Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species

Tetz, Lauren M.; Kamau, Patricia W; Cheng, Adrienne A; Meeker, John D.; Loch‐Caruso, Rita

doi:10.1016/j.vascn.2013.01.195

Cited by 63 publications

(54 citation statements)

References 41 publications

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“…24 Fluorescent chemical probes have several disadvantages including lack of ROS specificity, unspecific oxidation, photobleaching, and irreversible reaction with ROS, which makes a dynamic measurement of redox alterations impossible. 25,26 Genetically encoded reduction-oxidationsensitive protein probes are mostly YFP or GFP derivatives and were developed to serve as tools for real-time monitoring of the redox potential in living cells and tissues. 27 These biosensors mimic redox relays in which they exchange electrons with oxidoreductases, peroxidases, or other enzymes, which allows specificity and sensitivity of the obtained signals.…”

Section: Discussionmentioning

confidence: 99%

Redox Imaging Using Cardiac Myocyte-Specific Transgenic Biosensor Mice

Swain

Kesemeyer

Meyer-Roxlau

et al. 2016

Circulation Research

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Rationale: Changes in redox potentials of cardiac myocytes are linked to several cardiovascular diseases. Redox alterations are currently mostly described qualitatively using chemical sensors, which however do not allow quantifying redox potentials, lack specificity, and the possibility to analyze subcellular domains. Recent advances to quantitatively describe defined redox changes include the application of genetically encoded redox biosensors. Objective: Establishment of mouse models, which allow the quantification of the glutathione redox potential ( E GSH ) in the cytoplasm and the mitochondrial matrix of isolated cardiac myocytes and in Langendorff-perfused hearts based on the use of the redox-sensitive green fluorescent protein 2, coupled to the glutaredoxin 1 (Grx1-roGFP2). Methods and Results: We generated transgenic mice with cardiac myocyte–restricted expression of Grx1-roGFP2 targeted either to the mitochondrial matrix or to the cytoplasm. The response of the roGFP2 toward H 2 O 2 , diamide, and dithiothreitol was titrated and used to determine the E GSH in isolated cardiac myocytes and in Langendorff-perfused hearts. Distinct E GSH were observed in the cytoplasm and the mitochondrial matrix. Stimulation of the cardiac myocytes with isoprenaline, angiotensin II, or exposure to hypoxia/reoxygenation additionally underscored that these compartments responded independently. A compartment-specific response was also observed 3 to 14 days after myocardial infarction. Conclusions: We introduce redox biosensor mice as a new tool, which allows quantification of defined alterations of E GSH in the cytoplasm and the mitochondrial matrix in cardiac myocytes and can be exploited to answer questions in basic and translational cardiovascular research.

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Section: Discussionmentioning

confidence: 99%

Redox Imaging Using Cardiac Myocyte-Specific Transgenic Biosensor Mice

Swain

Kesemeyer

Meyer-Roxlau

et al. 2016

Circulation Research

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“…ROS stress was assessed using a fluorescent probe (carboxy-H2DCFDA; C400; Invitrogen, La Jolla, CA) as previously described (53). Briefly, cells seeded in a T75 tissue culture flask at a density of 2000 cells/cm 2 were maintained for 5 days before incubating with 10 lM C400 for 1 h. The ROS fluorescence intensity was analyzed using an FACSCalibur flow cytometer, and the ROS level was represented as the percentage of C400-stained cells.…”

Section: Detection Of the Intracellular Ros Levelmentioning

confidence: 99%

Senescence-Associated MCP-1 Secretion Is Dependent on a Decline in BMI1 in Human Mesenchymal Stromal Cells

