opa assay for accurate glutathione detection in cells

Misurazione GSH/GSSG - Un test OPA raffinato per la ricerca sugli antiossidanti

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 Several companies have also prepared proprietary kits for the detection of glutathione; however, they do not publish reagent incompatibilities, which presents issues dependent on the treatments used29.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.

Autore in primo piano: Innovazione nella quantificazione dei tioli e nel rilevamento dei biomarcatori per la ricerca sullo stress ossidativo 

Quantificazione rapida di forme ossidate e ridotte di glutatione utilizzando l'orto-ftalaldeide in cellule di mammifero in coltura in vitro

We aim to offer a novel means to quantify thiol concentration with a specific focus on the GSH and GSSG concentrations of the various cell lines that can be performed in situ. We consider this a useful protocol for the scientific community. Numerous efforts have been made to detect niche and specific biomarkers of interest of reactive oxygen species, and several have found great interest in detecting short-lived reactive oxygen species, such as radical detection and small molecules such as hydroxyls and singlet oxygen.One key challenge for several institutions has been the ability to accurately and specifically detect biomarkers of interest without the need of expensive equipment or laborious processes. This protocol allows for multiparametric analysis, allowing for normalization of data without the need for separate protocols, and remove several stages found in other assays that are highly time consumptive, present incompatible reagents, or use reagents hazardous to health. The Nanomaterial Safety Group will continue to assess various nanomaterials and polymers for the effect they may have on human health.We consider this protocol an essential part of our toolkit in achieving this goal as this protocol can potentially be multiplexed to detect additional biomarkers.

Saggio OPA per la rilevazione accurata del glutatione nelle cellule

Per iniziare, preparare tutti i materiali necessari per il test. Per gli standard GSH, aggiungere 40 microlitri di tampone glutatione totale a ciascun pozzetto. Quindi, aspirare il terreno da ciascun pozzetto di una piastra contenente il pellet della cella e lavare la cella tre volte con PBS ghiacciato.Preparare l'intervallo di calibrazione con 10 microlitri di glutatione e acqua distillata doppia in triplicato. Per la quantificazione del target desiderato, incubare le cellule lavate con le miscele appropriate in un agitatore orbitale a 300 giri/min per due minuti. Quindi aggiungere cinque microlitri di tris molare, soluzione di 2-carbossietilfosfina 0,01 molare in ciascun pozzetto e incubare nuovamente per 10 minuti.Quindi, centrifugare la piastra a 200 G per cinque minuti e trasferire 25 microlitri da ciascun pozzetto del campione a una piastra trasparente separata da 96 pozzetti per la quantificazione delle proteine. Quindi aggiungere 170 microlitri di soluzione di ortoftalaldeide funzionante a ciascun pozzetto contenente 30 microlitri di campioni. Per proteggere la piastra dalla luce, coprirla accuratamente con un foglio e incubarla nell'agitatore orbitale per 15 minuti.Infine, leggere la fluorescenza utilizzando un lettore di piastre a 340 nanometri di eccitazione e 450 nanometri di emissione. Le cellule A549 trattate con vari nanomateriali hanno mostrato diversi rapporti di glutatione disolfuro in questo test e il valore più alto è stato osservato per le cellule trattate con 125 microgrammi per millilitro di argento.

opa-assay-for-accurate-glutathione-detection-in-cells

Research

JoVE Journal

Biochemistry

OPA Assay for Accurate Glutathione Detection in Cells

326 Views.  Teesside University. Per iniziare, preparare tutti i materiali necessari per il test. Per gli standard GSH, aggiungere 40 microlitri di tampone glutatione totale a ciascun pozzetto. Quindi, aspirare il terreno da ciascun pozzetto di una piastra contenente il pellet della cella e lavare la cella tre volte con PBS ghiacciato.Preparare l'intervallo di calibrazione con 10 microlitri di glutatione e acqua distillata doppia in triplicato. Per la quantificazione del target desiderato, incubare le cellule lavate c...

Video: Saggio OPA per la rilevazione accurata del glutatione nelle cellule

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.

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.

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