| Assay Method Information | |
| | In Vitro Activity Assay |
| Description: | (1) Preparation of a reaction buffer (1×Stimulation buffer) required for the experiment: The 5×Stimulation buffer and ddH2O in the Cisbio IP-one kit were diluted in a ratio of 1:4 for later use.(2) Compound preparation: The compound was diluted with DMSO to obtain a 5 mM stock solution, followed by a 3.16-fold dilution to obtain 10 gradients, and then the prepared compound was diluted with the Stimulation buffer to the corresponding concentrations (4×) for later use.(3) Cell preparation: CHO-K1-OX1 and CHO-K1-OX2 cells on the culture dish were digested with trypsin, and the cells were washed off with culture medium and collected into 5 mL centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 3 mL of PBS was added, and the mixture was gently pipetted to be mixed well. The cells were centrifuged again at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 1×Stimulation buffer and counted using a Countstar cell counter, and the cell density was adjusted to 1.71×106 cells/mL for later use.(4) Cell addition: The cell suspensions were added to assay plates at 7 μL/well (i.e., about 12,000 cells/well).(5) Compound addition: The compound diluted with the Stimulation buffer was added to the assay plates described above at 3.5 μL/well.(6) Reaction and incubation: After gentle shaking, the assay plates were incubated at 37° C. for 30 min.(7) EC80 agonist addition: 4×Orexin A (OX1 receptor) and 4×Orexin 2 receptor agonist (OX2 receptor) solutions of EC80 were added at 3.5 μL/well.(8) Reaction and incubation: After gentle shaking, the assay plates were incubated at 37° C. for 45 min.(9) Detection reagent addition: IP1-d2 and Anti-IP1 cryptate were separately diluted in a 1:20 ratio with the Lysis & detection buffer in the Cisbio IP-one detection kit, and the diluted IP1-d2 and Anti-IP1 cryptate described above were added to the assay plates, each at 3 μL/well. After shaking, the assay plates were left to stand at room temperature for 60 min.(10) Experimental readings: The plate was read on Envision, readings from the 665 nm and 615 nm channels were taken, and the ratio of 665 nm/615 nm readings was calculated. |
| Affinity data for this assay | |
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