Reagent Details

Imaging information

For imaging at the 5-HT1A receptor use solutions up to 200 nM.

Pharmacological Validation

The CA200986 ligand was shown to antagonize the activity of the 5‑HT1A selective agonist, 8-OH-DPAT, in the ChanTest CHO cell line expressing the human 5-HT1A receptor, using the Ca⁺⁺ sensitive fluorescent indicator, Fluo4-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 0.1% bovine serum albumin, 0.5mM Brilliant Black BN, 2.5mM probenecid, 0.023% pluronic acid F-127 and 2.5µg/ml Fluo4‑AM at 37ºC for 30 – 45 mins, either in the presence of CA200986 or a DMSO vehicle control. During this time serial dilutions of the 5‑HT1A agonist, 8-OH-DPAT, were prepared in HEPES buffered saline containing 0.1% bovine serum albumin, 2.5mM probenecid and 0.5mM Brilliant Black BN.

To determine the antagonist activity of CA200986, serial dilutions of the agonist was added to wells with or without CA200986. The agonist Ca⁺⁺ response in the absence or presence of CA200986 was determined on a Molecular Devices FlexStation, recording the Fluo4 fluorescence intensity (excitation = 485 nm, emission = 520 nm).

The apparent K_D was calculated from the rightward shift of the agonist response curve in the presence of CA200986, compared to the response curve for the agonist alone.

The cell line used to generate the image above was a CHO-K1 line expressing the Human 5-HT1A receptor, supplied by ChanTest, Cat.#: A601.