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).