Luciferase-Based Growth Assay to Quantify Intracellular Parasite Reproduction

Abstract

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)

In this video, we demonstrate the luciferase assay to quantify the growth of the intracellular pathogen Toxoplasma gondii. Utilizing a strain of Toxoplasma that expresses luciferase enzyme after infecting the host cells, the luciferase activity is detected in the infected cell culture to determine the successful reproduction of the parasite.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Luciferase-based Toxoplasma growth assay

1. Seed human foreskin fibroblasts (HFFs) 1 week before parasite inoculation to ensure that host cells are fully confluent. Perform a mock assay in a transparent plate to ensure that parasites remain intracellular throughout the evaluation period.
   NOTE: Here, the assay is conducted in 96-well microplates. According to experimental needs, it can be scaled up to 384 or 1536-well microplates.
2. Pass Toxop....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biotek Synergy H1 Hybrid Multi-Mode Microplate Reader	BioTek Instuments
Coelenterazine h	Prolume	301-10 hCTZ
Software
GraphPad Prism software (8th version)