The rat CP cell line (The European Collection of Authenticated Cell Cultures, London, UK) were used for culturing. CP cells were seeded at a density of 0.8 × 104 cells/cm2 in a 24-well plate coated with type 1 collagen (Corning, Corning, NY, USA). Cells were cultured in DMEM/F-12 (Sigma-Aldrich, Saint Louis, MO, USA), 10% FBS (CCB, NICHIREI BIOSCIENCE, Tokyo, Japan), 10 mg/ml recombinant rat epidermal growth factor protein (R&D Systems), 1% Insulin/Transferrin/Selenium Solution (Thermo Fisher Scientific), and 1% penicillin streptomycin (PS) (Thermo Fisher Scientific) (Battle et al., 2000 (link)). Cells were maintained in a 5% CO2 incubator at 37 °C. At 3 days after seeding when each cultured cell type had reached 90–100% confluence, the conventional medium was replaced with 10% EXO-FBS-50A-1 (System Biosciences, Palo Alto, CA, USA) medium with several concentrations of recombinant rat IFN-γ (0 or 0.5 ng/ml; R&D Systems). At 24 h after incubation, the culture media were collected and the exosomes were isolated using the MagCapture Exosome Isolation Kit PS (FUJIFILM Wako Pure Chemical Corp, Osaka, Japan).
miRNA isolation, cDNA synthesis, and RT-PCR were performed as described above. The relative expression of miR-146a was calculated using 2−ΔΔCt with cel-miR-39 as an external spiked control.
miRNA isolation, cDNA synthesis, and RT-PCR were performed as described above. The relative expression of miR-146a was calculated using 2−ΔΔCt with cel-miR-39 as an external spiked control.