| Assay Method Information | |
| | Test for Evaluating Rat MC4 Receptor Activation, Using Cells Expressing Rat MC4 Receptor |
| Description: | Experiment Method(1) Construction of Rat MC4 Receptor-Expressing VectorA rat MC4 receptor gene (GenBank Accession No.: NM_013099.2) was introduced into an expression vector pcDNA 3.1/V5-His TOPO (registered trademark) (Thermo Fisher Scientific Inc.).(2) Construction of Cells Transiently Expressing Rat MC4 ReceptorAn expression vector for a rat MC4 receptor was introduced into FreeStyle 293-F cell (Thermo Fisher Scientific Inc.). For the introduction, electroporation was employed. That is, 1×107 FreeStyle 293-F cell were suspended in 80 μL of an electroporation buffer (Thermo Fisher Scientific Inc.), and 20 μg of the expression vector was added thereto. The resultant was put into a cuvette (OC-100 Processing Assembly, MaxCyte, Inc.) and electroporated with MaxCyte STX (registered trademark) (MaxCyte, Inc.). The cells were cultured over one day, suspended in a Cell Banker (registered trademark) 1 (JUJI FIELD Inc.), and stored frozen until use.(3) Measurement of Amount of cAMP ProducedMeasurement was carried out in accordance with the attached instructions, using a LANCE (registered trademark) Ultra cAMP Kit (PerkinElmer, Inc.). That is, after dissolution in DMSO, the test compound (a final concentration of 1 pM to 30 μM) diluted with an assay buffer (Hank's balanced salt solution, 5 mM HEPES, 0.5 mM IBMX, 0.1% bovine serum albumin, pH 7.4), or α-MSH (Bachem Inc., a final concentration of 1 pM to 30 μM) was added to OptiPlate-384 (PerkinElmer, Inc.). Furthermore, a suspension of cells transiently expressing the rat MC4 receptor, that had been prepared using the assay buffer, was added thereto at 1,000 cells/well, followed by being left to stand at room temperature for about 1 hour. Thereafter, an Eu-cAMP tracer solution and an ULight -anti-cAMP solution were added thereto, followed by being left to stand at room temperature for about 1 hour. The amount of cAMP was calculated using EnVision (registered trademark) (PerkinElmer Inc.).For the agonistic activity, an efficacy (EC50 (μM)) was calculated by a non-linear regression method with a Sigmoid-Emax model, by defining the maximum reaction with α-MSH as 100% and the reaction with the vehicle alone as 0%, respectively. |
| Affinity data for this assay | |
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