New Alternately Colored FRET Sensors for Simultaneous Monitoring of Zn2+ in Multiple Cellular Locations (original) (raw)
Figure 3
FRET Sensor calibration in the cytosol.
Representative calibrations of each sensor localized to the cytosol The background corrected FRET ratio (FRET Intensity ÷ Donor Intensity) is represented as a function of time. Calibrations were performed by adding 150 µM TPEN to achieve RTPEN, followed by washing of residual TPEN and addition of 135 µM ZnCl2 with 10 µM Digitonin to permeabilize the cell membrane and obtain RZn. A) NES-ZapSM2, FRET ratio goes slightly above resting; B) NES-ZapSR2 has a similar response as observed in the nucleus, Figure 2B; C) NES-ZapOC2 demonstrates a small decrease after TPEN compared to the same sensor in the nucleus; D) NES-ZapOK2 is observed with small changes in FRET ratio after TPEN and Zn2+/digitonin; E) NES-ZapCmR1 has an inverted response in which TPEN causes an increase in FRET ratio while Zn2+ with digitonin causes a decrease in the ratio; F) NES-ZapCmR1.1 and G) NES-ZapCmR2 exhibit a small decrease with TPEN and a larger increase in FRET ratio after addition of Zn2+ and digitonin. Representative traces are mean ± s.e.m. (n = 4 cells). Each experiment was repeated a minimum of three times.