Luciferase-Based Growth Assay to Quantify Intracellular Parasite Reproduction

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 Toxoplasma parasites into confluent HFFs 2 days prior to use by transferring ~0.3-0.4 mL of fully lysed parasites into a T25 flask. Incubate infected host cells at 37 °C with 5% CO₂ for 2 days.
3. Syringe 5 mL of freshly lysed parasites through a 21 G safety needle 5x to liberate intracellular parasites, then pass through a 3 µm filter to remove host cell debris. Rinse residual parasites out of the flask using 7 mL of phenol red-free D10 medium, then pass through the filter again.
4. Centrifuge parasites at 1000 x g for 10 min at room temperature (RT). Pour off the supernatant and resuspend the pellet in 10 mL of phenol red-free D10 media.
5. Count parasites using a hemocytometer to determine the concentration.
6. Dilute parasites to 1 x 10⁴ parasites/mL for the wild-type (WT) strain. For growth-deficient parasite strains, increase the concentration accordingly to observe a significant increase in luciferase signals.
7. Aspirate media carefully from 96 well microplates pre-seeded with HFFs and inoculate 150 µL of parasite resuspension into wells in a format of three columns and five rows, which represents three technical replicates and five time points.
8. Incubate the microplate at 37 °C and 5% CO₂ for 4 h.
9. Aspirate media carefully from the wells to remove non-invaded parasites, then fill the wells with RT phenol red-free media in each row (except for the first row).
10. Mix equal volumes of PBS and 2x luciferase assay buffer and dilute the luciferase substrate to 12.5 µM.
11. Add 100 µL of dilute luciferase substrate into each well of the top row. Incubate the microplates at RT for 10 min to allow the cells to fully lyse.
12. Measure the luciferase activity using a microplate reader. The plate reader settings are listed in Table 1. Each reading represents the initial number of invaded parasites at 4 h post-infection.
13. Repeat steps 1.9-1.12 for each row every 24 h for 4 days without changing the medium. These readings reflect the total number of replicated parasites at 24 h, 48 h, 72 h, and 96 h post-infection.
14. Calculate the average readings at each timepoint and divide them by the average readings at 4 h to determine the fold changes in parasite growth over time.
15. Plot the data using graphing software.
16. To calculate doubling time, plot the log2 values of fold changes at the individual time points over the incubation time. Use a linear regression function to calculate the slope, which represents the doubling time of each strain.

Table 1: Microplate reader settings for luciferase activity measurement during luciferase-based Toxoplasma growth assay