Cells were transiently transfected and incubated at 37°C for 24 hours. For hypoxia-induction studies, transfected cells were incubated for 5 hours under standard conditions followed by a 24-hour incubation in anaerobic or hypoxic chambers as indicated. For each construct, three separate wells were transfected in parallel for HEK 293T and MCF7 cells. In case of Jurkat cells, three wells were seeded in parallel originating from the same nucleofected culture. Luciferase activity was determined from diluted samples using the BioLux® Gaussia Luciferase Kit (New England Biolabs) in combination with a Modulus single-tube luminometer (Turner Biosystems, Sunnyvale, CA) or Synergy H1 hybrid plate reader (BioTek, Winooski, VT) with a 5-sec integration time. Background luminescence measured from the supernatant of mock-transfected cells was subtracted from the raw luminescence values. Fold-induction was calculated by dividing the background-subtracted luminescence values of each induced sample by the average value of the three corresponding uninduced samples.
Biolux gaussia luciferase kit
Manufactured by New England Biolabs
The BioLux® Gaussia Luciferase Kit is a laboratory tool that utilizes the bioluminescent properties of the Gaussia luciferase enzyme. This kit provides the necessary components to detect and measure Gaussia luciferase activity in various experimental settings.
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2 protocols using biolux gaussia luciferase kit
1
Transient Transfection Luciferase Assay
2
Evaluating IL-27's Anti-Viral Activity
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IL-27 anti-viral activity was tested against vaccinia virus and mouse cytomegalovirus (MCMV) infection. Resident peritoneal macrophages were treated overnight with 100 ng/ml IL-27 and then infected with a GFP-expressing vaccinia virus for 6hrs and infection was measured by flow cytometry. For MCMV experiments, resident peritoneal macrophages were first treated with 1, 10, or 100 ng/ml of IL-27 or 10 ng/ml of IFNγ for 6hrs. Then, media with the cytokines was removed, and macrophages were infected with MCMV-3DR, a gaussia luciferase-expressing MCMV (kindly provided by Martin Messerle, Hannover Medical School), at moi=0.1. Supernatants from these macrophage cultures were collected at 24, 48, and 72hrs post-infection and used to infect M2-10B4 fibroblasts. Luciferase activity in the supernatant of infected fibroblasts was determined 18 hr post-infection using the BioLux Gaussia Luciferase kit (New England Biolabs) and a Mithras LB 940 plate reader (Berthold).
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