Amphotericin b

The porcine intestinal organoids were kindly provided by Professor Xiaoliang Li at Zhejiang University. The cryopreserved organoids were placed in a 37 °C water bath for 2 min and then contents were transferred into DMEM/F-12 (ThermoFisher, Waltham, MA, USA) with 1% BSA (Biofroxx, Hesse, Germany). The suspensions were centrifuged at 200× g for 5 min to obtain the cell pellets, which were further resuspended in 100 μL complete organoid culture medium (STEMCELL Technologies, Vancouver, BC, Canada) and seeded in 100 μL Matrigel (BD Bioscience, Franklin, NJ, USA) on a prewarmed 24-well culture plate (Corning, Coring, NY, USA). Matrigel was incubated for 15 min at 37 °C to polymerize. Finally, 500 μL complete organoid culture medium supplemented with 1% antibiotics-antimycotic (10,000 IU/mL Penicillin + 10,000 μg/mL Streptomycin + 25 μg/mL Amphotericin B) (Beyotime, Shanghai, China) was added to every well. Intestinal organoids were cultured at 37 °C in a 5% CO₂ chamber. Culture medium was replaced every 2 days and the organoids were passaged every 5 or 7 days based on the growth of organoids. By dissociating the organoids with gentle cell dissociation reagent (GCDR) (STEMCELL Technologies, Vancouver, BC, Canada) on a shaker at 37 °C for 15 min, cells were obtained for seeding in fresh Matrigel. All the experiments were started after at least two passages.

Porcine alveolar macrophages (PAMs) were prepared from bronchoalveolar lavage and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Sigma, United States), 100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B (Beyotime Biotechnology, China) at 37°C with 5% CO₂. ASFV (CSTR: 16533.06. IVCAS 6.7494, genotype II) was stored at −80°C in the biosafety level 3 (BSL-3) facility of Wuhan Institute of Virology, Chinese Academy of Sciences (WIV-CAS). All the experiments involving infectious ASFV were performed in the BSL-3 laboratory. The titer of ASFV stocks were determined by the hemadsorbing (HAD) test. Briefly, 4 × 10⁴ cells/well of PAMs were seeded into 96-well plates and infected with 10-fold diluted ASFVs. After 1-day infection, 1% porcine erythrocyte cell suspensions stored in PBS (Gibco, United States) were added into each well. The phenomena of hemadsorption were observed over 7 days by a microscope. The 50% hemadsorbing dose (HAD₅₀) was calculated by the Reed and Muench method (Zhao et al., 2019 (link)).