This research was funded by the European projects GRACIOUS (GA760840) and SUNSHINE (GA952924). The authors would also like to acknowledge the efforts of all those who, in some way, assisted with the development of this protocol.

NOTE: The following protocol has been designed with the capacity to be utilized in conjunction with a bicinchoninic acid (BCA) protein assay and an adenylate kinase (AK) assay to normalize samples to treatments. Ensure the operator is wearing appropriate attire and necessary safety equipment, such as a Howie lab coat, nitrile gloves, and class I safety glasses, throughout the preparation and use of materials. The protocol is divided into several stages.
1. Stock and working solutions preparations
<ol>
	<li>Prepare stock solutions of 100 mM GSH standard in 1 mM HCl (prepared from 37% HCl in double distilled water (ddH2O)). 
	NOTE: If diluting from highly concentrated acid, such as 37% HCl, ensure the correct process of adding acid to water in a class I fume hood.</li>
	<li>Prepare a stock of 22.35 mM OPA in absolute ethanol. Perform this step in a class I fume hood. Prepare 25 mM N-ethylmaleimide (NEM) in ddH2O. Perform this step in a class I fume hood. 
	NOTE: These three solutions can be stored at -20 &#176;C for up to 3 months.</li>
	<li>Prepare 0.01 M Tris(2-carboxyethyl) phosphine (TCEP) to a total volume of 500 &#181;L, required for 100 wells. Prepare 100 &#181;L of 1 mM GSH standard, diluted from 100 mM stock using ddH2O.</li>
	<li>Either use immunoprecipitation (IP) lysis buffer or the following formulation: 394 mg of Tris-HCl (final concentration 25 mM), 877 mg of NaCl (final concentration 150 mM), 29 mg of EDTA (final concentration 1 mM), either 1 mL of 100% NP-40 or IGEPAL CA-630 (final concentration of 1% V/V), 5 mL of glycerol (final concentration 5% V/V), 84 mL of ddH2O. Mix components via gentle stirring and adjust pH to 7.4. Decant into a 100 mL volumetric flask and add the remaining volume of ddH2O to reach a final volume of 100 mL. Sterile filter through a 0.22 &#181;m filter and store at 2-8&#160;&#176;C for up to 6 months. 
	NOTE: Lysing solutions do act as a contaminant/interferent in this assay; hence, the above formulations are specified.</li>
	<li>Prepare 1 L of 0.1 M phosphate-buffered saline supplemented with ethylenediamine tetraacetic acid (PBS-EDTA) at 3 different pH values, specifically 7.2, 8.5, and 9.0. Use a commercial 0.1 M PBS buffer supplemented with 1 mM EDTA (292.24 mg/L) or the following 10x (1 L) formulation: 80 g NaCl, 2.0 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4, 800 mL of ddH2O. Add and mix; top up to 1 L. Autoclave the solution for sterility and store it for 12 months at room temperature. From the 10x stock solution, make a working solution of PBS and supplement it with EDTA (concentration as stated previously). 
	NOTE: pH is of critical importance; ensure pH is accurate before commencing protocol.</li>
	<li>Perform 1:2 serial dilution of 1 mM GSH using ddH2O (1 mM, 500 &#181;M, 250 &#181;M, 125 &#181;M, 62.5 &#181;M, 31.25 &#181;M, 15.625 &#181;M, and 7.8125 &#181;M). Add 10 &#181;L of each concentration to standard wells (performed in duplicate). Samples will be further diluted in lysis buffer; hence, concentrations will be 1/5th of their original concentration afterward (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 &#181;M). 
	NOTE: Final concentrations for calibration will be 30 &#181;m,15 &#181;m,7.5 &#181;m, 3.75 &#181;m, 1.875 &#181;m, 937.5 nm, 468.8 nm, and 234.4 nm.</li>
</ol>
2. Assay preparation
NOTE: This protocol uses human cell lines HepG2, A549, and J774, which were purchased commercially from ATCC. These cell lines were utilized under the approved guidelines outlined by the animal and tissue culture acts and regulations of the University.
<ol>
	<li>At 24 h prior to assay commencement, seed cells at concentrations shown in Table 1; however, depending on the cell line/ type and treatment used, adjust density as needed. Grow cells in complete growth medium (Eagles modified essentials medium (EMEM) with 10% Heat-inactivated fetal calf serum (HIFS), 1% Penstrep (10,000U/mL penicillin/ 10mg/mL streptomycin), and 1% non-essential amino acids.</li>
