96 well culture plate

Purified supernatants (1 mL) were inoculated on confluent LLC-MK2 cells in T 75 flasks with 9 mL of M199 medium at 37°C. After 1 h of contact, an aliquot of 100 μL was kept and stored frozen. After incubation for 7 days in an incubator at 37°C with gentle agitation, 100 μl was collected and compared to the 1-h control by rRT-PCR. Samples showing an increase of more than three Logs (9 C_T-values) were considered to be positive.
For titration of samples, 1 mL of purified supernatant was serially diluted (10^-1 to 10^-8) in medium and inoculated in 10-well for each dilution onto cells in 96-well culture plates (Thermo Fisher Scientific, France) under a volume of 200 μL, and incubated for 7 days at 37°C. For each experiment two 96-well culture plates were inoculated. CPE counts were converted into TCID₅₀ units using the Reed and Muench calculation method (Reed and Muench, 1938 ) and were expressed per g of DT. A negative control was included on each plate.

The effects of CME on the viability of CNE-1 and LO2 cells were evaluated by MTT assay [20 (link)]. Cells (3 × 10⁴ cells/mL) were seeded into the wells of 96-well culture plates (Thermo Fisher Scientific) in 100 μL of medium per well and then allowed to adhere for 24 h at 37 °C in a 5 % CO₂ atmosphere. After incubation, the cells were treated with the target concentrations of CME (15–50 μg/mL), 0.1 % DMSO as the vehicle control, and cisplatin (4 μg/mL) as a positive control, for 24 h. After incubation for 24, 48, and 72 h at 37 °C in a CO₂ incubator, 10 μL of MTT solution [5 mg/mL in phosphate-buffered saline (PBS)] was added to each well and incubated for a further 4 h. Then, excess medium was removed and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The optical density in each well was measured using a microplate spectrophotometer (BMG POLARstar Galaxy, Offenburg, Germany) at 490 nm. Triplicate experiments were performed for treatment with each concentration. The percentage cell viability (%) was calculated by comparison with a sample’s corresponding control. IC₅₀ values were calculated to evaluate the cytotoxic effects of treatments on CNE-1 cells.

The G2b PEDV-PT strain (GenBank: KY929405) was prepared as previously described [44 (link)]. The 6th passage of PEDV-PT (PEDV-PT-P6) was used as the antigen for the immunostainings in the present study. In brief, to prepare the virus-infected Vero cell plates with visible cytopathic effect (CPE), i.e. the presence of syncytial cells, the Vero cells were seeded onto 96-well culture plates (Thermo Fisher Scientific, Waltham, MA, USA) with a 90% confluence after 18 h. The cells were washed with PBS (Gibco, Gaithersburg, MD, USA) three times and inoculated with 1000 TCID₅₀/mL of PEDV-PT-P6 diluted in post-inoculation (PI) medium, containing DMEM (Gibco), 0.3% tryptose phosphate broth (Sigma, St. Louis, MO, USA), 0.02% yeast extract (Acumedia, Lansing, CA, USA), and 10 μg/mL of trypsin (Gibco). After 24 h of incubation, the CPE was observed, the supernatant was discarded and the PEDV-infected Vero cells with 10% area covered by cytopathic effect (CPE) were fixed with 80% acetone (Sigma) for 20 min. After removing the acetone and air-drying for 1 h, the plates were stored at − 20 °C until use.