Suspension culture flasks

SK cells were cultured in modified Eagle’s medium (MEM), supplemented with 10% (v/v) inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin and 0.05 mg/mL gentamycin (all from Gibco, Thermo Fisher Scientific, Waltham, MA, USA). SK cells grown in monolayers were detached from cell culture flasks (175 cm²) using trypsin, seeded in suspension culture flasks (25 cm², Sarstedt, Nümbrecht, Germany) at 1.2 × 10⁷ cells/8.5 mL, and put on a rocking platform at 37 °C as described before [25 (link)]. SK cells were either directly or after 8 h inoculated with PRV strain Kaplan [27 (link)] at a multiplicity of infection (MOI) of 10 and put back on the rocking platform at 37 °C for 14 h. To ensure that this inoculation procedure led to a 100% infection efficiency, flow cytometric detection of viral glycoproteins gB and gD was performed in every experiment (illustrated in Figure 1A). For the experiment using phosphonoacetic acid (PAA, Sigma-Aldrich, St. Louis, MO, USA, catalog number 284270), SK cells were treated with PAA at a concentration of 400 µg/mL from 30 min prior to infection onwards as described before [28 (link)]. For the experiment using cycloheximide (CHX, Sigma-Aldrich, catalog number C1988), SK cells were treated with CHX at a concentration of 5 µg/mL at 8 h of cultivation.

SK cells were used as target cells in cytolytic assays and were cultivated in Modified Eagle Medium (Life Technologies, Thermo Fisher Scientific) supplemented with 10% (v/v) fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.05 mg/ml gentamycin. SK cells were detached from cell culture flasks (175 cm²) using trypsin, seeded in suspension culture flasks (25 cm², Sarstedt, Nümbrecht, Germany) at 1.2 × 10⁷ cells/8.5 mL, inoculated at a multiplicity of infection of 10, and put on a rocking platform at 37°C for 12 h.

Six month-old, male Texel cross lambs that were raised under helminth-free conditions were infected per os either with 15,000 T. circumcincta (isolate MTci2) larvae (L₃) or 5000 H. contortus (isolate MHco3) L₃. Once a patent infection was detected by the observation of nematodes eggs in faeces (around 21 days post-infection), the lambs were fitted with a collection harness and bag to enable faecal collection. Pelleted faeces were collected from each bag 24 h later and placed in a covered seed tray, which was incubated at room temperature (>15 °C) for 10 days before larval recovery using the modified Baermann technique (Manual of Veterinary Parasitological Laboratory Techniques, 1986 ). The next day L₃ were recovered in ∼250 ml H₂O, number estimation performed then the larvae were stored in 100 ml tap water in 75 cm² surface area vented cap, suspension culture flasks (Sarstedt Ltd UK) at ∼ 5 °C for MTci2 and 8 °C for MHco3, respectively.