	<li>Seed cells using conventional cell seeding methods32. Seed cells in fully black plates if not using post-assay microscopy. Assess cells for confluency and general health in the T75 flask prior to use via microscopy.</li>
	<li>In a clean, sterile, class II biological safety cabinet and following a strict aseptic technique, discard cell media, gently wash cells with ~15 mL of sterile, room temperature (RT) PBS, and discard.</li>
	<li>To the cell flask, add 5 mL of sterile 1x trypsin, gently rock to ensure coverage of the cell monolayer, and place into an incubator at 37 &#176;C for 5 min to facilitate cell detachment.</li>
	<li>Stop trypsinization by addition of growth medium containing heat-inactivated fetal calf serum (10%), approximately 10 mL.</li>
	<li>Transfer cells to a 50 mL centrifuge tube and pellet at 200 x g for 5 min. Discard the media and replace it with 5 mL of the same media. Resuspend cells in the tube until homogenous and no clumping can be observed.</li>
	<li>Remove 20 &#181;L of cell suspension and place in a hemocytometer for counting. Once the number of cells required has been calculated, perform a dilution to make a solution with the correct cell density.</li>
	<li>Pipette media containing cells in 96-well plates (350 &#181;L capacity), with a maximum volume of 200 &#181;L per well. Place cells in a 37 &#176;C incubator with 5% CO2 for 24 h to adhere to the plate surface.</li>
	<li>To assess both total glutathione and GSSG, treat samples for both conditions. Seed and treat 6 wells to provide 2 sets each of GSH and GSSG with 3 technical repeats (Figure 1 demonstrates layout).</li>
	<li>Ensure all reagents are appropriately prepared prior to the commencement of the assay; ensure all buffer components and reagents are at RT before commencing construction of buffers or use in the assay, with the exception of pH 7.4 PBS (without EDTA), which should be kept on ice/ refrigerated until use. 
	NOTE: It is of critical importance that bubble formation be limited to a minimum to allow desired reactions to occur within the assay and allow accurate quantification via plate reader.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell line</td>
			<td>Seeding density (96-well plate)</td>
		</tr>
		<tr>
			<td>HepG2</td>
			<td>10,000 cells / well</td>
		</tr>
		<tr>
			<td>A549</td>
			<td>5,000 cells / well</td>
		</tr>
		<tr>
			<td>J774</td>
			<td>10,000 cells / well</td>
		</tr>
	</tbody>
</table>
Table 1: Suggested seeding densities for chosen cell lines. Demonstrated are different seeding densities for three different cell lines used in the represented data, specifically A549, J774, and HepG2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66267/66267fig01.jpg" /> 
Figure 1: Proposed layout for seeding of 96 well plates for the simultaneous determination of total Glutathione and Glutathione disulfide. Wells for calibration and controls are also demonstrated. Wells that are not utilized are represented with a cross. <a href="https://www.jove.com/files/ftp_upload/66267/66267fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Nanomaterial treatment
<ol>
	<li>Weigh nanomaterials (specifically ZnO, TiO2, CuO, and Ag) in a &#181;g capable weight scale. Perform a calculation to attain an initial concentration of 1 mg/mL per nanomaterial.</li>
	<li>Transfer nanomaterial solutions to a sonicator and sonicate for 16 min using a sonication bath (38 W) to produce a homogenous solution. Make a series of dilutions for each nanomaterial at 125, 62.5, 31.25, 15.625 &#181;g/mL.</li>
	<li>Remove cells from the incubator and lightly wash with RT PBS. After ensuring all PBS has been removed, add 100 &#181;L of treatments to the plate, with controls (culture media without HIFS). After applying treatment to cells, incubate with the nanomaterials in a 37&#176;C incubator (5% CO2) for 4 h; afterwards, use the protocol below to quantify GSH: GSSG, protein concentration, and AK release.</li>
</ol>
4. Assay protocol
<ol>
	<li>After exposure to treatment (Figure 2A), assess viability via adenylate kinase (AK) activity (optional). Use a commercial kit for this assay following the manufacturer&#39;s instructions.</li>
