Luciferase-Based Growth Assay to Quantify Intracellular Parasite Reproduction

Toxoplasma gondii - a single-celled protozoan - is an obligate anaerobic parasite, reproducing only inside host cells.

To quantify the parasite's intracellular growth using a luciferase-based assay, take a suspension of parasite cells expressing luciferase - an enzyme that produces luminescence upon reaction with its substrate. Add the suspension to a culture of human foreskin fibroblasts, and incubate for an adequate duration.

The parasite binds to host cell receptors and gets internalized via an endocytic process, forming a parasitophorous vacuole. The structure provides a niche for the parasite to reproduce.

Post-infection, aspirate the medium, removing the non-internalized parasites. Add a lysis buffer containing a detergent and luciferin - a substrate for luciferase. Detergent in the buffer creates pores in the cell membrane and completely lyses the cells, resulting in the release of luciferase.

The released enzyme oxidizes luciferin molecules, emitting light. Measure the luminescence intensity to determine luciferase activity. Using replicate wells containing infected host cells, measure luciferase activity at predetermined intervals post-infection.

Plot the normalized luminescence intensity versus the time intervals to obtain the fold changes in parasite growth over time. A temporal rise in intensity indicates successful parasite multiplication.

Plot the values in a logarithmic scale to calculate the curve's slope, representing the doubling time of the parasite.