| Assay Method Information | |
| | Radioligand Competition Binding Assay |
| Description: | Table 3: In the first step, a cell membrane component containing specific 5-HT1A receptors was prepared. A 10 cm culture dish with HEK-293T cells was used for transfection with 10 μg of 5-HT1A receptors and 40 μL of PEI. After 48 hours, the 10 cm culture dish was taken out from the incubator and the cultured cells had expressed the 5-HT1A receptors. A vacuum pump was used to suck off the culture medium, 3 mL of lysis buffer was added to each culture dish, and the cells were placed in a 4° C. cold room for 10 minutes. After the cells were detached, the cells were transferred to a 15 mL centrifuge tube and centrifuged at 1500 rpm for 5 minutes at 4° C., and the supernatant was discarded. The cell pellet was transferred to a tissue homogenizer, and 3 mL of lysis buffer was added and fully ground until the cells were broken. Then, cell suspension was equally aliquoted into several EP tubes, centrifuged at 12000 rpm for 5 minutes at 4° C., and the supernatant was discarded. The precipitate was the cell membrane component containing the 5-HT1A receptors.In the second step, a ligand-receptor binding assay was performed on 293T membrane component transiently expressing the 5-HT1A receptors. First, a standard binding buffer was added to the cell membrane component containing the 5-HT1A receptors, and the cell membrane was disrupted and resuspended with an electric tissue homogenizer. 30 μL of membrane protein suspension was added to each well of a 96-well plate. Then, 30 μL of different drugs were added to the 96-well plate sequentially from bottom to top to ensure that the final drug concentrations were 10−5 M, 10−6 M, 10−7 M, 10−8 M, 10−9 M, and 0 M, with two replicates per treatment. Next, 30 μL of [3H]-LSD was added to each well of the 96-well plate. The plate was incubated at room temperature in the dark for 2 hours. Detection was conducted. The machine readout value reflected the amount of [3H]-LSD bound on the membrane, and after further data processing, the binding affinity Ki values of different compounds for the 5-HT1A receptors were obtained. |
| Affinity data for this assay | |
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