An ICT-Based Approach to Ratiometric Fluorescence Imaging of Hydrogen Peroxide Produced in Living Cells

Srikun, Duangkhae; Miller, Evan W.; Domaille, Dylan W.; Chang, Christopher J.

doi:10.1021/ja711480f

Cited by 515 publications

(298 citation statements)

References 24 publications

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“…Accordingly, we developed a unique H 2 O 2 -specific fluorescent indicator, PY1-ME (Fig. S1A), which combines a boronate-masked phenol for the selective detection of H 2 O 2 (10,(33)(34)(35)(36)(37)(38)(39) and an appended methyl-ester group designed to increase cellular uptake and retention upon cleavage by intracellular esterases; this latter feature makes the dye suitable for both fluorescence imaging and flow cytometry experiments. Because this probe relies on a specific molecular reaction between H 2 O 2 and the boronate to generate a fluorescence signal, rather than thiol-based chemistry that can occur by non-specific ROS oxidation or disulfide exchange, our chemical method affords a direct measure of H 2 O 2 in cellular specimens.…”

Section: Resultsmentioning

confidence: 99%

Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling

Miller

Dickinson²,

Chang³

2010

Proc. Natl. Acad. Sci. U.S.A.

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622

466

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Hydrogen peroxide (H 2 O 2 ) produced by cell-surface NADPH Oxidase (Nox) enzymes is emerging as an important signaling molecule for growth, differentiation, and migration processes. However, how cells spatially regulate H 2 O 2 to achieve physiological redox signaling over nonspecific oxidative stress pathways is insufficiently understood. Here we report that the water channel Aquaporin-3 (AQP3) can facilitate the uptake of H 2 O 2 into mammalian cells and mediate downstream intracellular signaling. Molecular imaging with Peroxy Yellow 1 Methyl-Ester (PY1-ME), a new chemoselective fluorescent indicator for H 2 O 2 , directly demonstrates that aquaporin isoforms AQP3 and AQP8, but not AQP1, can promote uptake of H 2 O 2 specifically through membranes in mammalian cells. Moreover, we show that intracellular H 2 O 2 accumulation can be modulated up or down based on endogenous AQP3 expression, which in turn can influence downstream cell signaling cascades. Finally, we establish that AQP3 is required for Noxderived H 2 O 2 signaling upon growth factor stimulation. Taken together, our findings demonstrate that the downstream intracellular effects of H 2 O 2 can be regulated across biological barriers, a discovery that has broad implications for the controlled use of this potentially toxic small molecule for beneficial physiological functions.growth factor signaling | redox biology | reactive oxygen species | fluorescent sensor | membrane regulation H ydrogen peroxide (H 2 O 2 ) is garnering increased attention as a molecule involved not only in immune response and oxidative stress, but also as a physiological effector in essential cellular processes (1-5). Seminal contributions have elucidated ligand stimulants (6-10) and enzymatic sources (11-13) for cellular H 2 O 2 production as well as putative downstream targets (14-24), but principles of how this reactive oxygen species (ROS) is spatially and temporally regulated to promote redox signaling over oxidative stress pathways remain insufficiently understood. Because many of the signaling functions of H 2 O 2 rely on its generation by nonphagocytic forms of NADPH (Nox) proteins on the extracellular side of cell membranes, understanding how cells funnel H 2 O 2 toward beneficial pathways in these locales is of significant interest. Despite its reactive nature, H 2 O 2 has been long thought to be freely diffusible across biological membranes in a manner akin to the related canonical small-molecule signal nitric oxide (NO) (25). More recent studies implicate the role of AQP water channels, a class of membrane-spanning proteins that facilitate the diffusion of water and other substrates with varying specificity (26-28), in mediating H 2 O 2 passage across the plasma membrane of reconstituted yeast (29) and plant (30) cells. However, given that the experiments described in these reports utilized nonspecific chemical reagents for determination of the redox signal that was passed into the cell, direct evidence that aquaporins influence the cellular uptake of H 2 O 2 in a na...

