The construct and recombinant adenovirus for expressing cytosol-specific roGFP in cells were generated in the laboratory of Paul T. Schumacker, PhD, Freiberg School of Medicine, Northwestern University, and ViraQuest Inc., respectively. This study was supported by the Center for Studies of Host Response to Cancer Therapy grant P20GM109005 through the NIH National Institute of General Medical Sciences Centers of Biomedical Research Excellence (COBRE NIGMS), National Institute of General Medical Sciences Systems Pharmacology and Toxicology Training Program grant T32 GM106999, UAMS Foundation/Medical Research Endowment Award AWD00053956, UAMS Year-End Chancellor&#8217;s Awards AWD00053484. The flow cytometry core facility was supported in part by the Center for Microbial Pathogenesis and Host Inflammatory Responses grant P20GM103625 through the COBRE NIGMS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. ATA was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) 2214-A scholarship.

NOTE: This protocol was optimized for 70%&#8211;80% confluent MDA-MB-231 cells. For other cell lines, the number of cells and multiplicity of infection (MOI) should be reoptimized.
1. Preparation of cells (day 1)
<ol>
	<li>Maintain MDA-MB-231 cell line in 75 cm2 flasks with 10 mL of Dulbecco&#8217;s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 &#176;C in a 5% CO2 humidified atmosphere. 
	NOTE: DMEM supplemented with 10% FBS, 37 &#176;C, and a 5% CO2 humidified atmosphere are used for all attachment and treatment incubations throughout the entire protocol.</li>
	<li>Prepare the MDA-MB-231 cells for experiment.
	<ol>
		<li>Aspirate the medium within the flask, detach the cells with 2 mL of 0.25% trypsin-EDTA solution for 2 min, and inactivate the trypsin activity with 6 mL of complete medium (DMEM with 10% FBS). Centrifuge the cells at 150 x g for 5 min. Aspirate the supernatant and suspend the cells in 5 mL of complete medium.</li>
		<li>Mix an equal volume cell suspension and 0.4% trypan blue. Take 10 &#181;L of this mixture and count the cells with the automated cell counter. 
		NOTE: A Coulter counter or a hemocytometer can also be used for cell counting.</li>
		<li>Seed the cells into a 6 well plate for flow cytometry analyses and seed 150,000 cells in 1 mL of medium per well. Wait 16 h for cell attachment.</li>
		<li>Seed the cells into a 4 well chamber slide for fluorescent imaging and seed 25,000 cells in 0.5 mL of medium per well. Wait 16 h for cell attachment. 
		NOTE: Seed control wells in addition to treatment wells. Use one of the control wells to determine cell number (optional: if the attachment period for the cells is shorter than the doubling time, cell number can be assumed to be the same as the seeding density) and the other for a noninfected control (0 MOI).</li>
	</ol>
	</li>
</ol>
2. Adenoviral roGFP transduction (day 2 and 3)
CAUTION: Adenoviruses can cause diseases. While transducing the cells, use filtered tips and decontaminate tips, Pasteur pipettes, and microcentrifuge tubes with 10% bleach.
NOTE: This protocol was demonstrated with cytosol-specific roGFP, but other cellular compartments (e.g., mitochondria or mitochondrial intermembrane space) can be targeted with this same protocol.
<ol>
	<li>Generate a dose-response curve for the MOI to obtain the highest transduction efficiency by calculating the volume of adenovirus (mL) required for each MOI value for MDA-MB-231 cell line (Table 1): 
	<img alt="Equation 1" src="/files/ftp_upload/61229/61229equ01.jpg" /> 
	NOTE: Functional titer of each batch of adenoviral stock, which is expressed as plaque forming unit (PFU) per mL, is provided by the company. The optimum MOI for transduction differs between cell types. For most mammalian cells, the optimum MOI range is between 10 and 300. According to the cellular response, MOI values should be recalculated (e.g., MOI range should be reduced if cells have cytotoxic response, or range should be increased if cells have low transduction efficiency).</li>
	<li>Make 1:100 dilution of 6 x 1010 PFU/mL adenoviral roGFP solution with cell culture medium (DMEM with 10% FBS) for reliable pipetting.</li>
	<li>Pipette and add 0.0125 mL (12.5 &#181;L), 0.025 mL (25 &#181;L), 0.05 mL (50 &#181;L) of adenoviral roGFP dilution into each well of the 6 well plate in order to transduce the 150,000 cells with 50, 100, and 200 MOI respectively for flow cytometry analysis (Table 1).</li>
	<li>Pipette and add 0.0042 mL (4.2 &#181;L) of adenoviral roGFP dilution in the 4-chamber slide wells to transduce 25,000 cells with 100 MOI for fluorescence imaging (Table 1). 
