Firefly luciferase assay kit 2

The PEDSV.15 cells seeded in 96-well plates (3 × 10⁴ cells/100 µL/well) were treated with small-molecule compounds of the JAK/STAT Compound Library (Targetmol, Wellesley Hills, MA, USA, cat. No. L3700) at two concentrations (0.5 µM and 5 µM) for approximately one hour prior to stimulation with either LPS (100 ng/mL), pTNF (5 ng/mL) or the medium. After a stimulation period of six hours, the cells were infected with CSFV-luc at a multiplicity of infection (MOI) of 0.1 TCID₅₀/cell, and after 22 h of cultivation, the cell extracts were assayed for firefly luciferase activity (Firefly Luciferase Assay Kit 2.0, Biotium, Fremont, CA, USA) using a Centro LB 960 luminometer (Berthold Technologies, Bad Wildbad, Germany). Average relative luminescence units (RLU) with standard deviations from triplicate values were calculated. The data obtained from cytotoxic or antiviral compounds were eliminated from the analysis (RLU below 50% of non-stimulated infected cultures).

For the measurement of CSFV replicon-mediated firefly luciferase activity, 3 × 10⁶ PEDVS.15 cells were electroporated with 1 μg of replicon T7-RNA in 0.4 mL of ice-cold phosphate-buffered saline (PBS). Electroporations were performed using an ECM^® 830 electroporator (BTX, Holliston, MA, USA) with the following settings: 980V and two pulses of 100 µs pulse length at a 0.1 s interval. After 16 and 24 h, the cells were lysed through the addition of 1× Firefly Luciferase Lysis Buffer (Biotium, Fremont, CA, USA), and cell extracts were assayed for firefly luciferase activity (Firefly Luciferase Assay Kit 2.0, Biotium) using white 96-well microplates (Lumitrac, Greiner Bio-One, Kremsmünster, Austria) and a GloMax^® plate reader (Promega, Madison, WI, USA). To normalize firefly luciferase activity to the efficiency of replicon T7-RNA transfection, replica cultures of electroporated PEDSV.15 cells were subjected to E2 immunostaining and flow cytometry quantification.