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Figure 2 | BMC Research Notes

Figure 2

From: Improved molecular toolkit for cAMP studies in live cells

Figure 2

Reduced noise level of pm-Epac2-camps-GFP/mCherry compared to pm-Epac2-camps-CFP/YFP. (A) Normalized excitation (solid lines) and emission (dashed lines) of GFP, mCherry, CFP and YFP. Emission spectra of CFP and YFP largely overlap, making FRET measurement difficult. The large separation of GFP and mCherry emission spectra significantly reduces crosstalk between acceptor and donor. (B) ΔR/R0 was computed as (R-R0)/R0 where R is donor:acceptor ratio and R0 the mean value of this ratio before forskolin stimulation. ΔR/R0 of both pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry increase after 10 μM forskolin application. The lag between the pm-Epac2-camps-GFP/mCherry and pm-Epac2-camps-CFP/YFP signals falls under the precision of the perfusion system and is unlikely to reflect a difference between the sensors. No correction factor was applied. ≥ 15 cells were scored for each condition. (C) Acceptor and donor emission of pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry before and after 1000 frame acquisitions (excitation with a 442 nm laser line). Photobleaching is less pronounced for pm-Epac2-camps-GFP/mCherry. (D) Fluorescence intensity of acceptors and donors of pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry after 1000 frames, expressed as a fraction of the fluorescence intensity of the first frame. ** p < 0.01, *** p < 0.001. (E) Noise levels for pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry are equivalent for the first 50 frames. After 950 frames, only the noise level for pm-Epac2-camps-CFP/YFP has increased. Data are normalized to the value at the beginning of the recording as in B (ΔR/R0). (F) The standard deviation of ΔR/R0 over 40 frames, indicating the noise level, shows a progressive increase for pm-Epac2-camps-CFP/YFP but not for pm-Epac2-camps-GFP/mCherry. * p < 0.05 from frame 819 to 1000. C-F, 5 cells per condition. Error bars, sem. D, Kruskal-Wallis test. F, Mann-Whitney U test.

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