Cypridina luciferase assay kits

To evaluate the effect of WT and mutant PB2 and PB1 proteins on viral polymerase activity, a MG assay was performed as previously described (42 (link), 53 (link)). Briefly, human 293T cells (5 × 10⁵ cells/well, 12-well plate format, triplicates) were transiently co-transfected in suspension, using Lipofectamine 2000 (Invitrogen), with 125 ng of each ambisense pDZ plasmids (pDZ-PB2 or PB2_LAIV, -PB1 or PB1_LAIV or -PB1_L319Q or pDZ-PB1_LAIV+L319Q, -PA, - and NP), together with 250 ng of two reporter viral (v)RNA-like expression pPOL-I plasmids encoding GFP or Gluc driven by a human RNA polymerase I promoter (53 (link), 71 (link)). A Cluc-encoding plasmid under the simian virus 40 promoter (SV40-Cluc, 50 ng) was included to normalize transfection efficiencies (72 (link), 73 (link)). Cells transfected in the absence of pDZ NP were used as a negative control. At 6 h p.t., cells were placed at 33°C, 37°C, or 39°C, and viral replication and transcription were evaluated at 48 h p.t. GFP was imaged using a fluorescence microscope, while Gluc and Cluc expression levels were determined using Biolux Gaussia or Cypridina luciferase assay kits (New England BioLabs) and a microplate reader. The mean value and standard deviation (SD) were calculated using Microsoft Excel software.

We transiently transfected three plasmid DNA constructs, Gaussia luciferase construct containing putative ChREBP binding elements, cytomegalovirus promoter-driven Cypridina luciferase construct, and caChREBP construct or empty vector, to 832/13 cells in 96-well plates by Lipofectamine2000 (Life Technologies). We collected culture media at 48 hours after transfection. We then sequentially detected secreted luciferases using Gaussia luciferase and Cypridina luciferase assay kits (New England Biolabs). Luciferase expressed from Gaussia luciferase reporter plasmid is normalized by the activity of Cypridina luciferase.