| Assay Method Information | |
| | Biological Activity Test Assay |
| Description: | TRPC5 is a type of non-selective cation channel with permeability to calcium ions. Therefore, TRPC5 agonist Englerin A (EA) and TRPC5 inhibitor Pico145 were used as positive controls in this experiment. Fluo-4 AM fluorescence dye was used to indirectly reflect the effect of the compound on TRPC5 channel by detecting intracellular Ca21 in TRPC5-HEK 293 cells.1. Cell culture1.1 Revival of TRPC5-HEK 293 cellsRevival solution: 100% high glucose DMEMSelection medium: high glucose DMEM+10% FBS+1% P/S+1% HEPES+Blasticidin (5 μg/mL)+Hygromycine (50 μg/mL)Induction medium: high glucose DMEM+10% FBS+1% P/S+1% HEPES+Doxycycline (1 μg/mL).Revival process: A cryopreservation solution containing TRPC5-HEK 293 cells was taken out from the liquid nitrogen tank, and transferred from the ice box to the water bath, and then dissolved in circles to a small piece of ice, and the cryopreservation solution was transferred to the revival solution, and centrifuged at 1000 rpm for 5 minutes, and then the supernatant was discarded, then the residue was transferred to the selection medium, and expanded in a CO2 incubator (5% CO2, humidity of 95%, 37° C.).1.2 Seeding TRPC5-HEK 293 cells in plates14 hours before the real-time fluorescence experiment, cells were washed with PBS and digested with trypsin-EDTA (TE). Cells were diluted to 200,000 cells/mL with induction medium and seeded onto a 96-well black wall bottom transparent plate coated with poly-D-lysine (PDL) (stock solution of 10 mg/mL, final concentration of 10 μg/mL) at 100 μL/well, i.e., 20,000 cells/well.2. Preparation of buffer500 mL of external solution for detecting TRPC5 calcium signals: 4.0908 g of NaCl, 0.1864 g of KCl, 0.111 g of CaCl2, 0.0476 g of MgCl2, 0.9 g of glucose, 1.1915 g of HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid).Calcium fluorescent dye: an external solution for detecting TRPC5 calcium signals containing Fluo-4 with a final concentration of 4 μM (containing 0.5% BSA) |
| Affinity data for this assay | |
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