	NOTE: A minimal amount of medium should be used in the wells to ensure the highest interaction between the adenoviral roGFP construct and cells. The serum content of the culture medium may need to be decreased for different cell lines because high levels of serum can negatively affect transduction efficiency in some cell types.</li>
	<li>Incubate cells for 16&#8211;24 h under the cell maintenance conditions. The next day (day 3), change the medium to cell culture medium (DMEM with 10% FBS) to allow cell recovery for an additional 24 h. Visualize cells under a microscope to assess their morphology; cells can express roGFP even if they have morphological changes. 
	NOTE: On day 3, cells should start to express roGFP; therefore, transduction efficiency can be monitored using fluorescence microscopy (filters with ex. 488/em. 525). To obtain consistent assay results, be aware of and document the morphological changes under the phase contrast microscope and observe morphology while evaluating transduction efficiency.</li>
	<li>Construct a dose response curve using the 50, 100 and 200 MOI samples prepared in step 2.3 and their transduction efficiency results obtained from flow cytometry analysis (steps 3.1 and 4.1). Assess optimal transduction efficiency with documentation of morphological changes (step 2.5) and the dose-response curve of MOI. 
	NOTE: Although more than 98% of the cell population at 100 MOI and 200 MOI express roGFP (see representative results), 200 MOI group showed substantial changes in cell morphology of MDA-MB-231 cells. Consequently, the most efficacious MOI for MDA-MB-231 cells was determined to be 100 MOI.</li>
	<li>After optimal MOI (here, 100 MOI) was chosen for MDA-MB-231 cell line, conduct experiment with test materials (10 &#181;M H2O2 and its vehicle 0.1% deionized water).
	<ol>
		<li>Prepare and seed the cells according to section 1. Using the adenoviral transduction volume for 100 MOI calculated in step 2.1, repeat steps 2.2&#8722;2.4 for 100 MOI adenoviral transduction of cells. Then incubate the plate and chamber slides according to step 2.5.</li>
	</ol>
	</li>
</ol>
3. Acquisition of CyS/CySS balance
<ol>
	<li>Flow cytometry (day 4)
	<ol>
		<li>On day 4, incubate cells from step 2.7.1 with 10 &#181;M H2O2 for 1 h. 
		NOTE: 10 &#181;M H2O2 was used as the test substance and 0.1% deionized water was used as vehicle treatment in this protocol. Other oxidizing agents can be used as positive controls here.</li>
		<li>Aspirate media from the 6 well plate, replace with 750 &#181;L of 0.25% trypsin-EDTA solution and wait for 2 min for cells to detach. Inactivate trypsin with 2 mL of complete medium (DMEM with 10% FBS) and collect the volume into 15 mL conical tubes.</li>
		<li>Centrifuge the tubes at 150 x g for 5 min at 4&#186;C. Discard supernatant and suspend the cells in 500 &#181;L of phosphate-buffered saline (PBS).</li>
		<li>Repeat step 3.1.3</li>
		<li>Filter the cell suspensions into flow cytometry-compatible tubes using 40 &#181;m mesh. Keep the tubes on ice and away from the light and follow step 4.1 for data analysis.</li>
	</ol>
	</li>
	<li>Microscopic imaging (day 4)
	<ol>
		<li>On day 4, treat cells with 10 &#181;M H2O2, acquire images immediately (time point 0) and 1 h after treatment and follow step 4.2 for data analysis.</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Flow cytometry quantification
	<ol>
		<li>Set flow cytometry method for 3 different analyses via sample aquisition software (see Table of Materials): forward scatter (FCS) on x-axis and side scatter (SSC) on y-axis to assess cell size and complexity of cells (SSC can be used for rough identification of dead and live cells); ex. 488 nm/em. 525 nm (fluorescein isothiocyanate [FITC]) bandpass filter on x-axis and SSC on y-axis to assess CyS-roGFP; ex. 405 nm/em. 525 nm (Brilliant Violet 510 [BV510]) bandpass filter on x-axis and SSC on y-axis to assess CySS-roGFP.</li>
		<li>Acquire 0 MOI control&#160;and visualize cells with sample acquisition software. Repeat this step for remaining samples (50, 100, 200 MOI groups and later on 10 &#181;M H2O2 treated cells and vehicle treated cells). Save the files for data analysis.</li>
		<li>Open data analysis software (see Table of Materials) and open 0 MOI sample file. Assess cell population of interest (Gate 1). Set up the following gatings to minimize background fluorescence for ex. 488 nm/em. 525 nm (Gate 2) and ex. 405 nm/em. 525 nm (Gate 3) bandpass filters with the noninfected (0 MOI) control cells.</li>
