Refinement of the Dichlorofluorescein Assay for Flow Cytometric Measurement of Reactive Oxygen Species in Irradiated and Bystander Cell Populations

Hafer, Kurt; Iwamoto, Keisuke S.; Schiestl, Robert H.

doi:10.1667/rr1212.1

Cited by 80 publications

(45 citation statements)

References 40 publications

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“…Induction of DSBs in bystander cells has been documented extensively (16)(17)(18), and the involvement of ROS/RNS in this effect has been demonstrated (19,20). However, the time relationship between these different events had, as far as we know, never been investigated, and most reported experiments related to the RIBE include indirect evidence by the use of scavengers (10) and few directly show ROS generation (6,19,21).…”

Section: Discussionmentioning

confidence: 99%

Membrane-Dependent Bystander Effect Contributes to Amplification of the Response to Alpha-Particle Irradiation in Targeted and Nontargeted Cells

Hanot¹,

Hoarau²,

Carrière³

et al. 2009

International Journal of Radiation Oncology*Biology*Physics

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Section: Discussionmentioning

confidence: 99%

Membrane-Dependent Bystander Effect Contributes to Amplification of the Response to Alpha-Particle Irradiation in Targeted and Nontargeted Cells

Hanot¹,

Hoarau²,

Carrière³

et al. 2009

International Journal of Radiation Oncology*Biology*Physics

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“…3 While useful, these methods have specific limitations. Measurement of the GSH/GSSG ratio is notoriously difficult because of oxidation of GSH during cell disruption and sample preparation 1,4 ; ROS sensitive dyes exhibit promiscuous reactivity with multiple radical species 5 ; protein oxidation reflects accumulated damage, not current redox status. 6 Importantly, none of these measurements is well suited for in vivo experimentation.…”

Section: Introductionmentioning

confidence: 99%

A novel approach for in vivo measurement of mouse red cell redox status

Löhneysen

Soldau

et al. 2011

Blood

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Maintenance of a reducing redox balance is a critical physiologic function of red cells (RBC) that can be perturbed in variety of RBC pathologies. Here we describe a new approach to evaluate in vivo RBC redox status using a redox sensitive GFP (roGFP2) sensor under control of a ␤-globin mini-promoter, directing expression specifically to erythroid cells. RoGFP2 expressing RBCs demonstrate ratiometric and reversible shifts in fluorescence on exposure to oxidants and reductants. We demonstrate that roGFP2 expressing RBC can be used to monitor thiol redox status during in vitro phenylhydrazine treatment and over the course of in vivo RBC aging, where a shift to a more oxidized state is observed in older cells. Thus, roGFP2 transgenic mice are a new and versatile tool that can be used to probe how RBC redox status responds in the context of drug therapy, physiologic stressors and pathologic states. (Blood. 2011;118(13):3694-3697) IntroductionMany approaches for evaluation of RBC redox status have been established, including direct evaluation of the glutathione redox pair (GSH/GSSG), 1 and indirect approaches such as measuring rates of reactive oxygen species (ROS) production using oxidation sensitive dyes 2 and measurement of protein oxidation. 3 While useful, these methods have specific limitations. Measurement of the GSH/GSSG ratio is notoriously difficult because of oxidation of GSH during cell disruption and sample preparation 1,4 ; ROS sensitive dyes exhibit promiscuous reactivity with multiple radical species 5 ; protein oxidation reflects accumulated damage, not current redox status. 6 Importantly, none of these measurements is well suited for in vivo experimentation. Development of redoxsensitive green fluorescent protein variants (roGFPs) provides a new option for measuring cellular redox status. 7-12 RoGFP2 has been engineered as a redox sensor by replacement of S147 and Q204 positions of EGFP with 2 cysteine residues. These cysteines are either reduced or form an intra-molecular disulfide bond in equilibrium with local redox status. Rising levels of oxidized roGFP indicate a shift in cellular redox status that is monitored by following the ratio of GFP fluorescence emission after excitation at 405 versus 488 nm. [9][10][11]13 Here, we show how roGFP2-expressing RBC in transgenic mice can be used to follow, in vitro and in vivo, important changes in RBC redox status. MethodsGeneration of roGFP2 transgenic mice roGFP2 was sub-cloned into a ß-globin minigene expression plasmid p'LCR-␤pr-BglII-3Ј␤(int2-enh), 14 and micro-injected into pronuclei of fertilized C57BL/6J mouse oocytes. The best transgenic founder animal expressed GFP in ϳ 50% of peripheral RBC by FACS, and gave rise to progeny with the same expression pattern. Flow cytometryRBCs (1 ϫ 10 6 ) were washed and resuspended in 1 mL FACS buffer (PBS/0.2%BSA/2mM EDTA). Cells were incubated with oxidant (H 2 O 2 or t-butyl hydroperoxide), reductant (DTT) or Phenylhydrazine (PHZ) in the presence of 10mM glucose at 37°C for 60 minutes. Cells were analyzed o...

