The authors would like to thank Gregg Sealy and Alain Conti for excellent technical assistance and Prof. Zsuzsanna Izsv&#225;k from the Max-Delbr&#252;ck Center in Berlin for kindly providing the pSB100X&#160;and pT2-CAGGS-Venus&#160;plasmids. This work was supported by the Swiss National Sciences Foundation and the European Commission in the context of the Seventh Framework Programme. Z.I was funded by European Research Council, ERC Advanced [ERC-2011-ADG 294742].

<ol>
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The authors have nothing to disclose.

The protocol presented here offers an approach to analyze the anti-oxidative and protective function of PEDF and GM-CSF produced by transfected cells, which can be applied to cells transfected with any putative beneficial gene. In gene therapeutic strategies that have the objective to deliver proteins to tissue by transplanting genetically modified cells, it is critical to obtain information as to the level of protein expression, the longevity of expression, and the effectiveness of the expressed protein in a model of th...

The model described here, offers a useful approach to evaluate the efficiency ofbiopharmaceutical agents for reducing oxidative stress in cells. We have used the model to investigate the protective effects of PEDF and GM-CSF on the H2O2-mediated oxidative stress on retinal pigment epithelial cells, which are exposed to high levels of O2, and visible light, and the phagocytosis of photoreceptor outer segment membranes, generating significant levels of reactive oxygen species (ROS)1,2. They are considered a major contributor to the pathogenesis of avascular age-related macular degeneration (aAMD)3,4,5,6,7,8. Besides, there is a decrease in RPE-synthesized neuroprotective factors, specifically the pigment epithelium-derived factor (PEDF), insulin-like growth factors (IGFs), and granulocyte macrophage-colony-stimulating factor (GM-CSF) leading to the dysfunction and loss of RPE cells, followed by photoreceptor and retinal ganglion cell (RGC) death3,4,5. AMD is a complex disease that results from the interaction between metabolic, functional, genetic, and environmental factors4. The lack of treatments for aAMD is the major cause of blindness in patients older than 60 years of age in industrialized countries9,10. The reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of genetically modified RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation and supporting cell survival11,12,13,14,15,16. Even though there are several methodologies to deliver genes to cells, we have chosen the non-viral hyperactive Sleeping Beauty transposon system to deliver the PEDF and GM-CSF genes to RPE cells because of its safety profile, the integration of the genes into the host cells&#39; genome, and its propensity to integrate the delivered genes in non-transcriptionally active sites as we have shown previously17,18,19.
Cellular oxidative stress can be induced in cells cultured in vitro by several oxidative agents, including hydrogen peroxide (H2O2), 4-hydroynonenal (HNE), tertbutylhydroperoxide (tBH), high oxygen tensions, and visible light (full spectrum or UV irradiation)20,21. High oxygen tensions and light require special equipment and conditions, which limits transferability to other systems. Agents such as H2O2, HNE, and tBH induce overlapping oxidative stress molecular and cellular changes. We chose H2O2 to test the antioxidant activity of PEDF and GM-CSF because it is convenient and biologically relevant since it is produced by RPE cells as a reactive oxygen intermediate during photoreceptor outer segment phagocytosis22 and it is found in ocular tissues in vivo23. Since the oxidation of glutathione may be partially responsible for the production of H2O2 in the eye, we have analyzed the levels of GSH/glutathione in our studies, which are linked to H2O2-induced oxidative stress and the regenerative capacity of cells21,22. The analysis of glutathione levels is especially relevant since it participates in the anti-oxidative protective mechanisms in the eye24. Exposure to H2O2 is used frequently as a model to examine the oxidative stress susceptibility and antioxidant activity of RPE cells1,25,26,27,28,29,30, and, additionally, it shows similarities to light-induced oxidative stress damage, a &#34;physiological&#34; source of oxidative stress21.
To evaluate the functionality and the effectiveness of neuroprotective factors, we have developed an in vitro model that allows for the analysis to quantify the anti-oxidative effect of growth factors expressed by cells genetically modified to overexpress PEDF and GM-CSF. Here, we show that RPE cells transfected with the genes for PEDF and GM-CSF are more resistant to the harmful effects of H2O2 than are non-transfected control cells, as evidenced by cell morphology, density, viability, intracellular level of glutathione, and expression of UCP2 gene, which codes for the mitochondrial uncoupling protein 2 that has been shown to reduce reactive oxygen species (ROS)31.

