Figure 3.

FreSHtracer Can Report the GSH Concentration within Live Cells

(A and B) Determination of the F₅₁₀/F₅₈₀ ratio (FR) fraction attributable to GSH (FR_GSH). The FR within buthionine sulfoximine (BSO)-treated HeLa cells (3–4 × 10³ cells/cm²; 48 hr) in confocal imaging of each cell (n = 10 cells; A) or the FR within flow-cytometric analysis of BSO-treated cells (n = 3 independent biological replicates; B) were plotted against the GSH concentration determined by luminescence-based methods following cell lysis (n = 3 independent biological replicates; A and B, right).

(C–E) Effect of oxidants on the FR of GSH-depleted cells (FR_PSH). BSO-treated HeLa cells (48 hr, 80 μM), equilibrated with FreSHtracer (2 hr, 5 μM), were treated for 15 min with (E) or without (C and D) 50 μM 1-chloro-2,4-dinitrobenzene (CDNB), an irreversible inhibitor of thioredoxin reductase. Fluorescence intensities were measured every 10 s by confocal microscopy following treatment with H₂O₂ (C) or diamide (DA; D and E). Ratiometric pseudo-color images (C–E, upper) depict the FR (n = 4 cells/time point; C–E, lower). Arrows indicate time points of treatment of H₂O₂ or diamide.

All error bars represent mean ± SEM. Scale bars, 10 μm (A, C, D, E).