: Quantitation of β-galactosidase activity is used as an indirect way of determining the number of parasites. It is expected that growth will be inhibited in the presence of a trypanocidal compound, leading to a lower number of parasites compared to the untreated control, which will be reflected in a lower β-galactosidase activity and therefore lower absorbance.

1. Incubate the parasites with BZN
   1. Add 100 µL of corresponding 2x benznidazole (BZN) solution per well to reach a final concentration of BZN of 80, 40, 20, 10, 5, and 2.5 µM to 100 µL of epimastigote suspension, Vero cells with amastigotes (2 days post-infection), or trypomastigotes in a 96-well plate.
   2. Incubate the epimastigotes at 28 °C for 72 h, and the trypomastigotes or infected Vero cells with amastigotes for 24 h at 37 °C and 5% CO₂.
      NOTE: Each drug concentration should be evaluated at least in triplicate and include control cultures of epimastigotes, trypomastigotes, and infected Vero cells with dimethylsulfoxide (DMSO).
2. Colorimetric reaction
   1. After the treatment incubation period, if infected Vero cells or trypomastigotes are in Dulbecco's Modified Eagle Medium (DMEM) with phenol red, replace the medium with 100 µL of 1x PBS to avoid interference. Perform triplicate blank wells containing only 100 µL of corresponding medium (or 1x PBS as appropriate).
      NOTE: It is not necessary to remove the culture medium for epimastigotes in case of LIT medium or DMEM without phenol red. DMEM with phenol red can still be used; prepare a blank well with DMEM alone to measure the base absorbance and then subtract this value during data analysis. Schneider's insect medium, which is colorless, is an alternative for epimastigotes.
   2. Add 40 µL of chlorophenol red β-D-galactopyranoside (CPRG) substrate solution and 10 µL of the detergent solution to each well, obtaining a final concentration of 200 µM CPRG and 0.1% detergent in a final volume of 250 µL in each well.
      NOTE: The CPRG solution and detergent can be added together in a final volume of 50 µL per well.
   3. Incubate at 37 °C for 2 h and measure the absorbance at 595 nm in a microplate spectrophotometer.
      NOTE: The expected color change is yellow to reddish-brown upon β-galactosidase enzymatic cleavage (Figure 1A). Incubation time can be extended up to 4 h, and the absorbance spectra of chlorophenol red can be read between 570 and 595 nm with similar curve fitting. Incubation for up to 24 h in the presence of CPRG substrate has shown similar curve fittings.