	<li>Spin plates in a centrifuge at 200 x g for 5 min to lightly pellet treatment and cell debris (Figure 2B).</li>
	<li>Remove 20 &#181;L of media supernatant gently from each sample and control well (as specified above) and pipette into an adjacent 96-well white plate in the same layout format (Figure 2C).</li>
	<li>Add 100 &#181;L of working solution from the AK kit to each well, shield the plate from light, and leave to develop for 10 min at room temperature. Record luminescence using a plate reader at 1000 counts/s.</li>
	<li>For GSH standards, add 40 &#181;L of total glutathione buffer to each well (Figure 2D; optional, required for quantification).</li>
	<li>Aspirate remaining media from the plate and wash 3x with ice-cold 0.1 M PBS, pH 7.2, discarding each wash (Figure 2E) with the exception of standards. Leave the final wash in the plate until the GSH calibration has been loaded into the plate.</li>
	<li>Add 10 &#181;L of each glutathione concentration and blank (ddH2O) into wells in triplicate.</li>
	<li>Remove the final PBS wash and add the mixtures to each well as per Table 2 for desired target quantification (Figure 2E). Calculate required volumes prior to commencing this step due to time-sensitive loss of activity with complete buffers. Ensure these reagent mixes are made before starting the assay process, but do not allow them to sit for more than 30 min before use.</li>
	<li>Place on an orbital plate shaker and allow the plate to shake at 300 RPM for 2 min (Figure 2F).</li>
	<li>Remove from the shaker and add 5 &#181;L of 0.01M TCEP solution to each well, excluding the NEM control well. Return the plate to the shaker and incubate for 10 min (Figure 2F).</li>
	<li>Transfer plate to centrifuge and spin at 200 x g for 5 min. Transfer 25 &#181;L from each sample well to another 96-well plate (clear); this will be utilized for protein concentration (see step 4.15 ; Figure 2G). Do not transfer standards or assay controls.</li>
	<li>Ensure the final volume for each well is 30 &#181;L; remove volume from controls and standards to meet this requirement (Figure 2H).</li>
	<li>Add 170 &#181;L of working OPA solution to each well, shield plate from light and place on shaker for 15 min (Figure 2I).</li>
	<li>Read fluorescence using a plate reader at Ex340/Em450 (Figure 2J). Ensure no bubbles are present during the measurement stage; they will have a detrimental impact on both the reaction and the quantification via plate reader.</li>
	<li>To quantify the protein content of lysed cells, use a commercial BCA assay kit. Transfer samples taken from step 4.12 to a new 96-well plate (clear) at 25 &#181;L per well.</li>
	<li>Use a bovine serum albumin (BSA) standard diluted in IP lysis buffer and add to the plate in triplicate at 25 &#181;L per well. The exact concentrations are defined in the BCA kit protocol.</li>
	<li>Prepare a working solution consisting of 50:1 ratio of reagents A and B from the BCA kit and add 200 &#181;L to each well containing sample, standard, and control. Shield plates from light and incubate at 37&#176;C for 30 min (Figure 2K).</li>
	<li>Remove samples from the incubator, allow to equilibrate at room temperature for 5 min, then read absorbance via plate reader at 562 nm (Figure 2L).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">&#160;Total glutathione concentration lysis reagent mix</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
			<td>50&#181;L</td>
		</tr>
		<tr>
			<td>Total volume / well</td>
			<td>50&#181;L</td>
		</tr>
		<tr>
			<td colspan="2">&#160;Oxidised glutathione concentration lysis reagent mix</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
			<td>49.5&#181;L</td>
		</tr>
		<tr>
			<td>NEM (25mM)</td>
			<td>0.5&#181;L</td>
		</tr>
		<tr>
			<td>Total volume / well</td>
			<td>50&#181;L</td>
		</tr>
		<tr>
			<td colspan="2">USE BOTH SOLUTIONS WITHIN 30 MINUTES OF MIXTURE COMPOSITION</td>
		</tr>
		<tr>
			<td>&#160;OPA detection solution component</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>OPA 3mg/mL</td>
			<td>5&#181;L</td>
