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
- 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.
- 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.
- 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.
- 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.
- Count parasites using a hemocytometer to determine the concentration.
- 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.
- 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.
- Incubate the microplate at 37 °C and 5% CO2 for 4 h.
- 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).
- Mix equal volumes of PBS and 2x luciferase assay buffer and dilute the luciferase substrate to 12.5 µM.
- 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.
- 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.
- 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.
- 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.
- Plot the data using graphing software.
- 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
| Luciferase: | Endpoint |
| Integration time: | 1 s |
| Filter Set - Emission: | Full light |
| Optics: | Top |
| Gain: | 135 |
| Read speed: | Normal |
| Delay: | 100 ms |
| Read height: | 4.5 mm |
