Hi All,
I am currently trying to generate stable cell lines that contain the promega glosensor plasmid in cells already expressing Opioid Receptors (D, K & M). The principle should be that when a drug is applied, cAMP levels rise and luciferase is generated. However I am having some technical issues due to the nature of my lab and experience.
Currently I have tried to follow promegas protocol as best I can albeit with my cell lines generated, however I have had to use my own media with HEPES added for when the cells are out of the incubator for 2 hours during the incubation with the promega cAMP buffer, and my lab has no white cell culture plates for the luminosity reader. Therefore I am having to lyse the cells at the end and transfer them to a reusable plate to be read. However when trialling this today (and following the rest of the promega protocol as is) none of the cells read as anything other than background.
Therefore my question is two-fold. 1) Is there any glaring issues in what I am doing? and 2) Does anyone have any experience with this glosensor assay and have any troubleshooting tips that they might be able to offer?
Regards,
Luke
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