		</tr>
		<tr>
			<td>PBS (pH 9.0)</td>
			<td>165&#181;L</td>
		</tr>
		<tr>
			<td>Total volume / well</td>
			<td>170&#181;L</td>
		</tr>
	</tbody>
</table>
Table 2: Necessary volumes of reagents for performing the protocol. Volumes required per well for the determination of total glutathione, glutathione disulfide, and working reagent required. Ensure required volumes are calculated, and an excess is included to account for volume loss through transfer.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66267/66267fig02.jpg" /> 
Figure 2: Schematic representation of the protocol. (A) Initial seeding, incubation and treatment of cells. (B) Centrifugation to separate media from suspended solids. (C) Media transfer for Adenylate kinase assay. (D) Addition of glutathione concentrations for calibration range. (E) Washing stages and lysing reagent addition. (F) Buffer addition and tris(2-carboxyethyl) phosphine addition with shaking step. (G) Centrifugation of lysed cells for media removal for protein analysis. (H) Media removal to equalize volume across the plate. (I) Addition of ortho-phthalaldehyde working solution with shaking incubation. (J) Measurement of ortho-phthalaldehyde fluorescence via plate reader. (K) Incubation stages for Bicinchoninic acid assay for protein determination. (L) Measurement of protein concentration, allowing normalization of glutathione: glutathione disulfide values. <a href="https://www.jove.com/files/ftp_upload/66267/66267fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+intracellular+distribution+of+reduced+glutathione+in+cultured+rat+hepatocytes+using+monochlorobimane+and+confocal+laser+scanning+microscopy.">Measurement of the intracellular distribution of reduced glutathione in cultured rat hepatocytes using monochlorobimane and confocal laser scanning microscopy.</a> Toxicol in vitro. 16 (5), 609-619 (2002).</li><li>McBeth, C., Stott-Marshall, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+of+reversible+redox+compounds+in+enzyme+catalysed+assays&#8211;Electrochemical+limitations.">Interference of reversible redox compounds in enzyme catalysed assays&#8211;Electrochemical limitations.</a> Anal Biochem. 662, 114972 (2023).</li><li>Yu, Z., 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=Reactive+oxygen+species-related+nanoparticle+toxicity+in+the+biomedical+field.">Reactive oxygen species-related nanoparticle toxicity in the biomedical field.</a> Nanoscale Res Lett. 15 (1), 115 (2020).</li><li>Boyles, 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=Development+of+a+standard+operating+procedure+for+the+DCFH2-DA+acellular+assessment+of+reactive+oxygen+species+produced+by+nanomaterials.">Development of a standard operating procedure for the DCFH2-DA acellular assessment of reactive oxygen species produced by nanomaterials.</a> Toxicol Mech Meth. 32 (6), 439-452 (2022).</li><li>Segeritz, C. P., Vallier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture:+Growing+cells+as+model+systems+in+vitro.">Cell culture: Growing cells as model systems in vitro.</a> Basic Sci Meth Clin Res. , 151-172 (2017).</li><li>Ma, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nrf2+in+oxidative+stress+and+toxicity.">Role of nrf2 in oxidative stress and toxicity.</a> Ann Rev Pharmacol Toxicol. 53, 401-426 (2013).</li><li>Calabrese, V., Cornelius, C., Dinkova-Kostova, A. T., Calabrese, E. J., Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+stress+responses,+the+hormesis+paradigm,+and+vitagenes:+novel+targets+for+therapeutic+intervention+in+neurodegenerative+disorders.">Cellular stress responses, the hormesis paradigm, and vitagenes: novel targets for therapeutic intervention in neurodegenerative disorders.</a> Antioxid redox Signal. 13 (11), 1763-1811 (2010).</li><li>Ishkaeva, R. A., Zoughaib, M., Laikov, A. V., Angelova, P. R., Abdullin, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+cell+redox+state+and+glutathione-modulating+factors+using+a+monochlorobimane-based+microplate+assay.">Probing cell redox state and glutathione-modulating factors using a monochlorobimane-based microplate assay.</a> Antioxidants. 11 (2), 391 (2022).</li></ol>

