A protocol for in vivo detection of reactive oxygen species

Owusu-Ansah, Edward; Yavari, Amir; Banerjee, Utpal

doi:10.1038/nprot.2008.23

Cited by 144 publications

(137 citation statements)

References 6 publications

(6 reference statements)

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“…Thus, it is better to use the DHE stock within 10 days after dissolving from powder. If the color of the stock solution changes from gray to purple or pink, discard it and prepare fresh from powder 18 .…”

Section: Discussionmentioning

confidence: 99%

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Zhang

Soldati

2013

JoVE

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Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, mediumthroughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H 2 O 2 production, respectively. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Zhang

Soldati

2013

JoVE

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“…ROS production was also measured in sugar-fed flies, 1 day before infection/ blood-feeding. At each time point, five midguts were dissected for in vivo detection of ROS as described previously (35). Briefly, midguts were dissected in L-15 (Leibovitz) medium (Sigma) and incubated with 30 mol of dihydroethidium in L-15 medium for 5 min in a dark chamber on a mini orbital shaker (70 rpm) at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Reactive Oxygen Species-mediated Immunity against Leishmania mexicana and Serratia marcescens in the Phlebotomine Sand Fly Lutzomyia longipalpis

Diaz-Albiter

Sant’Anna

Genta

et al. 2012

Journal of Biological Chemistry

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Background: Reactive oxygen species are part of the sand fly innate immune system. Results: ROS production in the gut increases in response to a bacterial pathogen but not to Leishmania. Conclusion: Sand flies tolerate the presence of Leishmania by differential response of the ROS system. Significance: The successful use of sand flies as vehicles for Leishmania transmission relies partially on the parasite circumventing the ROS immune response.

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“…SH-SY5Y Cells were grown at 37°C in an incubator with 5% CO 2 . To investigate the effects of P. multiflorum and S. commixta extracts on cell viability, cells were pre-treated with P. multiflorum or S. commixta extract for 30 min, and then treated with 25 µM Aβ [25][26][27][28][29][30][31][32][33][34][35] (Sigma-Aldrich) for a further 24 h. Then, the cells were incubated with 2 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) at 37°C in a CO 2 incubator for 3 h. MTT medium was carefully aspirated, and the formazan dye was eluted using dimethyl sulfoxide (DMSO). The plate was shaken, and the absorbance was read using a spectrophotometer (Versamax) at a wavelength of 580 nm.…”

Section: Fly Stocks and Maintenancementioning

confidence: 99%

<i>In Vivo</i> Screening of Traditional Medicinal Plants for Neuroprotective Activity against Aβ42 Cytotoxicity by Using <i>Drosophila</i> Models of Alzheimer’s Disease

Liu

Lee

Kim

et al. 2015

Biological & Pharmaceutical Bulletin

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Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by progressive neuronal loss with amyloid β-peptide (Aβ) plaques. Despite several drugs currently used to treat AD, their beneficial effects on AD progress remains under debate. Here, we established a rapid in vivo screening system using Drosophila AD models to assess the neuroprotective activities of medicinal plants that have been used in traditional Chinese medicine. Among 23 medicinal plants tested, the extracts from five plants, Coriandrum sativum, Nardostachys jatamansi, Polygonum multiflorum (P. multiflorum), Rehmannia glutinosa, and Sorbus commixta (S. commixta), showed protective effects against the Aβ42 neurotoxicity. We further characterized the neuroprotective activity of ethanol extracts from P. multiflorum and S. commixta. Aβ42-expressing flies that we used showed AD neurological phenotypes, such as decreased survival and motility and increased cell death and reactive oxygen species level. However, feeding these flies extracts from P. multiflorum or S. commixta showed strong suppression of such phenotypes. Similar results were observed in human cells, so that the treatment of P. multiflorum and S. commixta extracts increased the viability of Aβ-treated SH-SY5Y cells. Moreover, 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside, one of the main constituents of P. multiflorum, also showed similar protective activity against Aβ42 cytotoxicity in both Drosophila and human cells. Taken together, our results suggest that both P. multiflorum and S. commixta have therapeutic potential for the treatment of neurodegenerative diseases, such as AD.Key words amyloid β-peptide 42 (Aβ42); Alzheimer's disease; Drosophila; Polygonum multiflorum; reactive oxygen species (ROS); Sorbus commixta Alzheimer's disease (AD) is a common neurodegenerative disease that is characterized by progressive neural loss caused by amyloid β-peptide (Aβ) plaques and cognitive deficits. 1,2) During disease progression, neurotoxic forms of Aβ cause neuronal damages via various cellular abnormalities such as increased oxidative damage, impaired energy metabolism, disrupted cellular calcium homeostasis, and increased inflammatory response.2,3) Several drugs have been approved to treat AD, given the dysfunction of cholinergic and glutamatergic neurotransmission; they function as acetylcholinesterase inhibitors or N-methyl-D-aspartate (NMDA) receptor antagonists. 4,5) However, the beneficial effects of these drugs during the progression of AD are still in question. 6) Due to its complex etiology, a combination therapy including the use of medicinal plants with effective components has been proposed as an alternative choice for treatment of AD. 7,8) Chinese traditional medicine has employed a variety of herbs to treat dementia, and a number of neuroprotective agents were isolated from these herbs. 7,8) For example, Polygonum multiflorum (P. multiflorum; syn. Reynoutria multiflora) has been known as a tonic and antiaging agent in many remedies for centuries, and...

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A protocol for in vivo detection of reactive oxygen species

Cited by 144 publications

References 6 publications

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive Oxygen Species-mediated Immunity against Leishmania mexicana and Serratia marcescens in the Phlebotomine Sand Fly Lutzomyia longipalpis

<i>In Vivo</i> Screening of Traditional Medicinal Plants for Neuroprotective Activity against Aβ42 Cytotoxicity by Using <i>Drosophila</i> Models of Alzheimer’s Disease

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