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Section: Resultsmentioning

confidence: 99%

Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling

Miller

Dickinson²,

Chang³

2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

622

466

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“…The Cell-IQ analyzer software utilizes machine vision to automatically identify and quantify cell types. Near-infrared fluorescence(NIRF)probe [49] Fluorescent protein Two-photon fluorescent probe [64,76] Quantum dot (QDs) probes [42,43] pH sensitive probe [51,52,95] Gold nanoparticles [83] Nanoparticles Magnetic nanocrystal [84] Proteins Direct observation of molecular dynamics can provide important information about cellular events that cannot be obtained by other methods. Thus imaging of protein dynamics in living cells becomes an important tool for cell biology to study molecular and cellular functions.…”

Section: Probe Sortingmentioning

confidence: 99%

“…BODIPY-alphaTocopherol adduct (B-TOH), a novel lipophilic fluorescent antioxidant indicator, is a sensitive and specific probe enabling the molecular imaging of peroxyl radicals in the lipid membranes of live cells [63]. Two-photon confocal microscopy experiments in live macrophages showed that Peroxy Lucifer 1 (PL1), which is a newly synthezied fluorescent probe for imaging hydrogen peroxide produced in living cells by a ratiometric response, can ratiometrically visualize localized hydrogen peroxide bursts generated in living cells at immune response levels [64]. Luminescence analysis has been recently proposed to measure free radical generation by isolated cells or organs, but it allowed only global tissue luminescence.…”

Section: Radicalsmentioning

confidence: 99%

Application of Probes in Live Cell Imaging~!2010-01-03~!2010-05-09~!2010-08-16~!

Chen¹,

Bai²,

Wang³

2010

JEBP

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Live cell imaging (LCI) is the approach of non-invasively analyzing dynamic processes in living cells using state-of-the-art microscopy and computer vision techniques. LCI provides exciting and novel insights into cell biology. The present review summarized LCI research in recent years and detailed the role of probes in LCI. We discussed the basic principle and development in LCI technology, including fluorecence resonance energy transfer, fluorescence lifetime imaging microscopy, atomic force microscopy, scanning ion conductance microscopy, confocal microscopy, two-photon excitation fluorescence microscopy and Cell-IQ® System . Then we listed several probes such as green fluorescent protein and quantum dots which are the most widely used probes. Proteins, organelles, nucleotides and other chemicals related to cell biology may all be potential targets. We also discussed the advantages and disadvantages of different probes. Although specific probes in LCI still need to be explored, the application of LCI may be attractive for better understanding of cellular functions.

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“…Whereas many of them only utilized the changes in emission intensity as detecting signals. A major limitation of the intensity-based fluorescent probe is that the signal output could be interfered by the factors such as environmental conditions, probe distribution, and instrumental efficiency [9,10]. By contrast, a ratiometric measurement, employing the ratio of two emissions at different wavelengths as the detecting signal, could provide a built-in correction for the above mentioned factors and thus allow more accurate analysis [11,12].…”

Section: Introductionmentioning

confidence: 99%

A Fluorescence Ratiometric Probe for Detection of Cyanide in Water Sample and Living Cells

Long¹,

Wang²,

Wu³

2013

AMPC

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In the present work, Compound 1 has been synthesized as a novel fluorescence ratiometric probe for CN − . Upon treatment with CN − , Probe 1 exhibited a fluorescence ratiometric response, with the emission wavelength shift from 570 nm to 608 nm. When 90 μM CN − was introduced, the emission ratios (I 570 /I 608 ) of the probe changed dramatically from 0.52156 to 4.21472. The detection limit was also measured to be 0.24 μM (S/N = 3). In addition, Probe 1 had a selective response to CN − , while other anions caused nearly no interference.

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An ICT-Based Approach to Ratiometric Fluorescence Imaging of Hydrogen Peroxide Produced in Living Cells

Cited by 515 publications

References 24 publications

Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling

Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling

Application of Probes in Live Cell Imaging~!2010-01-03~!2010-05-09~!2010-08-16~!

A Fluorescence Ratiometric Probe for Detection of Cyanide in Water Sample and Living Cells

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