The authors declare no conflicts of interest.

As stated, the need to understand cellular redox, monitor states of oxidative stress, and the antioxidant response has always been crucial in understanding and preventing a myriad of diseases, such as cancers and neurodegeneration33,34. Demonstrated here is a means to improve upon the translational landscape by increasing the accessibility of accurate GSH: GSSG detection with fast, minimal preparation.
This protocol demonstrates a multiparametric sequence of assays for the determination of intracellular glutathione/ thiol species (reduced and oxidized), with 2 means of normalization via BCA protein assay and/or AK assay. This assay can also be modified to detect various other markers through the initial mediator extraction step and can be simplified in a manner that will simply give an oxidized/reduced thiol ratio with the exclusion of the calibration range.
When considering the evaluation of the analytes, both mBCI and OPA were explored and compared for use. While mBCl initially demonstrated good signal potential, significant limitations in use were discovered. Primarily, the use of live cells demonstrates the best use of mBCl; however, after cell lysis, the signal was found to be quenched and is generally diminished in a multiwell format compared to OPA35. Another issue is the measurement of GSSG via mBCl, literature is sparse regarding this, and through protocol optimization/ exploration, accurate detection of GSSG through mBCl was not achieved.
We have demonstrated that OPA assay presents significantly reliable calibration ranges, with an R2 average of 0.9932 &#177; 0.007 (N=12) across a 0.234 - 30 &#181;M GSH concentration range. This range was chosen due to previous reference ranges found in the literature35. It is theoretically possible to detect glutathione outside of these ranges but will require modification to the concentration of reagents, incubation time, and potentially the equipment utilized in detection. It must be noted that each plate requires its own standard range for quantification; the slightest variance in time between plates performed on different days can have a significant effect on the values obtained during measurement.
Achieving accurate and reliable data from this protocol is dependent on several crucial steps being strictly adhered to. When constructing the various buffers required in the protocol, it is crucial that pH is accurate. Hence, buffers requiring a pH of 9 should have no deviation beyond &#177; 0.1 of this value. This is due to the potential for buffer components to precipitate out of the solution in the wrong pH; following this protocol exactly will prevent this issue.
Complete removal of treatment and accurate washing before lysing are also critical to prevent artifacts and inaccurate data acquisition during the plate reading stage. Once cells have been lysed (step 4.8), removal of treatment is not possible, and the plate will not be salvageable. Due to the volumes of buffers/reagents being added throughout the protocol varying between samples and standards, it is critical that the user is aware of the varied volumes, in step 4.12 and 4.13. The assay operator is also made aware of these varied volumes and instructed to ensure all volumes are the same to allow accurate measurement to be achieved. As the volumes between the samples and standards are not visibly significant, it can be an easy mistake to make with regard to having an excess solution in the sample well.
There are limitations to this protocol that rely on crucial steps, which are critical to acquiring accurate and reliable data. The users performing this assay need to possess a reasonable level of laboratory skills to prevent undesired problems, such as bubble formation. Bubble formation has a drastic impact on both the capacity for reactions to occur within the microplate and the measurement of fluorescence. The lysing agent used in this protocol contains a detergent, which presents difficulty to a novice researcher who may struggle to prevent bubble formation. Immediate centrifugation may salvage this error. The protocol is also potentially limited regarding cell type; cell lines A549, J774, and HepG2 were utilized to both optimize and produce data for this protocol. Other cell lines may require different seeding densities and optimization of the protocol to get accurate data.
This protocol offers numerous advantages over several existing assays. While the detection of thiols using Phthalaldehyde is not a novel concept, utilization in a combined assay format such as this, in a microplate, with limited required materials and equipment, offers great potential for all labs to access this protocol. Most Thiol/ GSH kits from commercial suppliers do not disclose the composition of their reagents. Hence, it can be difficult to foresee the potential for incompatibilities/ interference. Here, we present each component of all the utilized reagents to limit that potential.
This protocol is also performed rather rapidly upon completion of the initial treatment period. Accounting for user processing between incubation stages, the thiol quantification aspect of this protocol can be performed in under 1 h. Samples are simultaneously lysed and bound to prevent auto-oxidation of samples, which is optimal for these reaction species. While not specified in the protocol, samples can technically be lysed in a plate and sealed, allowing them to be frozen for future analysis. However, this alteration to the protocol has not been explored.

