Hek lucia rig 1

HEK-293T cells (cat. number 103, NIH AIDS Research and Reference Reagent Program) and Lucia luciferase reporter HEK-293 cells expressing human RIG-I, HEK-Lucia™ RIG-I, (hkl-hrigi, InvivoGen, San Diego, CA, USA) were grown at 37 °C in a humidified atmosphere with 5% CO₂ in DMEM (Lonza, Verviers, Belgium) supplemented with 10% foetal calf serum (FCS) (Lonza), 1% L-glutamine and 1% penicillin–streptomycin (Lonza, Basel, Switzerland). The cells were harvested and passaged every 3 days using trypsin-EDTA (L0930-100) (Biowest, Lakewood Ranch, FL, USA) or Versene 1× (15040-066; Gibco Chemicals, Thermo Fisher Scientific, MA, USA). HEK-Lucia™ RIG-I cells were cultured to 50–70% confluence in fresh supplemented DMEM 24 h before cell transfection with viral or human DNA constructs and before the induction of interferon (IFN) production. To maintain the stable expression of RIG-I and the luciferase reporter downstream of tandem interferon-stimulated gene 54 (ISG54) promoter elements, 30 μg/mL blasticidin and 100 μg/mL Zeocin™ were added to the HEK-Lucia™ RIG-I reporter cell line. Mycoplasma-free (Mycozap antibiotics, Lonza) or 100 μg/mL Normocin™ was routinely added to each HEK-293T and HEK-Lucia™ RIG-I cell split. Cell viability was quantitatively determined by light microscopic quantitation visualizing trypan blue-stained cells under each experimental condition.