Trout Spleen Cell Isolation

Rainbow trout were killed by benzocaine (Sigma-Aldrich) overdose and the spleen collected. Single cell suspensions were obtained as previously reported^{18 (link),24 (link)} using 100 µm nylon cell strainers (BD Biosciences) and Leibovitz medium (L-15, Invitrogen) supplemented with 100 I.U./ml penicillin and 100 µg/ml streptomycin (P/S, Life Technologies), 10 units/ml heparin (Sigma) and 5% foetal calf serum (FCS, Life Technologies). Cell suspensions were placed onto 30/51% discontinuous Percoll (GE Healthcare) density gradients and centrifuged at 500 × g for 30 min at 4 ºC. The interface cells were washed twice in L-15 with 2% FCS and cells were resuspended in L-15 with 5% FCS. The viable cell concentration was determined by Trypan blue (Sigma-Aldrich) exclusion, adjusting the concentration to 2 × 10⁶ cells/ml.