Reactive oxygen species (ROS) are a primary inducer of oxidative stress; oxidative stress has been well established in the generation of DNA mutations, cellular aging/ death, various cancers, diabetes, neurological diseases (such as Parkinson&#39;s and Alzheimers), and several other life-debilitating conditions1,2,3,4,5. A key defense against ROS are thiolic, non-enzymatic antioxidants, which are capable of reducing oxidants or radicals by acting as proton donors6,7. Glutathione (GSH) and cysteine are the two most prevalent thiols found in mammals8, while various other low molecular weight thiols exist (such as ergothioneine), GSH and cysteine are the most commonly measured non-enzymatic antioxidants found in literature9,10,11 and hold greatest relevance for combatting ROS8,12,13,14.
When GSH is utilized as an antioxidant, two molecules of GSH are covalently linked together via a disulfide bond to make glutathione disulfide (GSSG). Depletion of GSH is often used as an indicator of oxidative stress15,16. This assessment can also be combined with the detection of GSSG, although increases in GSSG in cells are often limited by active export processes since GSSG can be relatively reactive in cells, leading to disulfide bond formation with other protein thiols16.
Traditional methods for measuring GSH and GSSG are not simple processes and require numerous steps, including cellular extraction using lytic reagents17,18. The protocol outlined here simplifies these methods and allows the accurate measurement of non-enzymatic thiols and normalization using the cellular protein content or adenylate kinase release. In addition, it is possible to measure cellular viability prior to GSH/GSSG extraction. Several methods have previously attempted to target and quantify reduced and oxidized non-enzymatic thiols efficiently; methods including the use of HPLC19,20,21, plate assay (biochemical)22,23,24,25, and which use common reagents for thiol conjugation, such as 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB/ Ellman&#39;s reagent)19, Monochlorobimane (mBCI)26,27,28. Several companies have also prepared proprietary kits for the detection of glutathione; however, they do not publish reagent incompatibilities, which presents issues dependent on the treatments used29.
This protocol outlines a multiparametric assay that detects reduced thiols (such as GSH) via ortho-phthalaldehyde (OPA) conjugation to produce a fluorescent signal detectable at 340/450 Ex/Em, respectively. This assay facilitates the detection of both GSH and GSSG simultaneously (in plate), through the use of masking agents (N-ethylmaleimide) and GSSG reducing agents (tris(2-carboxyethyl) phosphine). This multi-biomarker protocol also provides an opportunity during the cellular lysing stage to quantify proteins via bicinchoninic acid assay for the normalization of samples on completion of the final measurement or via an adenylate kinase assay from the cell media. This assay can be performed utilizing several reagents readily available in most laboratories and only requires a few additional uncommon chemicals to perform. The process is simple, accessible, and can be performed without laborious stages in less than 2 h.
In this protocol, various nanomaterials were chosen that were either previously shown to induce ROS or suspected to induce oxidative stress30,31. A concentration range was explored to see the effects of exposure of these nanomaterials on various cell lines and the effectiveness of the assay in quantifying antioxidant thiols.

