The complexity of cancer systems biology requires innovative tools for interrogating the signaling pathways responsible for oncological transformation. Select each step in the workflow diagram to see how Promega integrated tools for reporter gene analysis work together to assure biologically relevant results in cancer research.
FuGENE® HD: Low-Toxicity, Efficient Transfection of Many Cell Types.
Efficient transfection is necessary to accurately measure small biological changes, and these changes should not be masked by toxic effects from the transfection reagent. FuGENE® HD allows transfection of optimized reporter genes into many model systems with minimal impact on physiology.
Comparison of Assay performance with Different Transfection Reagents
BaF3 murine pro-B cell lymphocytes were transfected with pGL4.32Luc2P/NFκB-RE/Hygro vector (Cat.# E849A) using the indicated transfection reagent and previously optimized conditions, and following the manufacturer's recommended protocol. Cells were transfected in a T75 flask at a density of 150,000 cells/ml. After overnight transfection, cells were harvested, counted, and resuspended in fresh growth media before plating in 96-well assay plates (90μl/well; ~90,000 cells/well). Cells were then stimulated for five hours with 10μl of 10X murine TNFα. The ONE-Glo™+Tox Luciferase Reporter and Cell Viability Assay was used according to the recommended protocol to assess reporter gene activation and to monitor cell health.
ONE-Glo™+Tox Assay: Sensitive Detection of Reporter Activity and Cell Viability in a Single Well
Luciferase activity can be detected at very low expression levels. This sensitivity and ease of detection make luciferase-based assays highly versatile and scalable for different uses.
- More Data: Measure cell viability and firefly luciferase gene expression in the same assay well.
- Better Biology: Understand reporter gene expression in the context of cell viability.
- Easy Assay Format: Simply add reagent, mix and read.
- Flexible and Automation-Friendly: Scale volumes to meet your throughput needs.
NF-κB inhibitor profiling studies in low-volume 384 format. TNF-α was applied for 6 hours to HEK 293 cells expressing firefly luciferase under control of the NF-κB response element. Optimal concentration of TNF-α (~EC80) was determined to be 25 ng/mL .NF-κB inhibitor profiles show dose dependent effects on NF-κB reporter gene expression, with different cytotoxicity profiles and treatment windows, following a 1 hour pre-incubation with compound.