Plaque Assay and Intracellular Growth Analysis

Plaque assays to compare the transgenic parasites with their parental strains were performed on HFFs cells in 6-well tissue culture plates (Corning costar, Beijing). Briefly, 500 parasites per well were seeded into confluent monolayers and infected cells were maintained in fresh DMEM containing 10% FBS and incubated undisturbed at 37°C in 5% CO₂ for 7 days. To stain the monolayers, media was aspirated and disassociated parasites were washed off using PBS. Cell monolayers were then fixed for 10 minutes in PBS with 4% formaldehyde and stained with crystal violet solution (12.5 g crystal violet dissolved in 125 mL ethanol and mixed with 500 mL 1% ammonium oxalate in water) at room temperature for 10 minutes, washed with deionized water, air dried and visualized by microscopy using image acquisition and plaque area measurement as previously described [42] (link).
To analyze the intracellular growth rate of the transgenic parasites compared with their parental strains, about 1×10⁵ parasites were inoculated on confluent HFFs in 24-well plates. The infected cells continuously incubated for 24 hours. Thereafter, cells were fixed in PBS with 4% formaldehyde, and RH and RHΔKU80 parasites were stained for IFA using rabbit anti-SAG1 polyclonal antibody as described above. TgHMGB1a overexpression and B box-deficient parasites were directly examined for fluorescence (GFP and eGFP, respectively). The numbers of parasites per vacuole for a minimum of 150 randomly chosen vacuoles were counted for each strain using a fluorescence microscope (IX71, Olympus, Japan) at 400× magnification.