Nunc 24 well plate

The titers of infectious ASFV in serum and clarified spleen suspensions kept in the insect incubator at 27 °C and in the Thermomixer in the laboratory 27 °C were determined by end-point titration in PPAM as described above.
The presence of infectious virus in six selected T. molitor homogenate samples from study T2 containing ASFV DNA (with Cq values from 29.8 to 32.8) was determined using PPAM. The cells were maintained in MEM with 5% fetal bovine serum (FBS) and seeded in 24 well NUNC 24-well plates (Thermo Fisher Scientific). Larval homogenate (100 µL) was mixed with 100 µL MEM with 10% FBS, streptomycin (Sigma-Aldrich), neomycin (Sigma-Aldrich), amphotericin (Sigma-Aldrich) and benzylpenicillin (Sigma-Aldrich) and inoculated onto 1 mL cells (1,600,000 cells/mL). The inoculum was removed after one hour incubation at 37 °C (in 5% CO₂) and the cells were washed twice using 1x PBS. Following washing, 1 mL MEM containing 5% FBS, streptomycin, neomycin, amphotericin (Sigma-Aldrich) and benzylpenicillin was added to the cells.
After three days of incubation at 37 °C (in 5% CO₂), the cells were harvested by freezing and 100 µL of the 1st passage material was inoculated onto 1 mL fresh PPAM. After three days of incubation, virus-infected cells were identified using the IPMA as described above (Section 2.1.1).

The T84 cell assay was performed as described previously [15 (link)]. Briefly, T84 cells (ATCC, Rockville, MD, USA) were seeded and grown to confluence on Nunc 24-well plates (Thermo Fisher Scientific) in Gibco™ Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 ((DMEM/F-12) (Thermo Fisher Scientific)), supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 0.2% gentamicin (Lonza, Basel, Switzerland). Cells were washed thrice with 500 µL DMEM/-F-12, and pre-incubated with 200 µL DMEM/F-12 containing 1 mM 3-isobutyl-1-methylxanthine (Sigma–Aldrich) for 10 min at 37 °C. A volume of 200 µL of sample was added to each well and incubated for 60 min at 37 °C. Following incubation, the reaction medium was aspirated, and cells were lysed with 0.1 M HCl at 20 °C for 20 min. The lysates were centrifuged at 16,000× g for 10 min and supernatants were collected for analysis. Levels of cGMP were determined using a cGMP enzyme immunoassay kit (Enzo Life Sciences, Inc, Farmingdale, NY, USA) according to the manufacturer's instructions. STh purified from ETEC was used as a control.

Samples were observed under a light microscope (Leica Microsystems GmbH, Germany) to isolate Gambierdiscus and Fukuyoa cells by the capillary method [86 ]. Each dinoflagellate cell was placed individually in one well of an untreated Nunc 24 well plate (Thermo Fisher Scientific) with 1 mL of modified ES medium [87 ]. The culture medium was constituted with seawater from L’Ametlla de Mar (Spain), Mediterranean Sea (40.8465°; 0.772432°), which was aged for two months in the dark and was filtered through an activated carbon filter of PTFE (Thermo Fisher Scientific) and after through a 0.22 µm cellulose acetate filter (Merck KGaA, Germany). The salinity was adjusted to 36 with Milli-Q water. After 2–3 weeks, fifty-two cultures achieved at least 20 cells mL⁻¹ and cells were transferred to fresh medium for maintenance in 28 mL round-bottom glass tubes (Thermo Fisher Scientific). Cells were cultured at 24 ± 1 °C, with a photon irradiance of 100 μmol photons m⁻² s⁻¹ and 12:12 light:dark cycle. Light was provided by fluorescent tubes with white light. Irradiance was measured by QSL-2100 Radiometer (Biospherical Instruments, San Diego, CA, USA).