Tc 100 insect medium

BSR-T7/5 cells are a clone of baby hamster kidney-21 cells that constitutively express T7 polymerase, and were used with permission from Ulla Buchholz, Department of Clinical Virology, Federal Research Centre for Virus Diseases of Animals, Tubingen, Germany [65 (link)]. The cells were cultured in Eagle’s minimum essential medium supplemented with Earle’s Balanced Salt Solution; 2 mM L-Glutamine (HyClone™, Logan, UT, USA), 1% non-essential amino acids (Sigma-Aldrich^®, Merck, Darmstadt, Germany), 5% foetal bovine serum (FBS, Gibco^®, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin and streptomycin (Lonza, Basel, Switzerland). Geneticin (Thermo Fisher Scientific, Waltham, MA, USA) was added every third passage at 1 mg/mL. Cells were grown at 37 °C in the presence of 5% CO₂. KC cells (derived from Culicoides variipennis) were obtained from the Onderstepoort Veterinary Institute (Pretoria, South Africa) and cultured at 28 °C in TC-100 insect medium (Lonza, Basel, Switzerland) with nonessential amino acids supplemented with 10% FBS (Gibco^®, Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin and streptomycin (Lonza, Basel, Switzerland), and antifungals (Fungizone, Sigma-Aldrich^®, Merck, Darmstadt, Germany).

Spodoptera frugiperda (Sf9) cells were maintained at 28 °C, suspended in TC-100 insect medium (Lonza, Basel, Switzerland) with nonessential amino acids supplemented with 10% (v/v) foetal calf serum and penicillin/streptomycin (Lonza, Basel, Switzerland), and fungizone (Sigma-Aldrich^®, Merck, Darmstadt, Germany). Recombinant baculoviruses were generated using the Bac-to-Bac^® Baculovirus expression system (Thermo Fisher Scientific, Waltham, MA, USA) and were propagated in Sf9 insect cells according to the manufacturer’s instructions.