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�� Six fish were infected with A. salmonicida (injected i.p with 1?��?108 colony forming units of live bacteria in PBS, overnight culture) and placed in the tank. All habitants (shedders) died at day 6�C8. Sampling was conducted 0, 5, 8, 10, 14, and 21?days after the shedders were added to the tank. Zero-day samples were controls. The fish started to die at day 14, about 1?week after the shedders died. After 41?days, ~15% of the fish were dead. The fish died from furunculosis as the bacterial subcultures made from kidney samples were verified as A. salmonicida, and no other bacterial species were detected after plating and inoculation Selleckchem Panobinostat from the head kidney samples. Skin, spleen, head kidney, and intestine were collected from four fishes at each time-point. learn more All organs were rapidly transferred to and kept in RNA-later (Ambion, Austin, TX, USA) and subsequently processed for RNA isolation, cDNA, and qPCR as described in Section ��Tissue Specific Expression of AsT-bet by Real-Time PCR (qPCR).�� Primary cell culture and stimulation of spleen leukocytes Three fish (1�C1.5?kg) were anesthetized with benzocaine, the spleen was aseptically removed and placed in universal tubes containing Leibowitz medium (L-15) containing 100?U/ml penicillin, 100?��g/ml streptomycin, 0.5% FBS, and 40?U/ml heparin (referred herein as incomplete medium, L-15i). The tissue was gently pushed through sterile 100-��m mesh screens and the screens were rinsed with L-15i. The resulting cell suspension was layered on 25/54% Percoll gradient (GE Healthcare, Oslo, Norway) and centrifuged at 400?��?g for 20?min at 4��C. Cells RHOBTB1 at the 25/54% density interface were collected, washed twice in L-15i by centrifugation at 400?��?g for 10?min at 4��C, and the leukocytes were then re-suspended in complete medium (the same as incomplete medium but with 5% FBS) at 2?��?106?cells/ml. One milliliter of cells were seeded in 12 well tissue culture plates and incubated at 14��C for 24, 48, and 72?h for each stimulant or combination of stimulants or no stimulants, i.e., only media control. The leukocytes were stimulated with known mitogens for T-cell stimulation, i.e., PHA (10?��g/ml)?+?ConA (10?��g/ml)?+?recombinant human IL-2 (rhIL-2) (1?ng/ml), and with recombinant Atlantic salmon IFN-��2 at two dose levels: 5?ng/ml and 0.5?��g/ml. Data analysis The log-transformed data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey��s test using SPSS 19.0 software. Differences were considered statistically significant when P?
