Invasion and Egress Assays for Parasites

Invasion assays were performed as previously described (69 (link)). Briefly, parasites were syringe-lysed through a 27.5-gauge needle, resuspended in Endo buffer and settled onto coverslips with HFF monolayers for 20 min. Endo buffer was then replaced with warm D1 media (DMEM, 20 mM HEPES, 1% fetal bovine serum) and incubated at 37°C for 10 min. Coverslips were fixed and blocked (3% bovine serum albumin [BSA] in PBS), and extracellular parasites were stained with anti-SAG1 antibodies. The samples were then permeabilized (3% BSA, 0.2% Triton X-100 in PBS), and all parasites were stained with the anti-F₁β ATPase antibody and incubated with appropriate secondary antibodies. Parasites were scored as invaded (SAG1–, F₁β ATPase+) or not (SAG1+, F₁β ATPase+) by fluorescence microscopy. These assays were performed in triplicate; at least 10 fields were counted for each replicate with a total of >500 individual parasites per replicate. Significance was determined using multiple two-tailed t tests and the Mann-Whitney test.
For egress assays, parasites were grown on a monolayer on coverslips for 24 to 36 h until most vacuoles contained 16 or 32 parasites. Coverslips were washed twice with prewarmed PBS and incubated with A23187 (or DMSO control) diluted in PBS at 37°C for 2 min. Coverslips were then fixed and stained with rabbit anti-IMC12 antibody. Three replicate coverslips were performed, with at least 10 fields counted for a total of ~200 vacuoles for each parasite strain. Significance was determined using two-way ANOVA of SD-centered means and Tukey’s multiple-comparison test.