		<li>Open 50, 100, and 200 MOI sample files within data analysis software to assess the dose-response curve. Analyze mean fluorescence intensities with Gates 2 and 3 for each sample. Repeat this step for test samples (10 &#181;M H2O2 treated cells and vehicle treated cells).</li>
		<li>Calculate the mean fluorescent intensity ratio between oxidized versus reduced forms of roGFP with the following equation.</li>
	</ol>
	</li>
</ol>
<img alt="Equation 2" src="/files/ftp_upload/61229/61229equ02.jpg" />
<ol start="2">
	<li>Image assessment
	<ol>
		<li>Use a microscope that contains fluorescence filters for CyS-roGFP and CySS-roGFP (ex. 488 nm/em. 525 nm and ex. 405 nm/em. 525 nm filters, respectively).</li>
		<li>In each well of the chamber slide, pick 4 random areas to acquire images, using the 4x objective to visualize larger areas. 
		NOTE: 20x objective can also be used for image displays.</li>
		<li>Open the image with ImageJ software11. Apply Analyze | Measure commands for each image and use the equation in step 4.1.5 to quantify the data. 
		NOTE: Quantification of the images is ratiometric; therefore, the protocol does not include subtraction of background. However, to be able to compare images, brightness, contrast, and saturation must be the same for each image. Statistical significance was assessed with one-way analysis of variance (ANOVA) and Tukey&#8217;s post hoc test.</li>
	</ol>
	</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol/disulfide+redox+states+in+signaling+and+sensing.">Thiol/disulfide redox states in signaling and sensing.</a> Critical Reviews in Biochemistry and Molecular Biology. 48 (2), 173-191 (2013).</li><li>Hansen, J. M., Go, Y., Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+and+Mitochondrial+Compartmentation+of+Oxidative+Stress+and+Redox+Signaling.">Nuclear and Mitochondrial Compartmentation of Oxidative Stress and Redox Signaling.</a> Annual Review of Pharmacology and Toxicology. 46 (1), 215-234 (2006).</li><li>Meyer, A. J., Dick, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein-based+redox+probes.">Fluorescent protein-based redox probes.</a> Antioxidants and Redox Signaling. 13 (5), 621-650 (2010).</li><li>Dooley, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+dynamic+redox+changes+in+mammalian+cells+with+green+fluorescent+protein+indicators.">Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators.</a> Journal of Biological Chemistry. 279 (21), 22284-22293 (2004).</li><li>Bj&#246;rnberg, O., &#216;stergaard, H., Winther, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+intracellular+redox+conditions+using+GFP-based+sensors.">Measuring intracellular redox conditions using GFP-based sensors.</a> Antioxidants and Redox Signaling. 8 (3-4), 354-361 (2006).</li><li>Bhaskar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reengineering+Redox+Sensitive+GFP+to+Measure+Mycothiol+Redox+Potential+of+Mycobacterium+tuberculosis+during+Infection.">Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of Mycobacterium tuberculosis during Infection.</a> PLoS Pathogens. 10 (1), 1003902 (2014).</li><li>Loor, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+oxidant+stress+triggers+cell+death+in+simulated+ischemia-reperfusion.">Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion.</a> Biochimica et Biophysica Acta - Molecular Cell Research. 1813 (7), 1382-1394 (2011).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Loor, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Menadione+triggers+cell+death+through+ROS-dependent+mechanisms+involving+PARP+activation+without+requiring+apoptosis.">Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.</a> Free Radical Biology and Medicine. 49 (12), 1925-1936 (2010).</li><li>Esposito, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox-sensitive+GFP+to+monitor+oxidative+stress+in+neurodegenerative+diseases.">Redox-sensitive GFP to monitor oxidative stress in neurodegenerative diseases.</a> Reviews in the Neurosciences. 28 (2), 133-144 (2017).</li><li>Meyer, A. 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S., Borowik, S., Zieseniss, A., Katschinski, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+organisms+meet+redox+bioimaging:+One+step+closer+to+physiology.">Transgenic organisms meet redox bioimaging: One step closer to physiology.</a> Antioxidants and Redox Signaling. 29 (6), 603-612 (2018).</li><li>Gutscher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity-based+protein+thiol+oxidation+by+H2O2-scavenging+peroxidases.">Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases.</a> Journal of Biological Chemistry. 284 (46), 31532-31540 (2009).</li><li>Morgan, B., Sobotta, M. C., Dick, T. 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The authors have nothing to disclose.