<table><tbody><tr><td>0.22&amp;micro;m filter (optional-For lysis buffer)</td><td>Fisher scientific</td><td>12561259</td><td></td></tr><tr><td>100mL volumetric flask</td><td>Fisher scientific</td><td>15290866</td><td></td></tr><tr><td>1L Volumetric flask&amp;nbsp;</td><td>Fisher scientific</td><td>15230876</td><td></td></tr><tr><td>250mL beaker (optional-For lysis buffer)</td><td>Fisher scientific</td><td>15409083</td><td></td></tr><tr><td>8-Channel micropipette (20-200&amp;micro;L)</td><td>SLS</td><td>FA10011D2</td><td></td></tr><tr><td>8-Channel micropipette (2-20&amp;micro;L)</td><td>SLS</td><td>B2B06492</td><td></td></tr><tr><td>96 well plates - black with clear bottom, TC treated</td><td>Fisher scientific</td><td>10000631</td><td>Preferred plate for seeding and fluoresence, use TC treated clear if unavailable</td></tr><tr><td>96 well plates - clear (TC treated and untreated)</td><td>Fisher scientific</td><td>10141161</td><td>If black plates with clear bottom is not available/ suitable use TC treated clear</td></tr><tr><td>96 well plates - white, Not TC treated</td><td>Fisher scientific</td><td>11457009</td><td></td></tr><tr><td>A549 (lung carcinoma) cell line</td><td>ATCC</td><td>CCL-185</td><td></td></tr><tr><td>Absolute ethanol</td><td>Merck (Sigma-Aldrich)</td><td>1.08543</td><td></td></tr><tr><td>Aluminium foil</td><td>Fisher scientific</td><td>11779408</td><td>For protecting plates from light</td></tr><tr><td>BCA Assay Kit</td><td>Thermo</td><td>23225</td><td></td></tr><tr><td>Benchtop Centrifuge (with 96 plate rotor)</td><td>Eppendorf</td><td>5804</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Merck (Sigma-Aldrich)</td><td>E9884</td><td></td></tr><tr><td>Glutathione (GSH)</td><td>Merck (Sigma-Aldrich)</td><td>G6013</td><td></td></tr><tr><td>Glutathione disulfide (GSSG)</td><td>Merck (Sigma-Aldrich)</td><td>G4501</td><td></td></tr><tr><td>Glycerol</td><td>Merck (Sigma-Aldrich)</td><td>G5516</td><td></td></tr><tr><td>HCl, 37%</td><td>Merck (Sigma-Aldrich)</td><td>258148</td><td>Dilute to 1mM for GSH stock, pH adjustment also</td></tr><tr><td>HepG2 (Hepatocarcinoma) cell line</td><td>ATCC</td><td>HB-8065</td><td></td></tr><tr><td>IGEPAL&amp;nbsp;CA-630&amp;nbsp;</td><td>Merck (Sigma-Aldrich)</td><td>18896</td><td>Use either IGEPAL CA-630 or NP-40 for solution, not both</td></tr><tr><td>IP lysis buffer&amp;nbsp;</td><td>Fisher scientific</td><td>11825135</td><td></td></tr><tr><td>J774 (monocyte, macrophage) cell line</td><td>ATCC</td><td>TIB-67</td><td></td></tr><tr><td>KCl</td><td>Merck (Sigma-Aldrich)</td><td>P3911</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Merck (Sigma-Aldrich)</td><td>P0662</td><td></td></tr><tr><td>Micropipette (20-200&amp;micro;L)</td><td>SLS</td><td>B2B06482</td><td></td></tr><tr><td>Micropipette (2-20&amp;micro;L)</td><td>SLS</td><td>B2B06478</td><td></td></tr><tr><td>Microplate shaker</td><td>VWR</td><td>444-0041</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck (Sigma-Aldrich)</td><td>S9763</td><td></td></tr><tr><td>NaCl</td><td>Merck (Sigma-Aldrich)</td><td>S9888</td><td></td></tr><tr><td>NaOH, 10M</td><td>Merck (Sigma-Aldrich)</td><td>72068</td><td>For pH adjustment only</td></tr><tr><td>N-Ethylmaleimide (NEM)&amp;nbsp;</td><td>Merck (Sigma-Aldrich)</td><td>E3876</td><td></td></tr><tr><td>NP-40</td><td>Merck (Sigma-Aldrich)</td><td>492016</td><td>Use either IGEPAL CA-630 or NP-40 for solution, not both. NP-40 alternative suggested</td></tr><tr><td>Ortho -Phthaldialdehyde (OPA)</td><td>Merck (Sigma-Aldrich)</td><td>P1378</td><td></td></tr><tr><td>PBS 0.1M</td><td>Merck (Sigma-Aldrich)</td><td>P2272</td><td>PBS can either be acquired pre-made or made in house, see notes</td></tr><tr><td>Plate reader (with fluoresence capacity)</td><td>Tecan</td><td>SPARK</td><td></td></tr><tr><td>Stir bar (optional-For lysis buffer)</td><td>Fisher scientific</td><td>16265731</td><td></td></tr><tr><td>Toxilight bioassay kit (AK assay)</td><td>Lonza</td><td>LT17-217</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) 0.5M&amp;nbsp; in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Alfa Aesar</td><td>H51864</td><td>Can also be purchased crystalised and suspended</td></tr><tr><td>TRIS-HCl</td><td>Merck (Sigma-Aldrich)</td><td>93363</td><td></td></tr><tr><td>X100 phosphatase and protease cocktail&amp;nbsp;</td><td>Fisher scientific</td><td>10025743</td><td></td></tr></tbody></table>

