Table 3. Example of how to compare ratiometric measurements.

The ratio of ratios provides a relative comparison to determine whether an NAD⁺-dependent fluorescence change occurred. This table contains a normalization step first, then a ratio is being determined from the normalized values. The statistical significance of the change is evaluated with experimental replicates, described in Basic Protocol steps 40–49. Because the comparison utilizes division, the order of the comparisons (either option 1, relative to cpVenus or option 2, relative to untreated control) does not matter.

Geometric mean 488/405 nm fluorescence values of each population
	nuclear cpVenus	nuclear sensor	cytoplasmic cpVenus	cytoplasmic sensor	mitochondrial cpVenus	mitochondrial sensor
0 nM FK866	6.83	2.26	7.79	2.51	22.1	5.37
10 nM FK866	6.85	3.34	7.46	3.78	22.8	7.30
Option 1: Compared to cpVenus
0 nM FK866	2.26/6.83 = 0.33		2.51/7.79 = 0.32		5.37/22.1 = 0.24
10 nM FK866	3.34/6.85 = 0.49		3.78/7.46 = 0.51		7.3/22.8 = 0.32
Fluorescence change	Nuclear		Cytoplasmic		Mitochondrial
Ratio of ratios (treated/untreated)	0.49/0.33 = 1.48		0.51/0.32 = 1.57*		0.32/0.24 = 1.32*
Option 2: Compared to untreated
	6.85/6.83 = 1.00	3.34/2.26 = 1.48	7.46/7.79 = 0.96	3.78/2.51 = 1.51	22.8/22.1 = 1.04	7.3/5.37 = 1.36
Fluorescence change	Nuclear		Cytoplasmic		Mitochondrial
Ratio of ratios (treated/untreated)	1.48/1.00 = 1.48		1.51/0.96 = 1.57		1.36/1.04 = 1.32*

^*values based on non-rounded numbers.