<table><tbody><tr><td>24-well plates</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>6-well plates</td><td>Greiner</td><td>7657160</td><td></td></tr><tr><td>96-well culture plate white with clear flat bottom&amp;nbsp;</td><td>Costar</td><td>3610</td><td>Allows to check the cells before measuring the luminescence (GSH-Glo Assay)</td></tr><tr><td>96-well plates</td><td>Corning&amp;nbsp;</td><td>353072</td><td></td></tr><tr><td>Acrylamid 40%</td><td>Biorad</td><td>161-0144</td><td></td></tr><tr><td>Amphotericin B</td><td>AMIMED</td><td>4-05F00-H</td><td></td></tr><tr><td>Antibody anti-GMCSF</td><td>ThermoFisher Scientific</td><td>PA5-24184</td><td></td></tr><tr><td>Antibody anti-mouse IgG/IgA/IgM&amp;nbsp;</td><td>Agilent</td><td>P0260</td><td></td></tr><tr><td>Antibody anti-PEDF&amp;nbsp;</td><td>Santa Cruz Biotechnology Inc</td><td>sc-390172</td><td></td></tr><tr><td>Antibody anti-penta-His&amp;nbsp;</td><td>Qiagen</td><td>34660</td><td></td></tr><tr><td>Antibody anti-phospho-Akt</td><td>Cell Signaling Technology</td><td>9271</td><td></td></tr><tr><td>Antibody anti-rabbit IgG H&amp;amp;L-HRP</td><td>Abcam</td><td>ab6721</td><td></td></tr><tr><td>Antibody donkey anti-rabbit Alexa Fluor 594&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>A11034</td><td></td></tr><tr><td>Antibody goat anti-mouse Alexa 488</td><td>ThermoFisher Scientific</td><td>A-11029</td><td></td></tr><tr><td>ARPE-19 cell line</td><td>ATCC</td><td>CRL-2302</td><td></td></tr><tr><td>BSA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9418-500G</td><td></td></tr><tr><td>chamber culture glass slides</td><td>Corning</td><td>354118</td><td></td></tr><tr><td>CytoTox-Glo Cytotoxicity Assay&amp;nbsp;</td><td>Promega</td><td>G9291</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542-5MG</td><td></td></tr><tr><td>DMEM/Ham`s F12&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8062</td><td></td></tr><tr><td>Duo Set ELISA kit</td><td>R&amp;amp;D Systems&amp;nbsp;</td><td>DY215-05</td><td></td></tr><tr><td>EDTA</td><td>ThermoFisher Scientific</td><td>78440</td><td></td></tr><tr><td>ELISAquant kit</td><td>BioProducts MD</td><td>PED613-10-Human</td><td></td></tr><tr><td>Eyes (human)</td><td>Lions Gift of Sight Eye Bank (Saint Paul, MN)</td><td></td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Brunschwig</td><td>P40-37500</td><td></td></tr><tr><td>Fluoromount Aqueous Mounting Medium</td><td>Sigma-Aldrich</td><td>F4680-25ML</td><td></td></tr><tr><td>FLUOstar Omega plate reader&amp;nbsp;</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>GraphPad Prism software (version 8.0)</td><td>GraphPad Software, Inc.</td><td></td><td></td></tr><tr><td>GSH-Glo Glutathione Assay</td><td>Promega</td><td>V6912</td><td></td></tr><tr><td>hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Merck</td><td>107209</td><td></td></tr><tr><td>ImageJ software (image processing program)</td><td>W.S. Rasband, NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/; 1997&amp;ndash;2014</td><td></td><td></td></tr><tr><td>Imidazol&amp;nbsp;</td><td>Axonlab</td><td>A1378.0010</td><td></td></tr><tr><td>Leica DMI4000B microscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>LightCycler 480 Instrument II&amp;nbsp;</td><td>Roche Molecular Systems</td><td></td><td></td></tr><tr><td>LightCycler 480 SW1.5.1 software</td><td>Roche Molecular Systems</td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71376-1000</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Axonlab</td><td>3468.1000</td><td></td></tr><tr><td>Neon Transfection System&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>MPK5000</td><td></td></tr><tr><td>Neon Transfection System 10 &amp;micro;L Kit</td><td>ThermoFisher Scientific</td><td>MPK1096</td><td></td></tr><tr><td>Neubauer chamber</td><td>Marienfeld-superior</td><td>640010</td><td></td></tr><tr><td>Ni-NTA superflow&amp;nbsp;</td><td>Qiagen</td><td>30410</td><td></td></tr><tr><td>Nitrocellulose&amp;nbsp;</td><td>VWR</td><td>732-3197</td><td></td></tr><tr><td>Omega Lum G Gel Imaging System</td><td>Aplegen Life Science</td><td></td><td></td></tr><tr><td>PBS 1X</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma-Aldrich</td><td>P0781-100</td><td></td></tr><tr><td>PerfeCTa SYBR Green FastMix</td><td>Quantabio</td><td>95072-012</td><td></td></tr><tr><td>PFA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>158127-100G</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>23227</td><td></td></tr><tr><td>Primers&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td></td><td>See Table 1 in Supplementary Materials</td></tr><tr><td>pSB100X (250 ng/&amp;micro;L)</td><td></td><td></td><td>M&amp;aacute;t&amp;eacute;s et al., 2009. Provide by Prof. Zsuzsanna Izsvak</td></tr><tr><td>pT2-CMV-GMCSF-His plasmid DNA (250 ng/&amp;micro;L)</td><td></td><td></td><td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td></tr><tr><td>pT2-CMV-PEDF-His plasmid DNA (250 ng/&amp;micro;L)</td><td></td><td></td><td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td></tr><tr><td>QIAamp DNA Mini Kit</td><td>QIAGEN</td><td>51304</td><td></td></tr><tr><td>recombinant hGM-CSF&amp;nbsp;</td><td>Peprotech</td><td>100-11</td><td></td></tr><tr><td>recombinant hPEDF&amp;nbsp;&amp;nbsp;</td><td>BioProductsMD</td><td>004-096</td><td></td></tr><tr><td>ReliaPrep RNA Cell Miniprep System</td><td>Promega</td><td>Z6011</td><td></td></tr><tr><td>RIPA buffer</td><td>ThermoFisher Scientific</td><td>89901</td><td></td></tr><tr><td>RNase-free DNase Set</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>QIAGEN</td><td>74204</td><td></td></tr><tr><td>SDS</td><td>Applichem</td><td>A2572</td><td></td></tr><tr><td>Semi-dry transfer system for WB&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>SuperMix qScript</td><td>Quantabio</td><td>95048-025</td><td></td></tr><tr><td>Tris-buffered saline (TBS)&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15504020</td><td></td></tr><tr><td>Triton X-100</td><td>AppliChem</td><td>A4975</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>Tween</td><td>AppliChem&amp;nbsp;</td><td>A1390</td><td></td></tr><tr><td>Urea</td><td>ThermoFisher Scientific</td><td>29700</td><td></td></tr><tr><td>WesternBright ECL HRP substrate</td><td>Advansta</td><td>K-12045-D50</td><td></td></tr><tr><td>Whatman nitrocellulose membrane</td><td>Chemie Brunschwig</td><td>MNSC04530301</td><td></td></tr></tbody></table>

