Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Kim, Hyeoncheol; Xue, Xiang

doi:10.3791/60682

Cited by 125 publications

(116 citation statements)

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“… 37 DCFH-DA, which has no fluorescence, passes through the cell membrane and enters into the cell freely and can be hydrolyzed by intracellular esterase to produce 2′,7′-dichlorodihydrofluorescein (DCFH). 38 DCFH cannot penetrate the cell membrane so that the probe can stay in the cell. Under the oxidation of intracellular ROS, nonfluorescent DCFH produces fluorescent 2′,7′-dichlorofluorescein (DCF).…”

Section: Results and Discussionmentioning

confidence: 99%

pH-Responsive Pluronic F127–Lenvatinib-Encapsulated Halogenated Boron-Dipyrromethene Nanoparticles for Combined Photodynamic Therapy and Chemotherapy of Liver Cancer

et al. 2021

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Combination therapy such as photodynamic therapy (PDT)-enhanced chemotherapy is regarded as a promising strategy for cancer treatment. Boron-dipyrromethene (BODIPY), as close relatives of porphyrins, was widely used in PDT. However, poor water solubility, rapid metabolism by the body and lack of targeting limits its clinical application. Lenvatinib, as the first-line drug for molecular-targeted therapy of liver cancer, restricted its clinical application for its side effects. Herein, to achieve the synergy between PDT and chemotherapy, we synthesized two halogenated BODIPY, BDPBr 2 and BDPCl 2 , which were prepared into self-assembly nanoparticles with lenvatinib, and were encapsulated with Pluronic F127 through the nanoprecipitation method, namely, LBPNPs (LBBr 2 NPs and LBCl 2 NPs). The fluorescence quantum yields of LBPNPs were 0.73 and 0.71, respectively. The calculated loading rates of lenvatinib for LBBr 2 NPs and LBCl 2 NPs were 11.8 and 10.2%, respectively. LBPNPs can be hydrolyzed under weakly acidic conditions (pH 5.0) to generate reactive oxygen species (ROS), and the release rate of lenvatinib reached 88.5 and 82.4%. Additionally, LBPNPs can be effectively taken up by Hep3B and Huh7 liver cancer cells, releasing halogenated BODIPY and lenvatinib in the acidic environment of tumor cells to enhance the targeting performance of chemotherapeutics. Compared with free lenvatinib and separate halogenated BODIPY, LBPNPs can inhibit tumor growth more effectively through pH-responsive chemo/photodynamic synergistic therapy and significantly promote the cascade of caspase apoptotic protease. This study shows that LBPNPs can be a promising nanotheranostic agent for synergetic chemo/photodynamic liver cancer therapy.

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Section: Results and Discussionmentioning

confidence: 99%

pH-Responsive Pluronic F127–Lenvatinib-Encapsulated Halogenated Boron-Dipyrromethene Nanoparticles for Combined Photodynamic Therapy and Chemotherapy of Liver Cancer

et al. 2021

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“…Intracellular ROS analysis by DCFH‐DA dye is widely used to quantify total ROS in cells. Relying on the biochemistry of acetyl groups cleavage by the cellular esterase to produce DCFH, in succession, DCFH undergoes two electron oxidation into DCF by the intracellular ROS, emitting green fluorescence at the excitation wavelength of 485 nm (Kim & Xue, 2020). Hence, in this relation, we have found an enhanced level of green fluorescence in the qualitative and quantitative analyses of ROS in HUVECs after exposure with 1000 μM of methomyl, carbaryl, metalaxyl, and pendimethalin.…”

Section: Discussionmentioning

confidence: 99%

Cytotoxicity and genotoxicity of methomyl, carbaryl, metalaxyl, and pendimethalin in human umbilical vein endothelial cells

