Heparin

The fish were divided into two groups (with three replicates in each group), and injected intramuscularly with purified LCDV (300 μL per fish) or the same volume of PBS as negative control. Six fish in each group were randomly sampled at 1, 3, 6, 12 and 36 hpi, and the peripheral blood was drawn from the caudal vein of flounder which were sacrificed via MS-222 (Sigma, St. Louis, MO, USA) overdose, and diluted 1:1 in 65% RPMI-1640 (Gibco, Darmstadt, Germany) containing 20 IU/mL heparin (Sangon Biotech, Shanghai, China). The peripheral blood from LCDV-infected fish and the PBS-control group was used for isolation of PBLs by Percoll density gradient centrifugation as previously described [33 (link)]. Briefly, the cell suspensions were laid over a discontinuous Percoll gradient at densities of 1.020 and 1.070 g/cm³ and centrifuged at 840× g for 30 min at 4 °C. The lymphocytes fraction was collected and washed twice with PBS containing 5% (v/v) newborn calf serum (Hyclone, Logan, UT, USA) at 640× g for 10 min, and then resuspended in PBS for next use.

Protoplast preparation and transformation were performed as described previously [15 (link)] with minor modifications. The mycelia were collected and washed with 0.6 M mannitol (Sangon Biotech) and digested with 2% (w/v) lywallzyme (Guangdong Institute of Microbiology, Guangzhou, China) for 3 h at 30 °C. After filtering insufficiently digested mycelia, the protoplasts were resuspended in MTC buffer (0.6 M mannitol, 100 mM CaCl₂, and 100 mM Tris-HCl at pH 7.5) and adjusted to a final concentration of approximately 10⁷ cells/mL. Then, the protoplasts in 160 μL MTC buffer were transformed with 20 μg linearized pXT-302-FfpyrG-1/3 plasmid, 10 μL 20 mM aurintricarboxylic acid (ATA) (Sigma-Aldrich, Shanghai, China), 5 μL 50 mM spermidine (Sigma-Aldrich), 2 μL 50 mg/mL heparin (Sangon Biotech), and 60 μL PTC buffer (40% polyethylene glycol (PEG) 3350, 100 mM CaCl₂, and 10 mM Tris-HCl at pH 7.5). Subsequently, the mixture was incubated on ice for 30 min, then 1 mL PTC buffer was added and incubated for another 30 min at 25 °C. After centrifugation at 4000 rpm for 10 min, protoplasts were resuspended in 1 mL liquid CYM medium containing 0.6 M mannitol for 24–30 h at 25 °C.