Measurement of Intracellular ROS in Caenorhabditis elegans Using  2’,7’-Dichlorodihydrofluorescein Diacetate

Yoon, Dong Suk; Lee, Myon‐Hee; Seok, Dong

doi:10.21769/bioprotoc.2774

Cited by 84 publications

(49 citation statements)

References 4 publications

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“…DCFH 2 -DA can easily penetrate the cellular membrane and is enzymatically deacetylated by esterases. This reaction converts DCFH 2 -DA into the nonfluorescent compound DCFH 2 , which is then rapidly oxidized to highly fluorescent DCF in the presence of ROS (Ren et al 2017;Yoon et al 2018). Since C. elegans can undergo experimental oxidative stress conditions upon exposure to certain pro-oxidant compounds (Zhou et al 2011) leading to increased ROS levels, this characteristic allowed us to investigate the in vivo antioxidant capacity of carotenoid powders as the in vitro and in vivo efficacies of antioxidants could differ (You et al 2015).…”

Section: Ros Measurementmentioning

confidence: 99%

Antioxidant activities of spray-dried carotenoids using maltodextrin-Arabic gum as wall materials

2021

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Background Carotenoids are natural pigments that are highly sensitive to light, heat, acids, oxygen, metals, and free radicals, which degrade the antioxidant activities of carotenoids. Microencapsulation techniques have been used to prevent carotenoid degradation and preserve their antioxidant activities. In this work, we aimed to encapsulate mamey (Pouteria sapota) and carrot (Daucus carota) carotenoids in mixtures of maltodextrin (10% w/v) and Arabic gum (5 or 10% w/v) by spray-drying. The obtained powders were stored at different temperatures (4 and 25 °C) with or without access to daylight, and changes in color, carotenoid content, and antioxidant activity were analyzed monthly for three months. Moreover, the in vivo antioxidant activities of spray-dried carotenoids were evaluated in Caenorhabditis elegans. Results The carotenoid and antioxidant activity losses of stored carotenoids were found in the following order: 25 °C in daylight > 25 °C without access to daylight > 4 °C without access to daylight. Moreover, the combination of maltodextrin and Arabic gum (10%) was more effective to prevent carotenoid loss compared to maltodextrin and Arabic gum (5%). In vivo antioxidant activity results showed that spray-dried carotenoids reduced approximately 30% of reactive oxygen species (ROS) production in nematodes, even after three months of storage. Conclusions Mamey and carrot carotenoids were successfully encapsulated by the spray-drying technique. The spray-dried carotenoids effectively reduced the intracellular ROS levels and neutralized the oxidative stress damage in C. elegans, even after three months of storage. Moreover, the antioxidant activities of mamey carotenoids were equally effective as those of carrot carotenoids, meaning that mamey carotenoids could be seen as an alternative source of carotenoids.

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Section: Ros Measurementmentioning

confidence: 99%

Antioxidant activities of spray-dried carotenoids using maltodextrin-Arabic gum as wall materials

2021

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“…First, the Amplex red hydrogen peroxide assay kit, 10-acetyl-3,7-dihydroxyphenoxazine, was used to selectively detect H 2 O 2 [18]. Second, using the 2′,7′-dichlorofluorescein diacetate (DCFDA) assay, fluorescent dye staining was used to detect all induced ROS [19]. In the result of the Amplex red hydrogen peroxide assay, treatment of B. xylophilus with 50 ppm juglone generated 1.5–2.5 times more H 2 O 2 than the 1% DMSO treatment, taken as the control ( p < 0.05).…”

Section: Resultsmentioning

confidence: 99%

Nematicidal Activities of Three Naphthoquinones against the Pine Wood Nematode, Bursaphelenchus xylophilus

Jea

Kim

2019

Molecules

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Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, is a serious forest pest, causing enormous economic losses in pine trees in Korea, China, Japan, and countries in Western Europe. To prevent pine wilt disease (PWD), trunk injection with nematicide is performed in Korea. Although these nematicidal agents are quite efficient, the development of new nematicidal agents is needed to prevent pesticide resistance and reduce pest management costs. The aim of this study was to investigate nematicidal activities of pure naphthoquinones (NTQs)–1,4-NTQ, juglone, and plumbagin—against B. xylophilus via in vitro and semi-in vivo assays to identify new candidate agents for trunk injection. Estimated LC50 values (48 h exposure) were 100.0 ppm, 57.0 ppm, and 104.0 ppm for 1,4-NTQ, juglone, and plumbagin, respectively. In the semi-in vivo assay on pine bolt of the Japanese black pine, Pinus thunbergii, the population of inoculated B. xylophilus was significantly decreased at two weeks after treatment with juglone when compared with the effects of treatment with 1,4-NTQ and plumbagin. We also observed that naphthoquinones could generate reactive oxygen species, which presumably indicated that naphthoquinones caused significant oxidative stress in B. xylophilus. The findings of this study suggest the nematicidal potential of naphthoquinones and their possible use in further in vivo assays to test their nematicidal efficacy against B. xylophilus when injected through trunk injection.

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“…2,7-Dichlorodihydrofluorescein diacetate (H 2 DCFDA; Sigma-Aldrich), a cell-permeable fluorogenic substance, was taken to analyze the amount of reactive oxygen species (ROS) in C. elegans and the influence of extracts on ROS levels according to Yoon et al, 2018 [ 45 ], with minor modifications. H 2 DCF is oxidized by ROS, leading to the formation of the fluorescent dye dichlorofluorescein (DCF).…”

Section: Methodsmentioning

confidence: 99%

Increased Stress Resistance and Lifespan in Chaenorhabditis elegans Wildtype and Knockout Mutants—Implications for Depression Treatment by Medicinal Herbs

2021

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Depression and anxiety disorders are widespread diseases, and they belong to the leading causes of disability and greatest burdens on healthcare systems worldwide. It is expected that the numbers will dramatically rise during the COVID-19 pandemic. Established medications are not sufficient to adequately treat depression and are not available for everyone. Plants from traditional medicine may be promising alternatives to treat depressive symptoms. The model organism Chaenorhabditis elegans was used to assess the stress reducing effects of methanol/dichlormethane extracts from plants used in traditional medicine. After initial screening for antioxidant activity, nine extracts were selected for in vivo testing in oxidative stress, heat stress, and osmotic stress assays. Additionally, anti-aging properties were evaluated in lifespan assay. The extracts from Acanthopanax senticosus, Campsis grandiflora, Centella asiatica, Corydalis yanhusuo, Dan Zhi, Houttuynia cordata, Psoralea corylifolia, Valeriana officinalis, and Withaniasomnifera showed antioxidant activity of more than 15 Trolox equivalents per mg extract. The extracts significantly lowered ROS in mutants, increased resistance to heat stress and osmotic stress, and the extended lifespan of the nematodes. The plant extracts tested showed promising results in increasing stress resistance in the nematode model. Further analyses are needed, in order to unravel underlying mechanisms and transfer results to humans.

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Measurement of Intracellular ROS in Caenorhabditis elegans Using 2’,7’-Dichlorodihydrofluorescein Diacetate

Cited by 84 publications

References 4 publications

Antioxidant activities of spray-dried carotenoids using maltodextrin-Arabic gum as wall materials

Antioxidant activities of spray-dried carotenoids using maltodextrin-Arabic gum as wall materials

Nematicidal Activities of Three Naphthoquinones against the Pine Wood Nematode, Bursaphelenchus xylophilus

Increased Stress Resistance and Lifespan in Chaenorhabditis elegans Wildtype and Knockout Mutants—Implications for Depression Treatment by Medicinal Herbs

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