Cultivation of Diverse Mammalian Cell Lines

Mammalian cells used in this study were murine C2C12 cells (muscle, myoblast), hamster BHK-21 cells (kidney, fibroblast), human Huh7 cells (liver, hepatocellular carcinoma), human SVG-A cells (brain, astroglia) and human RD cells (muscle, rhabdomyosarcoma), chosen as we had previously shown that these cells efficiently supported CHIKV replication [14 (link)]. Mammalian cells were maintained in a humidified incubator at 37°C with 5% CO₂ in complete Dulbeccos Modified Eagles Medium (DMEM) (Sigma) supplemented with 10% foetal calf serum (FBS) (Gibco) (or 20% FBS for C2C12 cells), 100 IU penicillin ml⁻¹ and 100 μg ml⁻¹ streptomycin (Gibco), 10% non-essential amino acids (NEAA) (Thermo Fisher Scientific) and 10 mM HEPES (Thermo Fisher Scientific). The mosquito cell line C6/36 (Ae. albopictus, embryonic) used in this study has a mutant Dcr2 gene that results in a defective RNAi response [15 (link)]. These cells were maintained at 28 °C in Leibovitz’s L-15 Medium (Thermo Fisher Scientific) supplemented with 10 % FBS, 100 IU penicillin ml⁻¹ and 100 μg ml⁻¹ streptomycin (Gibco), and 10% tryptose phosphate broth (Thermo Fisher Scientific).