Luciferase-Based Growth Assay to Quantify Intracellular Parasite Reproduction

To perform a luciferase-based Toxoplasma growth assay, begin by carefully aspirating the medium from pre-seeded 96-well microplate cultures of human foreskin fibroblasts, and inoculate 150 microliters of parasite-free suspension into the wells in a three-column, five-row format.

After a four-hour incubation in the cell culture incubator, carefully aspirate the medium from each well to remove the non-invaded parasites, and fill the wells in each but the first row with room temperature phenol red-free medium. Then, add 100 microliters of a 12.5 micromolar luciferase substrate solution in PBS into each well of the top row, and incubate the microplates for 10 minutes at room temperature to allow full cell lysis.

At the end of the incubation, measure the luciferase activity on a microplate reader. Each reading represents the initial number of invaded parasites at four hours post-infection. Repeat the analysis every 24 hours for the next four days without changing the medium.

To calculate the normalized fold change of parasite growth, each reading can be divided by the initial reading at four hours post-infection. The log2 of the normalized fold change of parasite growth can then be plotted against each timepoint, and subjected to a linear regression function to obtain the slope, which represents the doubling time.