Biolux gaussia luciferase reagent

To evaluate the effects of the different viral NS1 proteins on host protein synthesis, human 293T cells (24-well plate format, 2.5 × 10⁵ cells/well, triplicates) were transiently co-transfected, using LPF2000, with 1 μg/well of the indicated pCAGGS-HA COOH NS1 expression plasmids or an empty plasmid as control, together with 25 ng/well of pCAGGS plasmid expressing Gaussia luciferase (Gluc) (Capul and de la Torre, 2008 (link)). At 24 h post-transfection, Gluc activity was quantified from tissue culture supernatants using a Biolux Gaussia luciferase reagent (New England Bio-Labs) and a Lumicount luminometer (PacKard). The mean values and standard deviations (SD) were calculated using Microsoft Excel software.

To evaluate the effect of NS1 proteins on inhibition of host gene expression, HEK293T or DF-1 cells (24-well plate format, 2.5 × 10⁵cells/well, triplicates) were transiently co-transfected in suspension, using Lipofectamine2000 (Invitrogen), with 1 μg/well of pCAGGS-HA-NH2 NS1 protein expression plasmids, or an empty pCAGGS-HA-NH2 as internal control, together with 25 ng/well of pCAGGS plasmids expressing Gaussia luciferase (Gluc) (Capul and de la Torre, 2008 (link)) and the green fluorescent protein (GFP) (Kochs et al., 2007 (link)) and placed at 37°C. At 12 h post-transfection (hpt), the medium was replaced and cells were incubated 24 h at 37°C. Subsequently, cells were analyzed for GFP expression and photographed using a fluorescent microscope (Olympus IX81) and camera (QIMAGING, Retiga 2000R). Gluc activity was measured from the tissue culture supernatants (TCS) using a Biolux Gaussia luciferase reagent (New England Bio-Labs) and quantified with a Lumicount luminometer (Packard). The mean values and standard deviations (SDs) were calculated using Microsoft Excel software. Transfected cells were collected and cell lysates were prepared to evaluate levels of protein expression by Western blot assays.