Table 1. Assay Protocol.

Step	Parameter	Value	Description*
1	Reagent	6 µl	2000 cells/well in 6 µl/well DMEM with 10% FBS, 1× Penicillin/Streptomycin, 0.5 mg/ml G418
2	Incubation	16–24 hr	37°C, 5% CO2, 95% humidity
3	Aspirate	−5 µl	Leave 1 µl residual
4	Reagent	5 µl	DPBS, 1 mM CaCl2, 0.5 mM MgCl2, 0.05% BSA, 0.005% Tween 20
5	Aspirate	−4 µl	Leave 2 µl residual
6	Compound	23 nl	Columns 1–4 controls, columns 5–48 compounds
7	Reagent	1 µl	300 µM Ro-20174 in DMEM (no phenol) 10% FBS
8	Incubation	30 min	37°C, 5% CO2, 95% humidity
9	Reagent	1 µl	HTRF kit: cAMP-d2 in lysis buffer
10	Reagent	1 µl	HTRF kit: anti-cAMP-K in lysis buffer
11	Incubation	30 min	Room temperature
12	Detection		EnVision plate reader; HTRF mode (excitation at 320 nM, and emission at 615 nm and 665 nm)

*See Materials and Methods for more details, definitions, and non-standard abbreviations. Ro-20174 = 4-(3-Butoxy-4-methoxybenzoyl)-2-imidazolidine, HTRF = homogeneous time resolved fluorescence.