A dual-luciferase reporter assay was performed to verify the expression of FLUC and RLUC in transfected SCC VII tumor cells. Cells (1 × 106) were lysed in 200 μl of passive lysis buffer (Promega, Madison, WI) by gently shaking for 10 min at room temperature, and the whole cell lysate was centrifuged at 10, 000 rpm for 5 min. The cleared supernatant of 20 μl was mixed with 100 μl of LARII solution (Promega) and measured for 10 seconds on a GloMax-20/20-luminometer (Promega). Similarly, a 20 μl aliquot of lysate was mixed with 1 μg of coelenterazine in 100 μl of PBS for RLUC signal measurement by luminometer. The total protein content of each sample was used to normalize the results. The cells were also tested for the ARE-FLUC response by inducing with the known Nrf2-activator tert-butylhydroquinone (TBHQ) and with RRx-001 in various concentrations at different time points.
Larii solution
Manufactured by Promega
The LARII solution is a reagent used in molecular biology and biochemistry laboratories. It is designed to lyse cells and release their contents, including proteins, nucleic acids, and other cellular components. The LARII solution provides a fast and efficient way to extract and prepare samples for various analysis and experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Glomax 20 20 luminometer, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Glomax bioluminescence detector, by Promega (1 mentions)
Pmirglo reporter plasmid, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
2 protocols using larii solution
1
Dual-luciferase reporter assay for FLUC and RLUC expression in SCC VII cells
2
Investigating miR-4428 Regulation of FUT2 in Chondrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mRNA 3′UTR of FUT2 was amplified and sub-cloned into the pMIR-GLO reporter plasmid (Promega Corporation), and the resulting plasmids were named FUT2-WT and FUT2-MUT. Chondrocytes were cultured in a 96-well plate and co-transfected with miR-4428 mimics/NC and FUT2-WT/FUT2-MUT using Lipofectamine 2000 (Life Technologies; Thermo Fisher Scientific, Inc.). After 48 h, luciferase activity was detected using a dual-luciferase reporter assay system (Promega Corporation). The 20 µl cell lysate was added to the luminescent plate. Background values were read with a GloMax bioluminescence detector (Promega Corporation) and 100 µl LARII solution (Promega Corporation) were added to each sample. After reading, add 100 µl Stop&GloR Reagent (Promega Corporation) was added to each sample. Normalization was carried out according to Δ activity multiple=(F/R) sample (/F/R) for comparison (F, Firefly luciferase; R, Renilla luciferase).
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!