The thiol/disulfide balance in an organism reflects the redox status of cells. Living organisms have glutathione, cysteine, protein thiols, and low-molecular-weight thiols, all of which are affected by the level of oxidation and echo the redox status of cells4. Engineered roGFPs allow the non-disruptive quantification of the thiol/disulfide balance via their CyS residues7. The ratiometric property of roGFP provides reliable redox measurements for mammalian cells. roGFP can ...

Oxidative stress was defined in 1985 by Helmut Sies as &#8220;a disturbance in prooxidant&#8211;antioxidant balance in favor of the former&#8221;1, and a plethora of research has been conducted to obtain disease-, nutrition-, and aging-specific redox status of organisms1,2,3. Since then, the understanding of oxidative stress has become broader. Testing the hypotheses of using antioxidants against diseases and/or aging has shown that oxidative stress not only causes harm but also has other roles in cells. Furthermore, scientists have shown that free radicals play an important role for signal transduction2. All of these studies strengthen the importance of determining the changes in reduction-oxidation (redox) ratio of macromolecules. Enzyme activity, antioxidants and/or oxidants, and oxidation products can be assessed with various methods. Among these, methods that determine thiol oxidation are arguably the most used because they report on the balance between antioxidants and prooxidants in cells, as well as organisms4. Specifically, ratios between glutathione (GSH)/glutathione disulfide (GSSG) and/or cysteine (CyS)/cystine (CySS) are used as biomarkers for monitoring the redox status of organisms2.
Methods used for assaying the balance between prooxidants and antioxidants rely mainly on the levels of reduced/oxidized proteins or small molecules within cells. Western blots and mass spectrometry are used to broadly assess the ratios of reduced/oxidized macromolecules (protein, lipids etc.), and GSH/GSSG ratios can be assessed with spectrophotometry5. A common feature of these methods is the physical perturbation of the system by cell lysis and/or tissue homogenization. These analyses also become challenging when it is necessary to measure the oxidation status of different cellular compartments. All of these perturbations cause artifacts in the assay environment.
Redox-sensitive fluorescent proteins opened an advantageous era for evaluating the redox balance without causing a disturbance in the cells6. They can target different intracellular compartments, allowing the quantification of compartment-specific activities (e.g., assaying the redox state of mitochondria and the cytosol) to investigate crosstalk between cellular organelles. Yellow fluorescent protein (YFP), green fluorescent protein (GFP), and HyPeR proteins are reviewed by Meyer and colleagues6. Among these proteins, redox-sensitive GFP (roGFP) is unique due to different fluorescent readouts of its CyS (ex. 488 nm/em. 525 nm) and CySS (ex. 405 nm/525 nm) residues, which permits ratiometric analysis, unlike other redox-sensitive proteins such as YFP7,8. Ratiometric output is valuable because it counterbalances the differences between expression levels, detection sensitivities, and photobleaching8. Subcellular compartments of cells (cytosol, mitochondria, nucleus) or different organisms (bacteria as well as mammalian cells) can be targeted by modifying roGFP7,9,10.
roGFP assays are conducted using fluorescent imaging techniques, especially for real-time visualization experiments. Flow cytometric analyses of roGFPs are also possible for experiments with predetermined time points. The current article describes both the use of fluorescent microscopy and flow cytometry to perform a ratiometric assessment of redox status in mammalian cells overexpressing roGFP (targeted to cytosol) via adenoviral transduction.

