opa assay for accurate glutathione detection in cells

GSH/GSSG 测量 – 用于抗氧化剂研究的改进 OPA 检测

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 Several companies have also prepared proprietary kits for the detection of glutathione; however, they do not publish reagent incompatibilities, which presents issues dependent on the treatments used29.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

作者聚焦：创新巯基定量和生物标志物检测用于氧化应激研究 

在体外使用 邻 苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

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.

用于准确检测细胞中谷胱甘肽的 OPA 测定

首先，准备测定所需的所有材料。对于 GSH 标准品，向每个孔中加入 40 微升总谷胱甘肽缓冲液。接下来，从含有细胞沉淀的板的每个孔中吸出培养基，并用冰冷的PBS洗涤细胞三次。用 10 微升谷胱甘肽和双蒸水制备校准范围，一式三份作为空白。为了获得所需的靶标定量，将洗涤的细胞与适当的混合物在轨道振荡器中以 300 RPM 孵育 2 分钟。然后向每个孔中加入 5 微升 0.01 摩尔 tris，2-羧乙基膦溶液，并再次孵育 10 分钟。接下来，将板以 200 G 离心 5 分钟，并将 25 微升样品从每个样品孔转移到单独的透明 96 孔板中进行蛋白质定量。然后向每个含有 30 微升样品的孔中加入 170 微升工作邻苯二甲醛溶液。为了遮挡光线，用箔纸彻底覆盖它，并在轨道振荡器中孵育 15 分钟。最后，使用酶标仪在 340 纳米激发和 450 纳米发射下读取荧光。在该测定中，用各种纳米材料处理的 A549 细胞显示出不同的谷胱甘肽二硫化物比率，并且用 125 微克/毫升银处理的细胞观察到最高值。

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

Research

JoVE Journal

Biochemistry

OPA Assay for Accurate Glutathione Detection in Cells

326 次观看.  Teesside University. 首先，准备测定所需的所有材料。对于 GSH 标准品，向每个孔中加入 40 微升总谷胱甘肽缓冲液。接下来，从含有细胞沉淀的板的每个孔中吸出培养基，并用冰冷的PBS洗涤细胞三次。用 10 微升谷胱甘肽和双蒸水制备校准范围，一式三份作为空白。为了获得所需的靶标定量，将洗涤的细胞与适当的混合物在轨道振荡器中以 300 RPM 孵育 2 分钟。然后向每个孔中加入 5 微升 0.01 ...

视频: 用于准确检测细胞中谷胱甘肽的 OPA 测定

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 ...