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.

assessment-cellular-oxidation-using-subcellular-compartment-specific

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Here, we describe a technique, protein film infrared electrochemistry, which allows immobilized redox proteins to be studied spectroscopically under direct electrochemical control at a carbon electrode. Infrared spectra of a single protein sample can be recorded at a range of applied potentials and under a variety of solution conditions.

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological macromolecules. Until recently, a major requirement has been the availability of relatively large, well diffracting crystals, which are often challenging to obtain. However, the advent of serial crystallography and a renaissance in multi-crystal data collection methods has meant that the availability of large crystals need no longer be a limiting factor. Here, we illustrate the use of the automated MeshAndCollect protocol, which first identifies the positions of many small crystals mounted on the same sample holder and then directs the collection from the crystals of a series of partial diffraction data sets for subsequent merging and use in structure determination. MeshAndCollect can be applied to any type of micro-crystals, even if weakly diffracting. As an example, we present here the use of the technique to solve the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean.

We present the use of the MeshAndCollect protocol to obtain a complete diffraction data set, for use in subsequent structure determination, composed of partial diffraction data sets collected from many small crystals of the fluorescent protein Cerulean.

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein&#39;s solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Here, we characterize protein structure and interaction sites in living cells using a protein footprinting technique termed in-cell fast photochemical oxidation of proteins (IC-FPOP).

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...