Isolation and Culture of Fish Leukocytes

Cells from DI and HK were harvested and grown at 12°C in Leibovitz’s L-15 Medium (L-15; Sigma, Oslo, Norway) as described previously by Park, Zhang (8 (link)). Briefly, the isolated DI or HK leukocytes were allowed to adhere on a cell culture dish (Nunc EasYDish, Thermo Fisher Scientific, Oslo, Norway) with 2 mL L-15+ (L-15 medium with 50 U/mL penicillin, 50 μg/mL streptomycin, 2% fetal bovine serum and 10 U/mL heparin) for 2 days at 12°C. Thereafter the medium was removed and the adherent cells on the culture dish were detached by washing three times with 1.5 mL ice-cold PBS (Sigma) supplemented with 5 mM EDTA (Sigma). The cells were centrifuged (500 × g, 5 min, 4°C) and re-suspended with 2 mL L-15+. Then, the adherent intestinal cells or adherent head kidney cells were counted using a portable cell counter (Scepter™ 2.0 cell counter, EMD Millipore, Darmstadt, Germany) for further analysis.