Assay Method Information

Assay Name:  Antagonism of Humanized OX1 Receptor Assay
Description:  The capability of the compounds to the antagonism of humanized OX1 receptor transfected to the Chinese hamster ovary (CHO) cells was evaluated by the method of fluorescence detected free calcium concentration in the cytoplasm. The cells were suspended in a cell culture medium (invitrogen), and then plated to a microplate with an average density of 2×104 cells/well. The fluorescent probes (Fluo4 NW, Invitrogen) was mixed with probenecid Hank's balanced salt solution (invitrogen), followed by addition of 20 mM hydroxyethyl piperazine acetic sulfuric acid (invitrogen) (pH 7.4). The resulting mixture was eventually added to the microwells containing cells. The cells were incubated at 37° C. for 60 min, and then balanced at 22° C. for 15 min. The microplate was placed in a microplate reader (CellLux, PerkinElmer), and the solution of test compound with different concentrations or Hank's balanced salt solution was added. The microplate containing the solution was incubated for 5 min, followed by the addition of 3 nM of orexin A or a balanced salt solution (as control). The changes of fluorescence intensity proportional to the concentration of calcium ions in the cytoplasm were measured.
Affinity data for this assay
 

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