Table 1.

qHTS protocol.

Step	Parameter	Value	Description
1	Reagent	3 μL	HEK293-RXFP1 cells at 2,000 cells/well
2	Incubation	16 – 24 h	37 °C, 5% CO2 standard cell culture conditions
3	Reagent	1 μL	400 μM Ro 20-1724 in PBS+
4a	Library compounds	23 nL	Dispense library compounds via pintool transfer
4b	Controls	23 nL	Dispense control compounds via pintool transfer
6	Incubation	30 min	37 °C, 5% CO2 standard cell culture conditions
7	Reagent	1 μL	cAMP-d2 diluted 1:18 in HTRF lysis buffer
8	Reagent	1 μL	K-anti-cAMP antibody diluted 1:20 in HTRF lysis buffer
9	Incubation	30 min	Room temperature
10	Detector		EnVision (HTRF Eu/d2 protocl). ViewLux reader. Standard HTRF protocol