Figure 2.
FreSHtracer Visualizes GSH Levels within Subcellular Compartments in Living Cells
(A) Representative confocal images of F510 and F580, and pseudo-color images of the fluorescence ratio (FR) for HeLa cells, hBM-MSCs, and hES-MSCs. The cells were incubated with FreSHtracer (5 μM) for 2 hr. Images of a HeLa cell and hBM-MSCs are the same, with cells indicated by arrows in Figure 4A (0 min).
(B) A reversible reaction of FreSHtracer with intracellular thiols. In HeLa cells equilibrated FreSHtracer (5 μM, 2 hr), F510 and F580 were monitored at 5-s intervals after treatment with diamide (DA), followed by 0.5 mM DTT. (a) Confocal and pseudo-color images of FreSHtracer-loaded cells indicated by numbered arrowheads. (b and c) The F510, F580, and FR of the two cells (arrowheads) were monitored. (d) The average FR values in the whole cell, cytoplasm, and nucleoplasm (n = 4 cells/time point) are shown.
(C) The structure of MitoFreSHtracer, and changes in its fluorescence spectra when reacted with GSH.
(D) Confocal and ratiometric pseudo-color images of a MitoFreSHtracer-loaded (10 μM, 1.5 hr) HeLa cell.
Images are the same with the cell indicated by arrowhead 1 in E (0 min).
(E) Effect of DA (0.5 mM) on the FR within MitoFreSHtracer-loaded HeLa cells. Left: pseudo-color images of the FR. Right: time course of FR changes within the indicated cells (arrowheads).
(F) Effect of antimycin A (0, 100, 200 μM; 75 min) on FR values in MitoFreSHtracer-loaded HeLa cells. Left: pseudo-color images of the FR. Right: concentration-dependent decreases in the FR values within antimycin A-treated cells (n = 90 cells from n = 3 independent experiments).
Data represent the mean ± SEM of the FR; ∗∗∗p < 0.001. Scale bars, 10 μm (A, B, D, E, F). See also Figures S2–S4.