induction and analysis of oxidative stress in sleeping beauty transposon-transfected human retinal pigment epithelial cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction of oxidative stress in human Retinal Pigment Epithelial cells 
ARPE-19 and primary hRPE cells were treated with varying concentrations of H2O2 for 24 h and the intracellular level of the antioxidant glutathione was quantified (Figure 2A,B). H2O2 at 50 &#181;M and 100 &#181;M did not affect glutathione production, whereas at 350 &#181;M there was a significant decrease...

Watch this Scientific Journal Video about Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells at JoVE.com

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

induction-analysis-oxidative-stress-sleeping-beauty-transposon

Research

JoVE Journal

Medicine

2.5K Views.  University of Geneva. We presenteren een protocol voor de ontwikkeling en het gebruik van een oxidatieve stress-model door retinale pigmentepitheelcellen te behandelen met H2O2, waarbij celmorfologie, levensvatbaarheid, dichtheid, glutathion en UCP-2-niveau worden geanalyseerd. Het is een nuttig model om het antioxiderende effect te onderzoeken van eiwitten die worden uitgescheiden door getransfecteerde cellen om neuroretinale degeneratie te behandelen.

Video: Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Cancer Research

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H2DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H2DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H2DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H2DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H2DCFDA fluoresce was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.

A screening method to detect oxidative cellular environments is to measure the oxidation of CM-H2DCFDA. Once oxidized within a cell, CM-H2DCFDA changes from non-fluorescent into a fluorescent compound. This change in fluorescence is measured by flow cytometry and indicates the number of cells in an oxidative environment.

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

Neuroscience

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Developmental Biology

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Immunology and Infection

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Biology

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

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