I am working with Novus Biological HEK293_luciferase, in which the luciferase gene is driven by STAT3 . The cells were reporting robustly with IL-6, but lost the ability to report from one experiment to another , in a weeks time. The IL-6 was new, frozen down in 100ug/mL aliquots with minimal or no freeze thaw, and it is the same batch that worked one day and not a week later. The cells are kept under selective pressure with puromycin, and look robust and proliferate well. The luciferase assay system is new and also worked before. All I have now is background luminescence, 4 times in a row, I even used one of the original stock of cells with minimal passage...
Any idea on what could be going wrong?
Its hard to predict anything but could be due to following reasons.
1. Selective pressure due to Puromycin. Is there any specific reasons cells are selected using Puromycin. Try working with cells without selection.
2. Is it a stable cell line transfected with STAT3-Luciferase. Sometimes over the passage there is dilution and cells start loosing the stable transfection.
3. How about using the HEK293 cells and doing transient transfection with double luciferase Firefly driven by STAT3 and Renilla for normalization. You can also do single transfection with Firefly luciferase and normalize luciferase activity with protein amount.
Sandeep, thanks for the answer, but I bought these cells with stable transfected STAT3 driven luciferase, they have not been passaged more than 10 times. They actually worked on passage 5 but stopped at p7.... Meanwhile I tested the biological activity of the IL-6 and it works as well. so the mystery continues.
Quick update. This cell line is highly unstable, it is now off the market!
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