A palette of fluorescent probes with varying emission colors for imaging hydrogen peroxide signaling in living cells - PubMed (original) (raw)

A palette of fluorescent probes with varying emission colors for imaging hydrogen peroxide signaling in living cells

Bryan C Dickinson et al. J Am Chem Soc. 2010.

Abstract

We present a new family of fluorescent probes with varying emission colors for selectively imaging hydrogen peroxide (H(2)O(2)) generated at physiological cell signaling levels. This structurally homologous series of fluorescein- and rhodol-based reporters relies on a chemospecific boronate-to-phenol switch to respond to H(2)O(2) over a panel of biologically relevant reactive oxygen species (ROS) with tunable excitation and emission maxima and sensitivity to endogenously produced H(2)O(2) signals, as shown by studies in RAW264.7 macrophages during the phagocytic respiratory burst and A431 cells in response to EGF stimulation. We further demonstrate the utility of these reagents in multicolor imaging experiments by using one of the new H(2)O(2)-specific probes, Peroxy Orange 1 (PO1), in conjunction with the green-fluorescent highly reactive oxygen species (hROS) probe, APF. This dual-probe approach allows for selective discrimination between changes in H(2)O(2) and hypochlorous acid (HOCl) levels in live RAW264.7 macrophages. Moreover, when macrophages labeled with both PO1 and APF were stimulated to induce an immune response, we discovered three distinct types of phagosomes: those that generated mainly hROS, those that produced mainly H(2)O(2), and those that possessed both types of ROS. The ability to monitor multiple ROS fluxes simultaneously using a palette of different colored fluorescent probes opens new opportunities to disentangle the complex contributions of oxidation biology to living systems by molecular imaging.

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Figures

Figure 1

Figure 1

Fluorescence turn-on response of 5 µM PF2 (a), PF3 (b), PE1 (c), PY1 (d) or PO1 (e) to H2O2. Data were acquired at 25 °C in 20 mM HEPES, pH 7, with excitation at λ = 488 nm for PF2 and PF3, λ = 490 nm for PE1, λ = 514 nm for PY1, and λ = 540 nm for PO1. Emission was collected between 493 and 750 nm for PF2 and PF3, 495 and 750 nm for PE1, 520 and 750 nm for PY1 and 545 and 750 nm for PO1. Time points represent 0, 5, 15, 30, 45, and 60 minutes after the addition of 100 µM H2O2. Reactions are not complete at these time points. Full turn-on response of each probe is shown in Fig. S1.

Figure 2

Figure 2

Fluorescence responses of 5 µM PF2 (a), PF3 (b), PE1 (c), PY1 (d) and PO1 (e) to various reactive oxygen species (ROS). Bars represent relative responses at 0, 5, 15, 30, 45, and 60 min after addition of each ROS. Data shown are for 200 µM NO and 100 µM for all other ROS. Data were acquired at 25 °C in 20 mM HEPES, pH 7, with excitation at λ = 488 nm for PF2 and PF3, λ = 490 nm for PE1, λ = 514 nm for PY1, and λ = 540 nm for PO1. Emission was collected between 493 and 750 nm for PF2 and PF3, 495 and 750 nm for PE1, 520 and 750 nm for PY1 and 545 and 750 nm for PO1. Time points represent 0, 5, 15, 30, 45, and 60 minutes after the addition of 100 µM H2O2. Reactions are not complete at these time points.

Figure 3

Figure 3

Confocal fluorescence images of H2O2 in live A431 cells under oxidative stress with PF2, PF3-Ac, PY1 and PO1. A431 cells incubated with 10 µM PF2 for 40 min at 37 °C (a). A431 cells incubated with 10 µM PF2 for 40 min at 37 °C with 100 µM H2O2 added for the final 20 min (b) and quantification (c). A431 cells incubated with 5 µM PF3-Ac for 40 min at 37 °C (d). A431 cells incubated with 5 µM PF3-Ac for 40 min at 37 °C with 100 µM H2O2 added for the final 20 min (e) and quantification (f). A431 cells incubated with 5 µM PY1 for 40 min at 37 °C (g). A431 cells incubated with 5 µM PY1 for 40 min at 37 °C with 100 µM H2O2 added for the final 20 min (h) and quantification (i). A431 cells incubated with 5 µM PO1 for 40 min at 37 °C (j). A431 cells incubated with 5 µM PO1 for 40 min at 37 °C with 100 µM H2O2 added for the final 20 min (k) and quantification (l). Data were normalized to controls and statistical analyses were performed with a two-tailed Student’s _t_-test (n = 4). **P ≤ 0.005 and error bars are ± s.e.m.