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“…The production of reactive oxygen species (ROS) in ARPE-19 cells in the presence or absence of nano-curcumin or curcumin (5 or 50 μM) was determined by a FACS-based ROS detection kit (Enzo Life Sciences, Plymouth Meeting, PA, USA) using a modified protocol [35]. Untreated and unstained cells were used alongside a positive control with a ROS inducer (200 μM pyocyanine) and a negative control with a ROS inhibitor (5 mM N-acetyl-Lcysteine).…”

Section: Analysis Of Reactive Oxygen Species Production Of Arpe-19 Cementioning

confidence: 99%

Modulation of the Proteasome Pathway by Nano-Curcumin and Curcumin in Retinal Pigment Epithelial Cells

Carvalho¹,

Verwoert²,

Vogels³

et al. 2017

Ophthalmic Res

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Introduction: Curcumin has multiple biological effects including the modulation of protein homeostasis by the ubiquitin-proteasome system. The purpose of this study was to assess the in vitro cytotoxic and oxidative effects of nano-curcumin and standard curcumin and characterize their effects on proteasome regulation in retinal pigment epithelial (RPE) cells. Methods: Viability, cell cycle progression, and reactive oxygen species (ROS) production were determined after treatment with nano-curcumin or curcumin. Subsequently, the effects of nano-curcumin and curcumin on proteasome activity and the gene and protein expression of proteasome subunits PA28α, α₇, β₅, and β_5i were assessed. Results: Nano-curcumin (5-100 μM) did not show significant cytotoxicity or anti-oxidative effects against H₂O₂-induced oxidative stress, whereas curcumin (≥10 μM) was cytotoxic and a potent inducer of ROS production. Both nano-curcumin and curcumin induced changes in proteasome-mediated proteolytic activity characterized by increased activity of the proteasome subunits β₂ and β_5i/β₁ and reduced activity of β₅/β_1i. Likewise, nano-curcumin and curcumin affected mRNA and protein levels of household and immunoproteasome subunits. Conclusions: Nano-curcumin is less toxic to RPE cells and less prone to induce ROS production than curcumin. Both nano-curcumin and curcumin increase proteasome-mediated proteolytic activity. These results suggest that nano-curcumin may be regarded as a proteasome-modulating agent of limited cytotoxicity for RPE cells.

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Refinement of the Dichlorofluorescein Assay for Flow Cytometric Measurement of Reactive Oxygen Species in Irradiated and Bystander Cell Populations

Cited by 80 publications

References 40 publications

Membrane-Dependent Bystander Effect Contributes to Amplification of the Response to Alpha-Particle Irradiation in Targeted and Nontargeted Cells

Membrane-Dependent Bystander Effect Contributes to Amplification of the Response to Alpha-Particle Irradiation in Targeted and Nontargeted Cells

A novel approach for in vivo measurement of mouse red cell redox status

Modulation of the Proteasome Pathway by Nano-Curcumin and Curcumin in Retinal Pigment Epithelial Cells

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