We thank ReadiSorb Products for their generous support of these studies.

1. Cell culture of feline astrocytes and treatment with H2O2
<ol>
<li>Culture cell line G355-5 in a 75 cm2 flask with DMEM media plus nutrients at 37&deg;C and 5% CO2 until 60% confluent (&tilde; 5 days). Replace the media every 1-2 days or as needed to maintain a healthy culture.</li>
<li>Remove media and add 2 ml of 0.25% trypsin. Lift the cells by pipetting for 45 s - 1 min at room temperature (RT).</li> 
<li>Quickly transfer the suspension to a 15 ml conical tube containing 10 ml of media. Mix gently.</li>
<li>Centrifuge at 300 x g, 23&deg;C for 3 min to obtain a pellet. Discard the supernatant without disturbing the pellet.</li>
<li>Add 6-7 ml of fresh media and resuspend the cells.</li>
<li>Add &tilde; 1 ml of cells to each well of a 6-well plate. Add 2-3 ml of media to each well and incubate until 80-90% confluent.</li>
<li>Prepare a 100&mu;M solution of peroxide (H2O2) in media.</li>
<li>Remove media from each well and replace with either 2 ml fresh media (control), 2 ml of H2O2 (treatment) or 2ml of DPBS. Incubate at 37&deg;C, 5% CO2 for 3 h.</li>
<li>Remove solutions and add 1 ml of trypsin to lift the cells. Transfer cells to a 1.7 ml Eppendorf tube containing 0.5 ml DPBS. This step should be performed on each well individually in order to prevent cell death from prolonged trypsin incubation.</li>
<li>Spin at 300 x g for 3 min, discard the supernatant and resuspend in 1 ml of DPBS.</li>
</ol>
2. Staining for ROS
<ol>
<li>All steps for staining should be carried out with minimal light exposure to prevent bleaching.</li>
<li>Add 1 &mu;l of 50 mM CCCP to the positive control. Incubate at 37&deg;C, 5% CO2 for 5 min.</li>
<li>Add 5 &mu;l of a 10 mM CM-H2DCFDA solution to the H2O2 group. Incubate at 37&deg;C, 5% CO2 for 15-30 min. No stain is added to the saline sample.</li>
<li>Wash samples with 2 ml of DPBS to remove residual stain.</li>
<li>Spin at 300 x g for 3 min to obtain a pellet.</li>
<li>Resuspend cells in 0.5 ml of DPBS. Add 1 &mu;l PI to each sample, except the saline.</li>
<li>Transfer samples to a 5ml flow cytometer tube.</li>
<li>Run on the flow cytometer.</li>
</ol>
3. Flow cytometry
<ol>
<li>Run samples on a Beckman Coulter FC500 (or equivalent) equipped with a 488 nm argon laser and the following band passes: 525, 775 and 620 nm, all &plusmn; 20 nm.</li>
<li>Prepare the appropriate controls: unstained cells, CM-H2DCFDA stained only, PI only, double stained (negative control).</li>	
<li>Make a protocol that has the following histoplots: FS vs. SS, PI vs. CM-H2DCFDA. It should also have the following histograms: cell number vs. FL1, FL2 and FL3, which corresponds to the stains.</li>
<li>Before running the samples, check equipment, fluids and waste container. Warm up the equipment for at least 20 min.</li>
<li>Clean the equipment and perform a calibration with the appropriate beads according to the manufacturers standards.</li>
<li>Select your protocol and run the samples.</li>
<li>Clean the equipment as done previously.</li>
<li>Clean the vacuum line according to the manufacturers standards.</li>
<li>Turn off the flow and clean the head/vacuum line.</li>
<li>Turn off the software and the computer.</li>
</ol>
4. Representative Results:
Flow cytometry results were analyzed using FlowJo 7.6 and the stain controls were used to objectively set the gating. Based on this, healthy cells appear on the left of the histogram and ROS (oxidative stress) was detected as a shift of cells to the right. Figure 1 shows the results of the effect of hydrogen peroxide on healthy feline astrocytes. Data from FL1 was used to measure the intensity of CM-H2DCFDA, which indicates the presence of ROS. As expected, a higher amount of ROS was detected in the sample treated with H2O2. This is displayed on the histogram as a shift in fluorescent intensity from left to right. When comparing healthy cells treated with DMEM versus DPBS, there was no significant change in the amount of ROS (Fig 2).
<img src="/files/ftp_upload/2841/2841fig1.jpg" alt="Figure 1"/> 
Figure 1. Flow cytometry results of levels of oxidative stress in healthy feline astrocytes and the effect of H2O2 on healthy cells. The results indicate that H2O2 increases levels of ROS in healthy cells as compared to cells with no treatment (DMEM).
<img src="/files/ftp_upload/2841/2841fig2.jpg" alt="Figure 2"/> 
Figure 2. Flow Cytometry results of levels of ROS detected in healthy cells incubated for 3hrs with DPBS.