Jin

Lee

Heo

et al. 2016

Antioxidants & Redox Signaling

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Aims: Cellular senescence and its secretory phenotype (senescence-associated secretory phenotype [SASP]) develop after long-term expansion of mesenchymal stromal cells (MSCs). Further investigation of this phenotype is required to improve the therapeutic efficacy of MSC-based cell therapies. In this study, we show that positive feedback between SASP and inherent senescence processes plays a crucial role in the senescence of umbilical cord blood-derived MSCs (UCB-MSCs). Results: We found that monocyte chemoattractant protein-1 (MCP-1) was secreted as a dominant component of the SASP during expansion of UCB-MSCs and reinforced senescence via its cognate receptor chemokine (c-c motif) receptor 2 (CCR2) by activating the ROS-p38-MAPK-p53/p21 signaling cascade in both an autocrine and paracrine manner. The activated p53 in turn increased MCP-1 secretion, completing a feed-forward loop that triggered the senescence program in UCBMSCs. Accordingly, knockdown of CCR2 in UCB-MSCs significantly improved their therapeutic ability to alleviate airway inflammation in an experimental allergic asthma model. Moreover, BMI1, a polycomb protein, repressed the expression of MCP-1 by binding to its regulatory elements. The reduction in BMI1 levels during UCB-MSC senescence altered the epigenetic status of MCP-1, including the loss of H2AK119Ub, and resulted in derepression of MCP-1. Innovation: Our results provide the first evidence supporting the existence of the SASP as a causative contributor to UCB-MSC senescence and reveal a so far unappreciated link between epigenetic regulation and SASP for maintaining a stable senescent phenotype. Conclusion: Senescence of UCB-MSCs is orchestrated by MCP-1, which is secreted as a major component of the SASP and is epigenetically regulated by BMI1. Antioxid. Redox Signal. 24, 471-485.

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“…Production of intracellular ROS was evaluated using a 2'7'-dichlorofluorescein (DCF) assay (27), which was conducted using dichlorofluorescein diacetate (H 2 DCF-DA; Sigma-Aldrich; Merck Millipore). Briefly, following treatment, rBMSCs were washed and then incubated with 10 µM H 2 DCF-DA in serum-free culture medium (Gibco; Thermo Fisher Scientific, Inc.) for 30 min at 37˚C in the dark.…”

Section: Animals and Bbrmentioning

confidence: 99%

Protective effect of berberine against oxidative stress-induced apoptosis in rat bone marrow-derived mesenchymal stem cells

Wang

Nianhong

et al. 2016

Experimental and Therapeutic Medicine

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Abstract. Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to be used for the treatment of delayed union, nonunion or persistent bone defects in MSC-based cell therapy. However, implantation of BMSCs into the fracture site is confronted with apoptosis on account of harsh conditions and oxidative stress. In the present study, the anti-apoptotic effects of berberine (BBR) on BMSCs subjected to hydrogen peroxide (H 2 O 2 ) are investigated, and the potential underlying mechanisms are explored. Oxidative injury was induced by exposure to H 2 O 2 , and cell viability was assessed using a cell counting kit-8 assay. The apoptosis of BMSCs was measured by Hoechst 33258 and Annexin V-fluorescein isothiocyanate/propidium iodide assay. Reactive oxygen species staining and superoxide dismutase (SOD) assay were applied to assess the anti-oxidative effect of BBR. Finally, western blot was performed to measure the expression levels of phosphorylated (p)-Akt, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and cleaved caspase-3. In the present study, it was identified that BBR remarkably attenuated H 2 O 2 -induced apoptotic cell death via quenching ROS production and increasing SOD activity. Further studies indicated that BBR can reduce apoptosis by upregulating the expression level of p-Akt and Bcl-2, and downregulating the expression levels of Bax and cleaved caspase-3. Taken together, the results of the present study demonstrate that pretreatment with BBR could alleviate H 2 O 2 -induced apoptosis in rat BMSCs in vitro.

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Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species

Cited by 63 publications

References 41 publications

Redox Imaging Using Cardiac Myocyte-Specific Transgenic Biosensor Mice

Redox Imaging Using Cardiac Myocyte-Specific Transgenic Biosensor Mice

Senescence-Associated MCP-1 Secretion Is Dependent on a Decline in BMI1 in Human Mesenchymal Stromal Cells

Protective effect of berberine against oxidative stress-induced apoptosis in rat bone marrow-derived mesenchymal stem cells

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