Rtgill w1

Epithelial and fibroblastic cell lines were retrieved from American Type Culture Collection (ATCC), including: Fathead minnow (Pimephales promelas) Epithelioma papulosum cyprini (EPC) (ATCC: CRL-2872); Bluegill (Lepomis macrochirus) fry (BF-2) (ATCC: CCL-91); Rainbow trout (Oncorhynchus mykiss) gonad (RTG-2) (ATCC: CCL-55); and Rainbow trout gill (RTgill-W1) (ATCC: CRL-2523). Cell cultures were maintained in 25 cm² tissue culture flasks (CytoOne) at 20 °C, with L-15 Leibovitz media (HyClone) supplemented respectively with 1% Penicillin-Streptomycin (PS) solution (Corning), and with 2% (L15-2PS) or 10% (L15–10PS) fetal bovine serum (Corning). Before using, media were filtered through a 0.2 μm cellulose nitrate membrane (Nalgene). Confluent cell monolayers were split 1:2 or 1:3 to seed 5 × 10⁵ cells to each well of the 12-wells plate (CytoOne) in L15–10PS and grown for 72 h in standard conditions before each transfection experiment.

Cell line RTgill-W1 [42 (link)] was obtained from the American Type Culture Collection (CRL-2523, ATCC, Manassas, VA, USA). For culturing, gill cells were maintained in the dark at 19 °C in Leibovitz’s L-15 medium (L1518 Sigma) supplemented with an antibiotic–antimycotic solution (A5955, Sigma) containing amphotericin B (25 mg mL⁻¹), streptomycin (10 mg mL⁻¹), and penicillin (10,000 units mL⁻¹), and 10% (v/v) fetal bovine serum (FBS, 12003C, Sigma), in 25 cm² culture-treated flasks. TrypLE™ Express (Gibco™) was used to remove cells from the bottom of the flask. Subcultures were routinely set twice per week at a ratio of 1:2 with L-15 medium renewal.