6 well culture plates

Confluent IPEC-1 cells (2.5 × 10⁵ cells/well) grown in 6-well culture plates (VWR, Vienna, Austria) were infected with 5 × 10³C. suis sporozoites. The next day, cells were incubated with predetermined IC₅₀ (40 nM) or IC₉₅ (200 nM) concentrations of BKI 1369 (see above section on efficacy testing) at 40 °C for five days. Infected cells incubated with 0.001% DMSO served as controls. After 24 h, 72 h, 96 h and 120 h of incubation, medium supernatant was removed, the monolayers were washed twice with phosphate buffer saline (PBS) and fixed with 5% glutaraldehyde (Merck, Darmstadt, Germany) in 0.1 M sodium phosphate buffer (pH 7.2) for 10 min at 4 °C. Cells were carefully collected using a rubber scraper (TPP, Switzerland) and subjected to centrifugation at room temperature for 5 min at 390×g. The supernatant was removed and cells were postfixed with 1% osmium tetroxide (Merck) for 1 h at 4 °C. After dehydration in an alcohol series and propylene oxide (Merck), the cells were embedded in glycidyl ether 100 (Serva, Heidelberg, Germany). The ultra-thin sections were cut on a Leica Ultramicrotome (Leica Ultracut S, Vienna, Austria), stained with uranyl acetate (Sigma-Aldrich, Vienna, Austria) and lead citrate and examined in a Zeiss TEM 900 electron microscope (Carl Zeiss, Oberkochen, Germany) operated at 50 kV.