cAMP measurement was performed in NG108-15 cells transiently expressing 5-HT6R using the BRET sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc) (41). NG108-15 cells were cotransfected in suspension with the 5-HT6R and CAMYEL constructs using Lipofectamine 2000 according to the manufacturer’s protocol and plated in white 96-well plates (Greiner) at a density of 80,000 cells per well. Twenty-four hours after transfection, the cells were washed with PBS containing calcium chloride and magnesium chloride (Gibco, ref 14040-091). Coelenterazine h was added at a final concentration of 5 μM. Plates were incubated at room temperature for 5 min before BRET measurements. BRET was measured using a Mithras LB 940 plate reader. Expression of 5-HT6R in NG108-15 cells induced a decrease in the CAMYEL BRET signal compared to that in cells transfected with an empty vector. This decrease in CAMYEL BRET signal was used as an index of 5-HT6R constitutive activity through Gs signaling.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.