Figure 4

Figure 4

Confocal fluorescence images of PMA-induced H2O2 production in live RAW264.7 macrophages with PF2, PF3-Ac, PY1 and PO1. Macrophages incubated with 10 µM PF2 for 60 min at 37 °C (a). Macrophages incubated with 10 µM PF2 for 60 min at 37 °C with 1 µg/mL PMA added for the final 40 min (b) with a brightfield overlay (c) and quantification (d). Macrophages incubated with 5 µM PF3-Ac for 60 min at 37 °C (e). Macrophages incubated with 5 µM PF3-Ac for 60 min at 37 °C with 1 µg/mL PMA added for the final 40 min (f) with a brightfield overlay (g) and quantification (h). Macrophages incubated with 5 µM PY1 for 60 min at 37 °C (i). Macrophages incubated with 5 µM PY1 for 60 min at 37 °C with 1 µg/mL PMA added for the final 40 min (j) with a brightfield overlay (k) and quantification (l). Macrophages incubated with 5 µM PO1 for 60 min at 37 °C (m). Macrophages incubated with 5 µM PO1 for 60 min at 37 °C with 1 µg/mL PMA added for the final 40 min (n) with a brightfield overlay (o) and quantification (p). Data were normalized to controls and statistical analyses were performed with a two-tailed Student’s _t_-test (n = 4). **P ≤ 0.005 and error bars are ± s.e.m.

Figure 5

Figure 5

Confocal fluorescence images of EGF-induced H2O2 in live A431 cells with PF3-Ac, PY1 and PO1. A431 cells incubated with 5 µM PF3-Ac for 60 min at 37 °C (a). A431 cells incubated with 5 µM PF3-Ac for 60 min at 37 °C with 500 ng/mL EGF added for the final 40 min (b) and quantification (c). A431 cells incubated with 5 µM PY1 for 60 min at 37 °C (d). A431 cells incubated with 5 µM PY1 for 60 min at 37 °C with 500 ng/mL EGF added for the final 40 min (e) and quantification (f). A431 cells incubated with 5 µM PO1 for 60 min at 37 °C (g). A431 cells incubated with 5 µM PO1 for 60 min at 37 °C with 500 ng/mL EGF added for the final 20 min (h) and quantification (i). Data were normalized to controls and statistical analyses were performed with a two-tailed Student’s _t_-test (n = 4). *P ≤ 0.05, **P ≤ 0.005 and error bars are ± s.e.m.

Figure 6

Figure 6

Confocal fluorescence images of PMA-induced ROS production in live RAW264.7 macrophages with PO1 and APF simultaneously. Macrophages incubated with 5 µM PO1 and 5 µM APF for 40 min at 37 °C and imaged for PO1 (a) and APF (b). Macrophages incubated with 5 µM PO1 and 5 µM APF for 40 min at 37 °C with 50 µM H2O2 added for the final 20 min and imaged for PO1 (c) and APF (d). Macrophages incubated with 5 µM PO1 and 5 µM APF for 40 min at 37 °C with 100 µM HOCl added for the final 20 min and imaged for PO1 (e) and APF (f). Macrophages incubated with 5 µM PO1 and 5 µM APF for 40 min at 37 °C with 1 µg/mL PMA added for the final 20 min and imaged for PO1 (g) and APF (h). Quantification of a–h (i). Data were normalized to controls and statistical analyses were performed with a two-tailed Student’s _t_-test (n = 4). **P ≤ 0.005 and error bars are ± s.e.m.

Figure 7

Figure 7

Confocal fluorescence images of different types of phagosomes in live RAW264.7 macrophages distinguished by PO1 and APF. A macrophage producing mostly H2O2 as shown by the PO1 signal (a), APF signal (b), overlay (c) and brightfield (d). A macrophage producing mostly hROS as shown by the PO1 signal (e), APF signal (f), overlay (g) and brightfield (h). A macrophage producing a mixture of H2O2 phagosomes, hROS phagosomes, and H2O2 and hROS phagosomes. as shown by the PO1 signal (i), APF signal (j), overlay (k) and brightfield (l). 10 µm scale bar shown.

Scheme 1

Scheme 1

Boronate-based H2O2-specific fluorescent indicators.

Scheme 2

Scheme 2

Synthesis and activation of PF2, PF3, and PE1.

Scheme 3

Scheme 3

Synthesis and activation of PY1 and PO1.

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