Chicken Cell Line Culture

The chicken macrophage cell line HD11 (Klasing et al., 1987) and chicken T-cell line transformed by reticuloendotheliosis virus type T (REV-T) CU91 (Schat et al., 1992; Weinstock et al., 1989) were maintained in complete Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific) containing 100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific) in a humidified incubator with 5% CO₂ at 41°C. For exosome purification, HD11 cells (1.0 × 10⁷) were seeded in three 100-mm cell culture dishes (SPL Life Sciences, Pocheon, Korea) in complete RPMI 1640 medium. The next day, the medium was replaced with exosome-depleted fresh RPMI 1640 medium containing 100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% exosome-depleted fetal bovine serum (#EXO-FBSHI-250A-1; System Bioscience, Palo Alto, CA) with or without 50 µg/mL poly(I:C) (#P1530; Sigma-Aldrich) and incubated for 12 h. The cell culture supernatant was collected for exosome purification.