Table 1.

Comparison of different FRET measurement methods.

	EC50 (μM)	Hill Coefficient	Mean CV	Mean Error from Hill Equation
One filter set	0.98	1	8.43	0.198
Two filter set	0.65	0.72	0.13	0.019
Three filter set uncorrected*	1.73	0.89	18.89	0.140
Three filter set corrected	0.67	0.71	0.13	0.019
Peak radio	0.60	0.72	0.17	0.023
Linear unmixing	0.69	0.72	0.10	0.019
Linear unmixing corrected	0.60	0.71	0.16	0.022

All methods are measures of FRET efficiency except for those indicated by *, which are FRET indices that have been included as they have been used in prior literature. Linear unmixing produced the lowest coefficient of variation and second lowest error from fitting the Hill equation, while the three-filter set uncorrected method produced the highest coefficient of variation and the one-filter set method produced the highest error from fitting the Hill equation. EC50 represents the cAMP concentration required to achieve 50% of the full FRET response, CV is the coefficient of variation. n = 3 trials.