Source: Key, M., et al. Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay. J. Vis. Exp. (2020).
The video demonstrates the luciferase assay used to determine the efficacy of an antiparasitic chemical inhibitor against the intracellular pathogen Toxoplasma gondii. The study involves luciferase-expressing parasites invading the host cells to replicate and survive. The chemical inhibitor binds with the cysteine proteases of the parasite and inhibits replication. The luciferase activity detected is proportional to the fold changes in parasite growth, determining the drug's efficacy against the parasite.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Evaluation of chemical compound inhibition efficacy against Toxoplasma growth
NOTE: Here, evaluation of the inhibition of LHVS in Toxoplasma growth is presented as an example. Eight different concentrations of LHVS are tested, and three technical replicates are performed for each of the three biological replicates for both RHΔku80::NLuc and RHΔku80Δcpl::NLuc strains.
Figure 1: Inhibition efficacy assessment of LHVS and pyrimethamine using the luciferase-based growth assay. Parasites were inoculated into a 96-well microplate for 4 h to allow for invasion of host cells. Non-invaded parasites were washed away, and the plate was filled with media containing different concentrations of LHVS or pyrimethamine and incubated for an additional 96 h before determination of luciferase activity. The measured luciferase readings for parasites treated with individual inhibitor concentrations were normalized against the signal detected from untreated parasites. The data were plotted in a graphing program, and a regression analysis for IC50 determination was performed. The assay was repeated in three biological replicates with three technical replicates each. Data represent mean ± SEM, n = 3 biological replicates.
The authors have nothing to disclose.
Biotek Synergy H1 Hybrid Multi-Mode Microplate Reader | BioTek Instuments | ||
Coelenterazine h | Prolume | 301-10 hCTZ | |
Software | |||
GraphPad Prism software (8th version) |