| Assay Method Information | |
| | Test for Evaluating Human MC Receptor Activation, Using Cells Expressing Human MC4, MC1, or MC3 Receptor |
| Description: | Experiment Method(1) Construction of Human MC Receptor-Expressing VectorA human MC4 receptor gene (GenBank Accession No.: NM_005912.2), a human MC1 receptor gene (GenBank Accession No.: NM_002386.3), or a human MC3 receptor gene (GenBank Accession No.: NM_019888.3) was introduced into an expression vector pcDNA 3.1/V5-His TOPO (registered trademark) (Thermo Fisher Scientific Inc.).(2) Construction of Cells Transiently Expressing Human MC ReceptorAn expression vector for a human MC4, MC1, or MC3 receptor was introduced into FreeStyle 293-F cell (Thermo Fisher Scientific Inc., product number: R790-07). 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., product number: B201-100), 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 SIX (registered trademark) (MaxCyte, Inc.). The cells were cultured over one day, suspended in a Cell Banker (registered trademark) 1 (JUJI FIELD Inc.), product number: BLC-1), and stored frozen until their use.(3) Measurement of Amount of cAMP ProducedMeasurement was carried out by using a LANCE (registered trademark) Ultra cAMP Kit (PerkinElmer, Inc.) in accordance with the attached instructions. 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 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.5 mM 3-isobutyl-1-methylxanthine (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 human MC4, MC1, or MC3 receptor prepared by 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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