Firefly luciferase substrate

PC6–3 and HeLa cells transfected and kept under 660 nm (0.5 mW cm⁻²) or 780 nm (0.5 mW cm⁻²) light as described above were then lysed in 100 μl of lysis buffer (20 mM Tris-HCl, 10% glycerol, 0.1% Triton X-100, 1 mM PMSF, 0.1% β-mercaptoethanol, pH 8.0) for 30 min at room temperature on swinging platform shaker. Luciferase assay was performed in 96-well half-area white plates (Costar) by mixing 10 μl of cell lysate with 20 μl of firefly luciferase substrate (Nanolight Technology). Bioluminescence was measured immediately with a Victor X3 multilabel plate reader (Perkin Elmer), and data were analyzed with an OriginPro v. 8.6 software.

HEK-293T and HeLa cells were seeded in 96-well plates and when 70−80 % confluent were transfected with either the NF-KB-Luciferase reporter plasmid (pNF-KB-Luc) or the IFN-stimulated response element-Luciferase reporter plasmid (pISRE-Luc) and a plasmid constitutively expressing Renilla Luciferase (pTK-RL). Following stimulation, cells were lysed in passive lysis buffer (Promega) and Firefly luciferase and Renilla luciferase were measured with Firefly luciferase substrate and Renilla luciferase substrate (Nanolight Technology) using a FLUOstar luminometer (BMG). The Firefly Luciferase activity was normalised to the Renilla Luciferase activity first, and then data were normalised to non-stimulated EV group. At least three independent (technical replicates) measurements were taken per condition per experiment.