Methods to Monitor ROS Production by Fluorescence Microscopy and Fluorometry

Wojtala, Aleksandra; Bonora, Massimo; Malińska, Dominika; Pinton, Paolo; Duszyński, Jerzy; Wieckowski, Mariusz R.

doi:10.1016/b978-0-12-416618-9.00013-3

Cited by 230 publications

(127 citation statements)

References 39 publications

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“…Oxidative Stress and Antioxidant Levels-ROS production in neuronal cells was assessed using 2,7-dichlorohydrofluorescein diacetate and DHE dyes (49). Briefly, neuronal cells (1 ϫ 10 3 cells) were treated with BPA (100 M) for 12 h, followed by incubation with 2,7-dichlorohydrofluorescein diacetate (10 M) for 30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

Agarwal

Tiwari

Seth

et al. 2015

Journal of Biological Chemistry

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Background:The effects of xenoestrogen bisphenol-A on autophagy, and association with oxidative stress and apoptosis are still elusive. Results: Transient activation of autophagy protects against bisphenol-A-induced neurodegeneration via AMPK activation and mTOR down-regulation. Conclusion: Autophagy induction against bisphenol-A is an early cell's tolerance response. Significance: Autophagy provides an imperative biological marker for evaluation of neurotoxicity by xenoestrogen.

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Section: Methodsmentioning

confidence: 99%

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

Agarwal

Tiwari

Seth

et al. 2015

Journal of Biological Chemistry

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“…Recently, increases in reactive oxygen species (ROS) generation were found to be a key factor to activate NLRP3 inflammasomes (Bauernfeind et al, 2011). ROS, that can readily be measured in brain by multiple methodologies (Mattiasson 2004; Wojtala et al, 2014; Bar-Or et al, 2015), appear to be at the crossroads of inflammasomes and inflammation, and elevated markers of oxidative stress have been reported in TBI (Table 5). …”

Section: Methodologically Evaluating Neuroinflammation: What Can Be Mmentioning

confidence: 99%

Neuroinflammation in animal models of traumatic brain injury

Chiu

Liao

Yang

et al. 2016

Journal of Neuroscience Methods

200

121

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Traumatic brain injury (TBI) is a leading cause of mortality and morbidity worldwide. Neuroinflammation is prominent in the short and long-term consequences of neuronal injuries that occur after TBI. Neuroinflammation involves the activation of glia, including microglia and astrocytes, to release inflammatory mediators within the brain, and the subsequent recruitment of peripheral immune cells. Various animal models of TBI have been developed that have proved valuable to elucidate the pathophysiology of the disorder and to assess the safety and efficacy of novel therapies prior to clinical trials. These models provide an excellent platform to delineate key injury mechanisms that associate with types of injury (concussion, contusion, and penetration injuries) that occur clinically for the investigation of mild, moderate, and severe forms of TBI. Additionally, TBI modeling in genetically engineered mice, in particular, has aided the identification of key molecules and pathways for putative injury mechanisms, as targets for development of novel therapies for human TBI. This Review details the evidence showing that neuroinflammation, characterized by the activation of microglia and astrocytes and elevated production of inflammatory mediators, is a critical process occurring in various TBI animal models, provides a broad overview of commonly used animal models of TBI, and overviews representative techniques to quantify markers of the brain inflammatory process. A better understanding of neuroinflammation could open therapeutic avenues for abrogation of secondary cell death and behavioral symptoms that may mediate the progression of TBI.

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“…MitoSOX fluorescence was measured at 510nm excitation and 580nm emission on a SpectraMax® i3 microplate reader (Molecular Devices; Sunnyvale, CA). MitoSOX fluorescence was adjusted based on cell number as determined by the sulforhodamine B assay (as described in [27]) measured at an absorbance of 565nm.…”

Section: Methodsmentioning

confidence: 99%

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

et al. 2016

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Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.

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Methods to Monitor ROS Production by Fluorescence Microscopy and Fluorometry

Cited by 230 publications

References 39 publications

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

Neuroinflammation in animal models of traumatic brain injury

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

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