The cancer-related pathway reporters and the Gaussia luciferase assay were previously described [81 (link)–83 (link)]. The tested cancer-related pathways included NFAT, HIF-1, TCF/LEF, E2F/DP1, ELK1/SRF, AP1, NFκB, SMAD, STAT1/2, RBP-JK, CREB, MYC/MAX pathway reporters. Briefly, exponentially growing SKOV3CR cells were seeded in 60mm cell culture dishes and transfected with 5.0 μg reporter plasmid DNA/dish of each reporter plasmid by using the PEI transfection reagent (Polysciences, Warrington, PA). At 24 h after transfection, the cells were replated into 24-well cell culture plates, and treated with various concentrations of MBZ. At 24 and/or 48 h after treatment, culture media were taken for Gaussia luciferase assays using the Gaussia Luciferase Assay Kit (GeneCopoeia, Rockville, MD). Each assay condition was done in triplicate.
Gaussia luciferase assay kit
Manufactured by GeneCopoeia
The Gaussia Luciferase Assay Kit is a laboratory tool used to measure the activity of the Gaussia luciferase reporter gene. Gaussia luciferase is a secreted bioluminescent reporter enzyme that can be detected in cell culture media or other biological samples. The assay kit provides the necessary reagents and protocol to quantify Gaussia luciferase activity.
Automatically generated - may contain errors
4 protocols using gaussia luciferase assay kit
1
Pathway Reporters and Gaussia Luciferase Assay
2
Profiling Cancer Pathway Reporters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The transfection of the 12 cancer-associated pathway reporters and the Gaussia luciferase assay were performed as previously described.40 ,54 (link),55 (link) These cancer-associated pathways included NFAT, HIF-1, TCF/LEF, E2F/DP1, ELK1/SRF, AP1, NFκB, SMAD, STAT1/2, RBP-JK, CREB, MYC/MAX pathway reporters. Briefly, exponentially growing SKOV3CR cells were seeded in 60-mm cell culture dishes and transfected with 5 μg reporter plasmid DNA per dish of each reporter plasmid by using the PEI transfection reagent (Polysciences, Warrington, PA) as described.56 (link), 57 (link), 58 (link) At 18 h post-transfection, the transfected cells were replated into 24-well cell culture plates and treated with niclosamide at various concentrations or DMSO. At 24 h and/or 48 h after treatment, culture media were taken for Gaussia luciferase assays using the Gaussia Luciferase Assay Kit (GeneCopoeia, Rockville, MD) as previously described.59 (link), 60 (link), 61 (link), 62 (link) Each conditioned assay was done in triplicate.
3
Lgr5 Promoter Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells were co-transfected in 12 well plates with an Lgr5 promoter–reporter construct and secreted alkaline phosphatase (SEAP) plasmid as normalization control constructs using lipofectamine 2000. After 16 h, fresh media was added along with 1, 5 and 10 mM of sodium butyrate. A total of 50 μL of media was collected after 24 h and luciferase assay was performed using Gaussia Luciferase Assay kit (GeneCopoeia) according to the manufacturer’s instructions.
4
Suramin Inhibition of EBOV Polymerase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell-based assay for inhibitory activity of suramin against EBOV polymerase was performed on the stable EBOV-GLuc-Hyg replicon cell line55 (link). The cells were cultured overnight in Dulbecco’s modified Eagle medium (DMEM, Gibco) containing 100 μg ml−1 hygromycin and 10% fetal bovine serum (FBS, Gibco) at 37 °C, 5% CO2. The medium was replaced with FBS-free substrate before the addition of drugs. The cells were incubated with different concentrations of suramin and further cultured in 96-well plate. After 72 h incubation, a 30 μl volume of supernatant from each well was pipetted after centrifugation and then added to a new 96-well white plate and mixed with same volume of Gluc assay solution (Gaussia Luciferase Assay Kit, GeneCopoeia) immediately. The values of luminescence were measured by Glmax Reader (Promega).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!