| Assay Method Information | |
| | Measurement of TRPA1 Antagonist Activity |
| Description: | Human TRPA1 Expression PlasmidUsing cDNA encoding human TRPA1 (GenBank accession No. NM_007332) (manufactured by Kazusa DNA Research Institute, item No. FHC07217) as a template, primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2), PCR by PfuUltra High-Fidelity DNA Polymerase (Stratagene) was performed, and full-length human TRPA1 gene was amplified.primer 1: (SEQ ID NO: 1) 5′-AACTTTTAGTAAGCTTCGATCGCCATGAAG-3′ primer 2: (SEQ ID NO: 2) 5′-GTACCGATCTAGAATTCGTTTTACTAAGGCTCAAG-3′A recognition site (underlined) of restriction enzyme HindIII was added to the 5′ end of human TRPA1 gene, and XbaI site (underlined) was added to the 3′ end of human TRPA1 gene, and GTT of the template sequence was changed to termination codon TAG (bold). The obtained double stranded DNA was enzyme-digested with HindIII and XbaI, and introduced into a multicloning site of expression plasmid pcDNA3.1/zeo(+) (manufactured by Invitrogen) to give a human TRPA1 expression plasmid.Cell PreparationHuman embryonic kidney-derived 293T cells were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum, 10 unit penicillin, and 10 μg streptomycin. One day before assay, 3×106 of 293T cells were plated on a petri dish having a diameter of 10 cm, and cultured in a CO2 incubator for 24 hr. OPTI-MEM I Reduced Serum Media (Invitrogen) (600 μL), Mirus TransIT-293 (Mirus Bio) (18 μL), and human TRPA1 expression plasmid (6 μg) were mixed, the total amount of the mixture was added to the cells on the petri dish to allow for gene transfer. The cells were recovered about for 8 hr later, plated on a poly-D-lysine coated 384 well black/clear bottom plate at 12,000 cells/well, and cultured overnight.Measurement of Intracellular Calcium IncreaseThe medium was removed from the 384 well plate, calcium indicator (Molecular Device, trade name: FLIPR Calcium4 Assay Kit) dissolved in HBSS (Thermo Fisher Scientific) (pH 7.2) containing 20 mM HEPES was added (38 μL/well), and the cells were stained in a CO2 incubator for 1 hr. The 384 well plate was stood at room temperature for not less than 15 min, set on FDSS7000 (Hamamatsu Photonics K.K.), and a test substance solution was added at 10 μL/well. After 10 min, allylisothiocyanate solution (12 μL/well) was added, the relative fluorescence intensity was measured for 5 min after addition of the allylisothiocyanate solution.Test Substance PreparationPreparation of Test Substance Solution and Allylisothiocyanate SolutionA test substance solution was prepared to have a composition of HBSS (Thermo Fisher Scientific) (pH 7.2) containing 0.48% dimethyl sulfoxide, a test substance at 4.8-fold concentration of the evaluation concentration, 0.1% bovine serum albumin and 20 mM HEPES. An allylisothiocyanate solution was prepared to have a composition of HBSS (Thermo Fisher Scientific) (pH 7.2) containing 0.1% dimethyl sulfoxide, 100 μM allylisothiocyanate, 0.1% bovine serum albumin and 20 mM HEPES.Calculation of Antagonist ActivityThe activity rate of a test substance at each concentration was calculated, wherein the relative fluorescence intensity change of a well free of a test substance and containing allylisothiocyanate is 100% activity rate, and the relative fluorescence intensity change of a well free of a test substance and allylisothiocyanate is 0% activity rate. The inhibitory rate of a test substance at each concentration was calculated by subtracting the activity rate of the test substance from 100% activity rate, and the concentration of a test substance showing 50% inhibitory rate was calculated as IC50 from the sigmoid approximate curve by XLfit (idbs). |
| Affinity data for this assay | |
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