Saquib

Siddiqui

Ansari

et al. 2021

J of Applied Toxicology

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Pesticides have adverse effects on the cellular functionality, which may trigger myriad of health consequences. However, pesticides‐mediated toxicity in the endothelial cells (ECs) is still elusive. Hence, in this study, we have used human umbilical vein endothelial cells (HUVECs) as a model to quantify the cytotoxicity and genotoxicity of four pesticides (methomyl, carbaryl, metalaxyl, and pendimethalin). In the MTT assay, HUVECs exposed to methomyl, carbaryl, metalaxyl, and pendimethalin demonstrated significant proliferation inhibition only at higher concentrations (500 and 1000 μM). Likewise, neutral red uptake (NRU) assay also showed proliferation inhibition of HUVECs at 500 and 1000 μM by the four pesticides, confirming lysosomal fragility. HUVECs exposed to the four pesticides significantly increased the level of intracellular reactive oxygen species (ROS). Comet assay and flow cytometric data exhibited DNA damage and apoptotic cell death in HUVECs after 24 h of exposure with methomyl, metalaxyl, carbaryl, and pendimethalin. This is a first study on HUVECs signifying the cytotoxic–genotoxic and apoptotic potential of carbamate insecticides (methomyl and carbaryl), fungicide (metalaxyl), and herbicide (pendimethalin). Overall, these pesticides may affect ECs functions and angiogenesis; nonetheless, mechanistic studies are warranted from the perspective of vascular biology using in vivo test models.

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“…Often probes are used that show neither a specificity for a ROS subspecies nor a defined cellular compartment, which leads to the frequently used terms “intracellular ROS” or “total cellular ROS,” which implicate that ROS once produced are equally distributed in the cell. Common examples for diffusible ROS probes are luminol ( Caldefie-Chezet et al, 2002 ; Pavelkova and Kubala, 2004 ), 2′,7′-dichlordihydrofluorescein-diacetat (H2DCF-DA) ( Ushijima et al, 1997 ; Hempel et al, 1999 ; Kim and Xue, 2020 ; Kim et al, 2021 ; Wang et al, 2021a ) or dihydroethidium (DHE) ( Gatliff et al, 2017 ; Wang and Zou, 2018 ; Zeller et al, 2021 ), which are regarded as compartment-specific but in fact they are not ( Lundqvist and Dahlgren, 1996 ; Ushijima et al, 1997 ; Hempel et al, 1999 ; Wang and Zou, 2018 ). There are compartment-specific derivates available for these ROS probes, namely Isoluminol ( Lundqvist and Dahlgren, 1996 ; Dahlgren and Karlsson, 1999 ; Caldefie-Chezet et al, 2002 ; Gluschko et al, 2018 ; Herb et al, 2019b ; Wolf et al, 2020 ), 5-(and −6)-carboxy-2′,7′-dihydrochlorofluorescein-diacetat (5/6-Carboxy-DCF) ( Hempel et al, 1999 ; Mak et al, 2017 ; Herb et al, 2019b ; Wolf et al, 2020 ) and MitoSOX Red (MitoSOX) ( Robinson et al, 2006 ; Mukhopadhyay et al, 2007 ) as alternatives, whose combined usage gives a much more confluent picture of the cellular ROS production.…”

Section: Reactive Oxygen Species: Handle With Care!mentioning

confidence: 99%

Reactive Oxygen Species: Not Omnipresent but Important in Many Locations

Herb

Gluschko

Schramm

2021

Front. Cell Dev. Biol.

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Reactive oxygen species (ROS), such as the superoxide anion or hydrogen peroxide, have been established over decades of research as, on the one hand, important and versatile molecules involved in a plethora of homeostatic processes and, on the other hand, as inducers of damage, pathologies and diseases. Which effects ROS induce, strongly depends on the cell type and the source, amount, duration and location of ROS production. Similar to cellular pH and calcium levels, which are both strictly regulated and only altered by the cell when necessary, the redox balance of the cell is also tightly regulated, not only on the level of the whole cell but in every cellular compartment. However, a still widespread view present in the scientific community is that the location of ROS production is of no major importance and that ROS randomly diffuse from their cellular source of production throughout the whole cell and hit their redox-sensitive targets when passing by. Yet, evidence is growing that cells regulate ROS production and therefore their redox balance by strictly controlling ROS source activation as well as localization, amount and duration of ROS production. Hopefully, future studies in the field of redox biology will consider these factors and analyze cellular ROS more specifically in order to revise the view of ROS as freely flowing through the cell.

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Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Cited by 125 publications

References 9 publications

pH-Responsive Pluronic F127–Lenvatinib-Encapsulated Halogenated Boron-Dipyrromethene Nanoparticles for Combined Photodynamic Therapy and Chemotherapy of Liver Cancer

pH-Responsive Pluronic F127–Lenvatinib-Encapsulated Halogenated Boron-Dipyrromethene Nanoparticles for Combined Photodynamic Therapy and Chemotherapy of Liver Cancer

Cytotoxicity and genotoxicity of methomyl, carbaryl, metalaxyl, and pendimethalin in human umbilical vein endothelial cells

Reactive Oxygen Species: Not Omnipresent but Important in Many Locations

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