rapid quantification of oxidized and reduced forms of glutathione using ortho -phthalaldehyde in cultured mammalian cells in vitro

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

Following this protocol, A549 and J774 cell lines were seeded at densities of 5,000 cells/ well and 10,000 cells/well, respectively and cultured at 37&#160;&#176;C in 5% CO2 for 48 h. The AK analysis after nanomaterials treatment is shown in Supplementary Table 1, and the protein concentration is shown in Supplementary Table 2.
Calibration graph 
Shown in Figure 3 are three calibrations using the stated concentration range (0.234 - 30 &#181;M final concentration) from three separate plates from three different cell types (though should not impact calibration) on three different, non-consecutive, days. While 3 samples are shown, an N of 12 was observed, and demonstrated similar linear regressions with an average R2 value of 0.9932 &#177; 0.007.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66267/66267fig03.jpg" /> 
Figure 3: Glutathione calibration graphs for assay. Three calibration graphs from separate in-plate glutathione calibration ranges, each performed a week apart; error bars &#177; SD (n=3, N=12) n=technical replicates, N=Biological replicates. <a href="https://www.jove.com/files/ftp_upload/66267/66267fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Sample results 
HepG2, A549, and J774 cells were utilized in the assessment of various nanomaterials suspected of inducing changes to cellular mechanisms via oxidative stress. The detection and quantification protocol described were utilized.
The data received from the 3 measurements (AK, BCA, and GSH/GSSG) was handled as follows. The AK and BCA assay was implemented for normalization; the AK assay, using the recommended kit, will give the fastest, simplest data for the amount of AK released into the cell media. An increase in AK values is expected for increasing cell death. Hence, a -ve (Alive) and +ve (Dead) control is required. This will allow for normalization based on percentage.
The BCA assay is a longer process but will allow for quantifiable results to be acquired via protein quantification (mg/mL). This does not require a -ve or +ve control as in the AK but will still require a general -ve control (untreated cells) to allow for the normalization of values to be achieved.
In this representative results section, it was found that the treatment (nanomaterials) had the potential to cause interference with the AK assay. Hence, all normalization was performed using the BCA data. Therefore, information is presented as the concentration of detected species (GSH or GSH+GSSG (however, a subtraction of GSH concentration from total GSH+GSSG concentration is performed to get GSSG concentration) per mg/mL of protein (via BCA assay). If desired, this can then be converted into a ratio to assess the change in GSH: GSSG from the desired treatment.
Shown in Figure 4 is the GSH: GSSG ratio data from three different cell lines (A549, J774, and HepG2) acquired using the OPA protocol and normalized to protein expression via BCA (&#181;g/mL), further data specifying additional GSH and GSSG values can be found in Supplementary Figure 1.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66267/66267fig04.jpg" /> 
Figure 4: Glutathione: Glutathione disulfide ratiofrom performing the assay. Shown are the glutathione: Glutathione disulfide ratios of 3 cell lines, namely (A) A549, (B) J774, and (C) HepG2. Cells were incubated with treatments (various nanomaterials in serum-free media) for 4 h. Cells were processed using this protocol to quantify changes in glutathione and glutathione disulfide and normalized via protein quantification, error bars &#177; SE (n=3, N=3) <a href="https://www.jove.com/files/ftp_upload/66267/66267fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The plate also contains a series of controls to ensure the assay has run correctly. NEM is added as an individual component to demonstrate a lack of interaction with OPA detection media. The calibration standard demonstrates a linear increase with GSH concentration, which demonstrates the effective capacity for the OPA detection reagent to effectively bind to increasing concentrations of GSH.
It must be noted that this assay specifically targets free sulfhydryl groups commonly found in thiols (such as GSH, which are commonly considered antioxidants). One potential interaction is the binding of OPA to protein thiols, which would result in inaccurate data gathering. Hence, the BCA assay is a crucial stage to normalize data to protein and allow accurate reflection of free GSH.
Supplementary Figure 1: Figures demonstrating glutathione, glutathione disulfide, and glutathione: glutathione disulfide ratio from 3 cell lines, namely, (A) A549, (B) J774, and (C) HepG2. Cells were incubated with treatments (various nanomaterials in serum-free media) for 4 h. Cells were processed using this protocol to quantify changes in glutathione and glutathione disulfide and normalized via protein quantification, error bars &#177; SE (n=3, N=3) <a href="https://www.jove.com/files/ftp_upload/66267/Supplemetary Figure 1_RE.zip" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Metadata of adenylate kinase values for A549 and J774 cells. <a href="https://www.jove.com/files/ftp_upload/66267/Supplementary data 1_AK values_RE.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: Metadata of Bicinchoninic acid values with calibration for A549, J774, and HepG2 cells. <a href="https://www.jove.com/files/ftp_upload/66267/Supplementary data 2_BCA values_RE.xlsx" target="_blank">Please click here to download this File.</a>

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

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

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

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

rapid-quantification-oxidized-reduced-forms-glutathione-using-ortho

Research

JoVE Journal

Biochemistry

1.2K Views.  Teesside University. Quantification of both oxidized and reduced forms of glutathione (GSSG and GSH, respectively) has been achieved through the use of Ortho-phthalaldehyde (OPA). OPA becomes highly fluorescent once conjugated to GSH but is unable to conjugate GSSG until reduced. Here, we describe a multiparametric assay to quantify both using protein quantification for normalization.

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

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2&#39;-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.

The goal of this protocol is the detection of the DNA oxidation marker, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo) by HPLC-ED, in DNA from cultured cells or animal tissues.

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

Chemistry

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

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Medicine

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.

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. ...

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. 
The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Biology

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 ...

Neuroscience

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 ...

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

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

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

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

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

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

Summary

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HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2’-deoxyguanosine, in Cultured Cells and Animal Tissues

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

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

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

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

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

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