opa assay for accurate glutathione detection in cells

GSH / GSSG Ölçümü - Antioksidan Araştırmaları için Rafine Bir OPA Testi

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 Several companies have also prepared proprietary kits for the detection of glutathione; however, they do not publish reagent incompatibilities, which presents issues dependent on the treatments used29.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

Yazar Gündemi: Oksidatif Stres Araştırmaları için Tiyol Kantitasyonu ve Biyobelirteç Tespitinde Yenilik 

İn Vitro Kültürlenmiş Memeli Hücrelerinde Orto-ftalaldehit Kullanılarak Oksitlenmiş ve İndirgenmiş Glutatyon Formlarının Hızlı Miktar Tayini

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.

Hücrelerde Doğru Glutatyon Tespiti için OPA Testi

Başlamak için, tahlil için gerekli tüm malzemeleri hazırlayın. GSH standartları için, her kuyucuğa 40 mikrolitre toplam glutatyon tamponu ekleyin. Daha sonra, hücre peletini içeren bir plakanın her bir oyuğundan ortamı aspire edin ve hücreyi buz gibi soğuk PBS ile üç kez yıkayın.Kalibrasyon aralığını 10 mikrolitre glutatyon ve çift damıtılmış su ile üç nüsha halinde boş olarak hazırlayın. İstenen hedef miktar tayini için, yıkanmış hücreleri uygun karışımlarla 300 RPM'de bir orbital çalkalayıcıda iki dakika inkübe edin. Daha sonra her bir oyuğa beş mikrolitre 0.01 molar tris, 2-karboksietil fosfin çözeltisi ekleyin ve tekrar 10 dakika inkübe edin.Daha sonra, plakayı beş dakika boyunca 200 G'de santrifüjleyin ve protein miktar tayini için her numune kuyucuğundan 25 mikrolitreyi ayrı bir şeffaf 96 oyuklu plakaya aktarın. Daha sonra, 30 mikrolitre numune içeren her bir oyuğa 170 mikrolitre çalışan orto-ftalaldehit çözeltisi ekleyin. Plakayı ışıktan korumak için bir folyo ile iyice örtün ve orbital çalkalayıcıda 15 dakika inkübe edin.Son olarak, 340 nanometre uyarma ve 450 nanometre emisyonda bir plaka okuyucu kullanarak floresansı okuyun. Çeşitli nanomalzemelerle muamele edilen A549 hücreleri, bu tahlilde farklı glutatyon disülfür oranları gösterdi ve en yüksek değer, mililitre gümüş başına 125 mikrogram ile muamele edilen hücreler için gözlendi.

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

Research

JoVE Journal

Biochemistry

OPA Assay for Accurate Glutathione Detection in Cells

326 Görüntüleme.  Teesside University. Başlamak için, tahlil için gerekli tüm malzemeleri hazırlayın. GSH standartları için, her kuyucuğa 40 mikrolitre toplam glutatyon tamponu ekleyin. Daha sonra, hücre peletini içeren bir plakanın her bir oyuğundan ortamı aspire edin ve hücreyi buz gibi soğuk PBS ile üç kez yıkayın.Kalibrasyon aralığını 10 mikrolitre glutatyon ve çift damıtılmış su ile üç nüsha halinde boş olarak hazırlayın. İstenen hedef miktar tayini için, yıkanmış hücreleri uygu...

Video: Hücrelerde Doğru Glutatyon Tespiti için OPA Testi

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 ...