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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22&#181;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&#160;</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&#181;L)</td>
			<td>SLS</td>
			<td>FA10011D2</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Channel micropipette (2-20&#181;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&#160;CA-630&#160;</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&#160;</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>KH2PO4&#160;</td>
			<td>Merck (Sigma-Aldrich)</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette (20-200&#181;L)</td>
			<td>SLS</td>
			<td>B2B06482</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette (2-20&#181;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>Na2HPO4</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)&#160;</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&#160; in H2O</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&#160;</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

1.1K 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 

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 ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

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 ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

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 ...

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies &#8212; small, monomeric, high-affinity protein binders &#8212; that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters&#39; sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...