For imaging at the 5-HT1A receptor use solutions up to 200 nM.
The CA200992 ligand was shown to antagonize the activity of the 5‑HT1A agonist, 5-HT, in the ChanTest CHO cell line expressing the human 5-HT1A receptor, using the Ca++ sensitive fluorescent indicator, Fura-2 AM.
Cells were plated in clear bottom black 96-well tissue culture plates and grown to confluence for 24 hours. Medium was then removed and cells incubated in loading buffer comprising HEPES buffered saline with 0.5mM Brilliant Black BN, 2.5mM probenecid, 0.023% pluronic acid F-127 and 2.5µg/ml Fura-2 AM at 37ºC for 30 – 45 mins, either in the presence of CA200992 or a DMSO vehicle control. During this time serial dilutions of the 5‑HT1A agonist, 5-HT, were prepared in HEPES buffered saline containing 2.5mM probenecid and 0.5mM Brilliant Black BN.
To determine the antagonist activity of CA200992, serial dilutions of the agonist was added to wells with or without CA200992. The agonist Ca++ response in the absence or presence of CA200992 was determined on a Molecular Devices FlexStation by exciting at 340 nm and 380 nm and ratioing the fluorescence intensity of Fura-2 signal collected at 320 nm.
The apparent KD was calculated from the rightward shift of the agonist response curve in the presence of CA200992, compared to the response curve for the agonist alone.
|The cell line used to generate the images above was a CHO-K1 line expressing the Human 5-HT1A receptor, supplied by ChanTest, Cat.#: A601.