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

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.

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,....

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 add.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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