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

Published: April 30, 2023

Abstract

Source: Alonso, V. L. et al. In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase. J. Vis. Exp. (2021)

In this video, we describe a simple and robust method to determine the cytotoxic potential of a trypanocidal drug against a parasite, Trypanosoma cruzi, expressing the β-galactosidase enzyme. β-galactosidase serves as a cell lysis indicator, and its activity can be used to assess the effects of the drug.

Protocol

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. Incubate the parasites with BZN</st…

Representative Results

Figure 1: Calculation of IC50 value of benznidazole for epimastigote form Dm28c. (A) A 96-well plate with epimastigotes treated with different BZN concentrations (2.5, 5, 10, 20, 40, and 80 µM) before adding CPRG (1), after initial addition of CPRG and detergent (2), and after incubation with CPRG and detergent showing the change of color (3). (B) XY-plot of…

Disclosures

The authors have nothing to disclose.

Materials

96-well plates Corning 3599
Benznidazole Sigma Aldrich 419656
5 mL serological pipette sterile Jet Biofil GSP010005
10 mL serological pipette sterile Jet Biofil GSP211010
Biosafety Cabinet Telstar Bio II A/P
CPRG Roche 10 884308001 Chlorophenol Red-β-D-galactopyranoside
DMEM, High Glucose Thermo Fisher Cientific 12100046 Powder
DMSO Sintorgan SIN-061 Dimethylsulfoxide
Fetal Calf Serum Internegocios SA FCS FRA 500 Sterile and heat-inactivated
Vero cells ATCC CRL-1587
Microplate Spectrophotometer Biotek Synergy HTX
Microcentrifuge tube 1.5 mL Tarson 500010-N
G418 disulphate salt solution Roche G418-RO Stock concentration: 50 mg/mL

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Cite This Article
β-Galactosidase Assay to Evaluate Drug-Induced Cytotoxicity in Parasites. J. Vis. Exp. (Pending Publication), e21278, doi: (2023).

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