Ccl 91

From each sampled fish a skin section, and a pool of head kidney, spleen, heart and brain were homogenized 1:10 in L-15 media supplemented with 1 mM L-glutamine, 10% fetal bovine serum, 1% antibiotic-antimycotic solution (Gibco, Life Technologies, Paisley, GB), 5% glutamax, 0.16% Trizma base solution (Sigma, Poole, GB) and 0.48% sodium bicarbonate. clarified by centrifugation for 10 min at 2500g and inoculated onto four different fish cell lines: chinook salmon embryo (CHSE-214) (ATCC^®: CRL-2872^™); a salmonid cell line derived from head kidney (TO) [16 (link)]; epithelioma papulosum cyprini (EPC) (ATCC^®: CRL-2872^™); and bluegill Lepomis macrochirus fry (BF-2) (ATCC^®: CCL-91^™; [17 (link)]).
Prior to inoculating the cells, a fraction of each sample was pre-treated with infectious pancreatic necrosis virus(IPNV) neutralizing antisera (Polyclonal goat anti-Serotype Sp IPNV serum; Harlan Sera-lab). Then, each sample, with and without IPNV antisera, was inoculated at 1/10 and 1/100 and incubated at 15°C for 7 days with regular observation for development of cytopathic effects (CPE). Monolayers were blind passaged onto fresh cells and observed for a further seven days.
Samples developing CPE were subjected to an IPNV Ag ELISA test (TestLine Clinical Diagnostics, Czech Republic).

A NCTC1469 cell line (normal mouse hepatocytes) and a human embryonic kidney HEK293T cell line were purchased from American Type Culture Collection (CCL9.1, ATCC, Manassas, VA). Cells were routinely cultured in DMEM medium. Upon cell confluence reaching 90%, the cell monolayer was detached with 1 mL of 0.25% trypsin for 3–4 min. Then 3 mL of complete medium was added to the cells to terminate the digestion and suspend the cells. Cells were incubated for 24 h with or without palmitic acid (PA) for following experiment. The presence of PA could induce lipid accumulation in hepatocytes.
ADSCs were transduced with lentiviral vectors containing miR-223-3p mimic or miR-223-3p inhibitor or the corresponding negative control (NC), referred to as LV-miR-223-3p mimic, LV-miR-223-3p inhibitor, LV-NC and LV-inhibitor (Table S1). One day prior to transduction, ADSCs were seeded into 75 cm² culture flasks at a concentration of 1 × 10⁶ cells/mL. When the cell fusion reached 50% – 70%, the ADSCs in flask were cultured with the above-mentioned LVs (purchased from GenePharma, Suzhou, Jiangsu, China). After stably transduced cell lines were obtained, ADSC-EVs were extracted. NCTC1469 cells were transduced with LV-sh-E2F1 or LV-oe-E2F1 before co-culture with EVs isolated from the ADSCs transduced with LV-miR-223-3p mimic or LV-miR-223-3p inhibitor.