Table 1.
Example of Gonadotropin Releasing Hormone Receptor High Throughput Screening Assay Protocol
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Plate cellsa | 20 μL | 8,500 HeLa GnRHR E90K cells |
| 2 | Pin compounds and control | 100 nL | IN3, DMSO |
| 3 | Incubate | Overnight | 37°C, 5% CO2 |
| 4 | Reporter dye | 20 μL | Fluo-2 dye |
| 5 | Incubate | 1 h | 37°C, 5% CO2 |
| 6 | Incubate | 30 min | Ambient temperature |
| 7 | Assay readout | 490ex/530em | Kinetic read for 140 sec |
Step Notes
1. Greiner 384-well part 781091 (Frickenhausen Germany).
2. Column 2 receives IN3 (high control); Column 23 receives DMSO (low control); columns 3–22 receive test compound; columns 1 and 24 receive cells.
3. Plates covered with stainless steel gasket lined lids containing pinholes for gas exchange.
4. Single tip dispense reagent all wells.
5. Plates covered with stainless steel gasket lined lids containing pinholes for gas exchange.
6. Plates lidded until moved to FLIPR Tetra (Molecular Devices).
7. Add 5 μL of GnRH (500 nM final) in assay buffer, or DMSO equivalent, and read using kinetic read modality on FLIPR tetra. Read settings include 470_495 LED excitation, 515_575 emission, gain of 140, exposure time of 0.40 and excitation intensity of 70. Export the data as Max signal obtained divided by the Min signal=Ratio (Max/Min).
For the primary assay, cells are not treated with doxycycline and hence are induced to express the GnRHR (per the Tet-off system). The counterscreen assay uses doxycycline-treated cells.
IN3, ((2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl] 2-(3,5-dimethylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl) propan-1-amine); DMSO, dimethyl sulfoxide; GnRH, gonadotropin releasing hormone; GnRHR, GnRH receptor.