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Gibco by Life Sciences</td><td>25200-056</td><td>Cell culture</td></tr><tr><td>4-well chamber slide</td><td>Thermo Scientific</td><td>154526</td><td>Cell seeding material for fluorescent imaging</td></tr><tr><td>5 ml tubes with cell strainer cap</td><td>Falcon</td><td>352235</td><td>Single cell suspension tube for flow cytometry analysis</td></tr><tr><td>6-well plate</td><td>Corning</td><td>353046</td><td>Cell seeding material for flow cytometry analysis</td></tr><tr><td>15 ml conical tubes</td><td>MidSci</td><td>C15B</td><td>Cell culture</td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; ventilated cap tissue culture flasks</td><td>Corning</td><td>4306414</td><td>Cell culture</td></tr><tr><td>Adenoviral cytosol specific roGFP</td><td>ViraQuest</td><td>VQAd roGFP</td><td>roGFP construct kindly provided by Dr. Schumaker</td></tr><tr><td>Class II, Type A2 Safety Hood Cabinet</td><td>Thermo Scientific</td><td>1300 Series A2</td><td>Cell culture</td></tr><tr><td>Countess automated cell counter</td><td>Invitrogen</td><td>C10227</td><td>Cell counting</td></tr><tr><td>Countess cell counter chamber slides</td><td>Invitrogen</td><td>C10283</td><td>Cell counting</td></tr><tr><td>DMEM</td><td>Gibco by Life Sciences</td><td>11995-065</td><td>Cell culture</td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>S11150</td><td>Cell culture</td></tr><tr><td>Filtered pipette tips, sterile, 20 &amp;micro;l</td><td>Fisherbrand</td><td>02-717-161</td><td>Cell culture</td></tr><tr><td>Filtered pipette tips, sterile, 1000 &amp;micro;l</td><td>Fisherbrand</td><td>02-717-166</td><td>Cell culture</td></tr><tr><td>Flow Cytometer</td><td>BD Biosciences</td><td>LSRFortessa</td><td>Instrument equipped with FITC and BV510 bandpass filters for flow cytometry analyses</td></tr><tr><td>Fluorescent Microscope</td><td>Advanced Microscopy Group (AMG)</td><td>Evos FL</td><td>Fluorescent imaging</td></tr><tr><td>Hydrogen Peroxide 30%</td><td>Fisher Scientific</td><td>H325-100</td><td>Positive control</td></tr><tr><td>Light Cube, Custom</td><td>Life Sciences</td><td>CUB0037</td><td>Fluorescent imaging of roGFP expressing cells (ex 405 nm)</td></tr><tr><td>Light Cube, GFP</td><td>Thermo Scientific</td><td>AMEP4651</td><td>Fluorescent imaging of roGFP expressing cells (ex 488 nm)</td></tr><tr><td>MDA-MB-231</td><td>American Tissue Culture Collection</td><td>HTB-26</td><td>Human epithelial breast cancer cell line</td></tr><tr><td>Microcentrifuge tubes, 2 ml</td><td>Grenier Bio-One</td><td>623201</td><td>Cell culture</td></tr><tr><td>PBS</td><td>Gibco by Life Sciences</td><td>10010-023</td><td>Cell culture</td></tr><tr><td>Pipet controller</td><td>Drummond</td><td>Hood Mate Model 360</td><td>Cell culture</td></tr><tr><td>Serologycal pipet, 1 ml</td><td>Fisherbrand</td><td>13-678-11B</td><td>Cell culture</td></tr><tr><td>Serologycal pipet, 5 ml</td><td>Fisherbrand</td><td>13-678-11D</td><td>Cell culture</td></tr><tr><td>Serologycal pipet, 10 ml</td><td>Fisherbrand</td><td>13-678-11E</td><td>Cell culture</td></tr><tr><td>Tissue Culture Incubator</td><td>Thermo Scientific</td><td>HERACell 150i</td><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator for cell culture</td></tr><tr><td>Trypan blue stain 0.4%</td><td>Invitrogen</td><td>T10282</td><td>Cell counting</td></tr></tbody></table>

assessment of cellular oxidation using a subcellular compartment-specific redox-sensitive green fluorescent protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

The redox state of CyS/CySS is easily assayed with transduced roGFPs. The fluorescent probe quantifies the ratio between the reduced and oxidized forms (excitation wavelengths 488 nm and 405 nm, respectively). Fluorescence data can be obtained by both flow cytometry and microscopy.
A large number of cells can consistently and conveniently be acquired using flow cytometry. The analysis consists of 3 main steps: 1) select the cell population of interest with the FSC area filter (<strong class="x...

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Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

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7.2K Views.  University of Arkansas for Medical Sciences. This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

Video: Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

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

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

Bioengineering

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Biology

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

Neuroscience

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

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 ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

Detecting Reactive Oxygen Species

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are many deleterious effects associated with unchecked ROS production, including structural damage to DNA and other biological molecules. However, ROS can also be mediators of physiological signaling. There is accumulating evidence that ROS play significant roles in everything from activation of transcription factors to the mediation of inflammatory toxicity that kills foreign pathogens and defend the body.In this video we will delve into the associations between ROS, metabolism and disease. After establishing their significance, we will discuss the principles and a protocol of a commonly used methodology for measuring ROS levels in cells: the use of non-fluorescent probes that become fluorescent upon oxidation. Lastly, we will review some current applications of this technique in cell biology research.

Reactive Oxygen Species and Oxidative Stress

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are ...

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

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

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

ROS Live Cell Imaging During Neuronal Development

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Quantification of Reactive Oxygen Species Using 2′,7′-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells

Cellular Redox Profiling Using High-content Microscopy

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Detecting Reactive Oxygen Species