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An often suggested mechanism of virus induced neuronal damage is oxidative stress 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31. Basically, it is proposed that through viral exposure the glia (astrocytes and microglia) release reactive oxygen radicals such as, hydroxide, superoxide anion, nitric oxide, and/or hydrogen peroxide, which are toxic to neurons. Similarly, oxidative stress has also been suggested as a major pathologic mechanism in methamphetamine-induced neurotoxicity 2, 6, 17. Since our group is interested in the effects of both virus and drugs of abuse on the CNS, we elected to use a quick screening test to determine the present of excess ROS in astrocytes. The majority of neuronal glutathione is produced by the astrocytes and then delivered to the neurons 5, 8, 10-12, 24, 26, 29, 30. Thus, the astrocytes role in oxidative stress is important in maintaining a homeostatic environment for neurons as well as protecting neurons from ROS, and is the reason that astrocyte cell cultures were chosen for this study.
CM-H2DCFDA is also known as 2',7'-dichlorofluorescein and H2DCF. CM-H2DCFDA is a cell-permeable indicator for presence of reactive oxygen species. CM-H2DCFDA enters the cell as a non-fluorescent compound, which becomes fluorescent after cellular esterases remove the acetate groups, and the compound becomes oxidized. Once inside the cell and the acetate groups are removed, the CM-H2DCFDA can be oxidized by a number of ROS species, including nitric oxide, peroxynitrite anions, hydrogen peroxide, and organic hydroperoxides 3, 14, 25, resulting in a green fluorescent product 23, 28. This lack of specify in the ROS, which can oxidize CM-H2DCFDA, makes it a valuable reagent in the early stages of a pathogenesis investigation, in which an oxidative stress mechanism is believed to be playing a role, but it is unknown which oxygen radical might be involved, rather than test for each ROS separately. 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 present study demonstrate that with the addition of hydrogen peroxide an increase in CM-H2DCFDA fluoresce was detected when compared to the saline control, indicating that that this assay is valuable test for detecting an oxidative environment within the G355-5 cells, a feline astrocyte cell line.

<table><tbody><tr><td>DMEM</td><td>Cellgro</td><td>15-013-CV</td><td></td></tr><tr><td>MEM Vitamins</td><td>Cellgro</td><td>25-020-cl</td><td>1% (v/v)</td></tr><tr><td>L-glutamine</td><td>Hyclone</td><td>17-605E</td><td>1% (v/v)</td></tr><tr><td>Non Essential Amino Acids</td><td>Hyclone</td><td>25-025-cl</td><td>1% (v/v)</td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30071.03</td><td>20% (v/v)</td></tr><tr><td>Essential Amino Acids</td><td>Cellgro</td><td>25-030-cl</td><td>0.2% (v/v)</td></tr><tr><td>500ml vacuum filtration system</td><td>VWR international</td><td>87006-076</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Falcon BD</td><td>352097</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flasks</td><td>Falcon BD</td><td>353136</td><td></td></tr><tr><td>6 well tissue culture plate</td><td>Falcon BD</td><td>353224</td><td></td></tr><tr><td>CM-H&lt;sub&gt;2&lt;/sub&gt;DCFDA</td><td>Invitrogen</td><td>C6827</td><td></td></tr><tr><td>Propidium Iodide (PI)</td><td>Sigma-Aldrich</td><td>P4170-10mg</td><td></td></tr><tr><td>Trypsin</td><td>Lonza Inc.</td><td>17-160F</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>H6520</td><td></td></tr><tr><td>HyQ Antibiotic</td><td>Hyclone</td><td>SV30079.01</td><td>0.1% (v/v)</td></tr><tr><td>G355-5 cells</td><td>ATCC</td><td>CRL-2033</td><td>Normal feline brain</td></tr></tbody></table>

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.

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

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 ...

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14.4K Views.  Western University of Health Sciences. 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.

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

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

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biology

GSH/GSSG Measurement &#8211; A Refined OPA Assay for Antioxidant Research

Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30&#181;M (R2=0.9932&#177;0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.

Author Spotlight: Innovating Thiol Quantification and Biomarker Detection for Oxidative Stress Research 

Quantification of both oxidized and reduced forms of glutathione (GSSG and GSH, respectively) has been achieved through the use of Ortho-phthalaldehyde (OPA). OPA becomes highly fluorescent once conjugated to GSH but is unable to conjugate GSSG until reduced. Here, we describe a multiparametric assay to quantify both using protein quantification for normalization.

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive ...

Biochemistry

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Novel ROS Quantification Using Dihydroethidium in Liver Cancer Cells

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

Author Spotlight: High-Throughput Measurement of Intracellular ROS Levels in Hepatocellular Lines

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...

Bioengineering

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 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.

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

Medicine

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 &#181;M) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Based on in vitro experiments, this study revealed the mechanism of crocetin in repairing oxidative stress damage of cardiomyocytes by influencing mitophagy, in which the PINK1/Parkin signaling pathway plays an important role.

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and ...

Fluorescence-Based Monitoring of Mitochondrial Stress in Astrocytes

Source: Espourteille, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62957">Live-imaging of Mitochondrial System in Cultured Astrocytes.</a> J. Vis. Exp. (2021)
This video demonstrates the evaluation of mitochondrial health in genetically modified astrocytes, which express a fluorescent protein within their mitochondria. The protein helps to track changes in mitochondrial health as its fluorescence changes from green to red, indicating stress or damage. Images are captured and analyzed using various parameters.

1. Rat hippocampal astrocyte primary culture

	Sacrifice five rat pups (Wistar IGS Rat) by decapitation at postnatal days 1-2.
	Remove the brain and keep ...

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

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Chapters in this video

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

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Viability Assays for Cells in Culture

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

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

SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Fluorescence-Based Monitoring of Mitochondrial Stress in Astrocytes