Name: assessment of cellular oxidation using a subcellular compartment-specific redox-sensitive green fluorescent protein
Uploaded: 2020-06-18T14:30:00
Description: Watch this Scientific Journal Video about Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein at JoVE.com

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 ...

<ol>
	<li>Sies, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress:+A+concept+in+redox+biology+and+medicine.">Oxidative stress: A concept in redox biology and medicine.</a> Redox Biology. 4, 180-183 (2015).</li><li>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=Redefining+Oxidative+Stress.">Redefining Oxidative Stress.</a> Antioxidants &amp; Redox Signalling. 8 (9-10), (2006).</li><li>Pizzino, 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=Oxidative+Stress:+Harms+and+Benefits+for+Human+Health.">Oxidative Stress: Harms and Benefits for Human Health.</a> Oxidative Medicine and Cellular Longevity. 20117, 8416763 (2017).</li><li>Go, Y. M., 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=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. J., 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+in+Arabidopsis+thaliana+is+a+quantitative+biosensor+for+the+redox+potential+of+the+cellular+glutathione+redox+buffer.">Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer.</a> Plant Journal. 52 (5), 973-986 (2007).</li><li>Galvan, D. L., 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=Real-time+in+vivo+mitochondrial+redox+assessment+confirms+enhanced+mitochondrial+reactive+oxygen+species+in+diabetic+nephropathy.">Real-time in vivo mitochondrial redox assessment confirms enhanced mitochondrial reactive oxygen species in diabetic nephropathy.</a> Kidney International. 92 (5), 1282-1287 (2017).</li><li>Swain, L., Nanadikar, M. 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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+EGSH+and+H2O2+with+roGFP2-based+redox+probes.">Measuring EGSH and H2O2 with roGFP2-based redox probes.</a> Free Radical Biology and Medicine. 51 (11), 1943-1951 (2011).</li><li>Dey, S., Sidor, A., O'Rourke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartment-specific+control+of+reactive+oxygen+species+scavenging+by+antioxidant+pathway+enzymes.">Compartment-specific control of reactive oxygen species scavenging by antioxidant pathway enzymes.</a> Journal of Biological Chemistry. 291 (21), 11185-11197 (2016).</li></ol>

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 cm2 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 &#181;l</td>
			<td>Fisherbrand</td>
			<td>02-717-161</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Filtered pipette tips, sterile, 1000 &#181;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>CO2 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...

Watch this Scientific Journal Video about Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein at JoVE.com

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. ...

Current protocol assessors in textile, redox state changes in an intracellular compartment-specific manner. Our protocol enables a better understanding and monitoring all the physiological or pathophysiological mechanisms of oxidative stress. This technique allows non-disruptive quantification of the tiled eye sulfide ratio in intact cells using a ratio metric output to counterbalance the differences between signal expression levels, photo bleaching, and detection sensitivities.This protocol can be applied to various organisms, including bacteria in plants. On day one of the experiment treat the cells from a confluent breast cancer cell line for two minutes, with two milliliters of 0.2, 5%trips in EDTA. When the cells begin to detach arrest the reaction with six milliliters of complete medium and sediment the cells by centrifugation.Re suspend the pellet in five milliliters of fresh, complete medium, count the cells and dilute them to 1.5 times 10 to the fifth cells per one milliliter of complete medium. Then seed 1.5 times 10 to the fifth cells per milliliter of cells per well in a 6 well plate, and incubate the cells in the cell culture incubator overnight. Or add no viral row GFP transduction.The next morning, add 12.5, 25, and 50 microliters of a one to 100 diluted six times 10 to the 10th plaque forming units per milliliter of add no viral erode GFP solution, in complete medium. Add that to the appropriate wells or the 6 well plate to transduce the cells with 50, 100, and 200 multiplicities of infection, respectively. When all of the sample Wells have been transduced with virus, return the cells to the cell culture incubator for 16 to 24 hours.The next day, visualize the cells by fluorescence microscopy and replaced the supernatant with medium, without virus to allow the cells to recover for an additional 24 hours. To determine the optimal transduction efficiency, assess the fluorescence intensity and morphology of the cells by flow cytometry. Be sure to conduct all of the procedures that can cause infection from aerosols or splashes within a biosafety level two certified biological safety cabinet.To assess the redox status of the cells, the next morning, incubate the cells and the appropriate Wells of the 6 well plate with 10 micromolar hydrogen peroxide for one hour, before detaching the cells with 750 microliters of 0.2 5%drips in EDTA per well, as demonstrated. Stop the reaction with two milliliters of complete medium per well and pull the supernatants for each condition in individual 15 milliliter conical tubes. Sediment the cells by centrifugation and wash the pellets in 500 microliters of PBS per well by centrifugations two times.After the second wash, filter the cell suspensions through 40 micro meter meshes into flow cytometry compatible tubes on ice, protected from light. In the Flow cytometer software, run the zero multiplicity of infection control sample. Then analyze the hydrogen peroxide and untreated cells.It will allow calculation of the mean fluorescent intensity ratio between the oxidized versus reduced forms of row GFP, using the formula as indicated. Here are the gating strategy for selecting a large number of cells by flow cytometry is shown. Flow cytometry can also be used to determine the dose response curve for row GFP analysis and the most efficient multiplicity of infection input.According to the multiplicity of infection, dose response curve in this representative analysis, a 200 multiplicity of infection gave the highest road GFP expression, but the cell morphology was effected suggesting cytotoxicity. Therefore, the optimum transduction efficiency was determined to be a multiplicity of infection of 100. In this redox status analysis, oxidized and reduced row GFP mean fluorescence intensities were obtained from flow cytometry analysis for vehicle and four 10 micromolar positive control hydrogen peroxide treatments.The overlaid histograms represent the shifts in the cell number in the 10 micromolar hydrogen peroxide and vehicle treated groups or reduced and oxidized erode GFP. Calculation of the ratio between the oxidized and reduced row GFP for this analysis revealed that 10 micromolar hydrogen peroxide caused a threefold increase in oxidation of row GFP compared to the vehicle treatment. Having accurate cell numbers is important for MRI calculations and reproducibility.In order to have reproducible results, researchers should thoroughly mix the antiviral solutions to obtain homogeneous suspensions. This protocol allows researchers to study rapid redox changes as an integral part of the broad range of normal cellular processes and of disease progression in real time.

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