2.1. Culture of fin cells

The isolation of fin cells was followed by reported protocol with minor modification. Two healthy S. fuscescens fishes (approximately 15 g in weight) were collected from an aquaculture farm (Shenzhen, China): the fishes were maintained in an aquarium equipped with seawater recirculation system. The fishes were anesthetized with 2-penoxyethanol (1:10,000) and then washed with diluted bleach (1:100), wiped with 70% ethanol, to remove surface contamination. The fishes were decapitated, and the fins were subsequently removed and placed in HBSS medium with antibiotics (penicillin, 100 U/mL; streptomycin, 100 μg/mL; amphotericin-B 0.01 μg/mL) (Thermo Fisher Scientific, Waltham, MA) for washing. Then, the fin fragments were minced into small pieces (approximately 2 mm²) using surgical scissors. Tissue pieces were put into DMEM (FBS free) (Thermo Fisher Scientific) with antibiotics, then 5 mL collagenase A (0.4 mg/mL; Sigma-Aldrich, St Louis, MO) was added. The tissue mixture was kept in an incubator with orbital shaking at 28 °C at speed of 90 rpm for 60 min. The solution was centrifuged and washed with DMEM to remove collagenase. Then, 5 mL of 0.25% trypsin-EDTA solution were added and maintained for 20 min at 37 °C. Three mL FBS (Thermo Fisher Scientific) was added for trypsin neutralization and centrifuged at 200 X g for 10 min. The cell pellet was resuspended in DMEM and filtered with 180 μm-nylon to remove undigested tissues. Then, centrifuged at 200 X g for 10 min. Cell pellets were suspended and seeded into 25-cm² flasks and cultured in 3 mL of DMEM (supplemented with 20% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, amphotericin-B 0.01 μg/mL; pH 7.4) in an incubator at 28 °C with 5% CO₂. Afterwards, the medium was replaced with fresh medium by half every 2–3 days. The subculture was carried out at 1:2 split subsequently by trypsinization when primary cell cultures grew to 90–100% of confluence. The culture was designated RFF cell line.