Cell-trappable fluorescent probes for nitric oxide visualization in living cells

Abstract

Two new cell-trappable fluorescent probes for nitric oxide (NO) are reported based on either incorporation of hydrolyzable esters or conjugation to aminodextran polymers. Both probes are highly selective for NO over other reactive oxygen and nitrogen species (RONS). The efficacy of these probes for the fluorescence imaging of nitric oxide produced endogenously in Raw 264.7 cells is demonstrated.

Figure 1

Preparation of FL1E, FL1A, and FL^Dex.

Figure 2

Fluorescence turn-on of CuFL1A (1 μM FL1A, 1 μM CuCl₂, 1300 equiv NO, 50 mM PIPES, 100 mM KCl, pH 7, 37 °C). Inset: Time dependence of fluorescence turn-on.

Figure 3

Comparison of the selectivity for RONS with CuFL1E, CuFL1A, and CuFL^Dex. (50 mM PIPES, 100 mM KCl, pH 7, 37 °C, 60 min, 100 equiv RONS, 1300 equiv NO).

Figure 4

pH dependence of λ_max(emission) for FL1A and CuFL1A (5 μM FL, 100 mM KCl, 25 °C)

Figure 5

Visualization of NO produced endogenously in Raw 264.7 cells using CuFL1E (left) and CuFL^Dex (right) after stimulation with LPS and INF-γ for 14 h. Top: DIC images, Bottom: fluorescence. (a) Probe only, (b) Probe, LPS, INF-γ, and L-NNA (iNOS inhibitor), (c) Probe, LPS, and INF-γ. [Probe] = 4 μM, [LPS] = (1.25 μg/mL), [INF-γ] = (625 – 6250 U/mL), [L-NNA] = 10 μM. Scale bars = 25 μm.

Scheme 1

Fluorescence turn-on for the FL1₅ NO probe.