使用2'，7'-二氯荧光素双乙酸探针和流式细胞术在穆勒神经胶质细胞中定量活性氧
Summary
在这里，我们提出了一种系统化，可访问和可重复的方案，以使用Müller神经胶质细胞（MGCs）中的2’，7′-二氯荧光素二乙酸探针（DCFH-DA）检测细胞活性氧（ROS）。该方法用流式细胞仪量化总细胞ROS水平。该协议非常易于使用，适用且可重复。
Abstract
氧化还原平衡在维持细胞稳态方面具有重要作用。活性氧（ROS）的产生增加促进了蛋白质，脂质和DNA的修饰，最终可能导致细胞功能的改变和细胞死亡。因此，通过激活像Keap1 / Nrf2这样的抗氧化途径或通过改善氧化还原清除剂（维生素A，C和E，β胡萝卜素和多酚等）来增加其抗氧化防御能力对细胞的有害侮辱是有益的。炎症和氧化应激与视网膜病变的发病机制和进展有关，例如糖尿病视网膜病变（DR）和早产儿视网膜病变（ROP）。由于Müller神经胶质细胞（MGCs）在神经视网膜组织的稳态中起着关键作用，因此它们被认为是研究这些细胞保护机制的理想模型。从这个意义上说，用可重复和简单的方法量化ROS水平对于评估参与抗氧化细胞防御机制的途径或分子的贡献至关重要。在本文中，我们提供了使用DCFH-DA探针和MGC中的流式细胞术测量ROS所需的程序的完整描述。此处提供了使用该软件进行流式细胞术数据处理的关键步骤，因此读者将能够测量ROS水平（FITC的几何手段）并分析荧光直方图。这些工具不仅对评估细胞损伤后ROS的增加非常有帮助，而且对研究某些分子的抗氧化作用非常有帮助，这些分子可以对细胞提供保护作用。
Introduction
神经视网膜是一个非常有组织的组织，呈现出定义明确的神经元层。在这些中，神经元（神经节，腺素，双极，水平和光感受器细胞）彼此相互连接，并且还与Müller神经胶质细胞（MGCs）和星形胶质细胞相互连接，从而导致视觉信息的充分光转导和处理1，2。众所周知，MGCs在维持视网膜稳态方面具有重要作用，因为它们穿过整个视网膜部分，因此，它们可以与调节多个保护过程的所有细胞类型相互作用。据报道，MGCs具有维持和存活视网膜神经元的几个重要功能，包括糖酵解为神经元提供能量，去除神经元废物，回收神经递质以及释放神经营养因子等3，4，5。
另一方面，炎症，氧化和亚硝化应激参与许多人类疾病的发病机制和进展，包括视网膜病变6，7，8，9，10，11。细胞中的氧化还原平衡取决于ROS水平的严格调节。ROS主要是由于有氧呼吸而在生理条件下不断产生的。ROS家族的主要成员包括反应性自由基，如超氧阴离子（O2͘͘͘͘•−），羟基自由基（•OH），各种过氧化物（ROOR′），氢过氧化物（ROOH）和无自由基过氧化氢（H2O2）12，13。在过去的几年中，很明显，ROS通过控制基本过程在细胞中起着重要的信号作用。MGCs具有很强的抗氧化防御能力，通过激活转录核因子红系2相关因子2（Nrf2）和随后表达抗氧化蛋白以消除病理条件下ROS的过量产生14，15，16。当细胞由于ROS的夸大产生或去除ROS的能力缺陷而失去氧化还原平衡时，氧化应激的积累会促进蛋白质，脂质和DNA的有害修饰，从而导致细胞应激或死亡。视网膜抗氧化防御系统的增加改善了视网膜病变的消退和预防，例如ROP和RD17，18，19，20，21，22，23，24。因此，实时测量ROS产量是一个强大而有用的工具。
有几种方法可以测量细胞中的ROS产生或氧化应激。其中，2’，7′-二氯荧光素二乙酸酯（DCFH-DA）探针是直接量化细胞25，26，27，28氧化还原状态的最广泛使用的技术之一。该探针是亲脂性的和非荧光的。该探针在细胞膜上的扩散允许其通过细胞内酯酶在两个酯键处裂解，产生相对极性和细胞膜不渗透的产物2’，7′-二氯荧光素（H2DCF）。这种非荧光分子在细胞内积聚，随后通过ROS氧化产生高荧光产物DCF。探针的氧化是多种类型的ROS（过氧亚硝酸盐，羟基自由基，一氧化氮或过氧化物）作用的产物，可以通过流式细胞术或共聚焦显微镜（在530nm处发射并在485nm处激发）来检测。该技术的局限性在于超氧化物和过氧化氢不与H 2 DCF25，29发生强烈反应。在本文中，我们使用DCFH-DA探针通过流式细胞术测量和量化ROS。出于这个原因，我们通过用ROS诱导剂A或B刺激MGCs来诱导ROS的产生，然后再用荧光探针加载细胞。此外，我们使用抗氧化化合物。最后，我们展示了使用该协议获得的具有代表性和可靠的数据。
Protocol
Representative Results
Discussion
几种病理状况，如癌症，炎症性疾病，缺血/再灌注，缺血性心脏病，糖尿病和视网膜病变，以及衰老等生理状况，导致ROS生产过剩6，7，8，9，10，11。因此，检测、测量和了解ROS调节所涉及的途径是许多疾病的重要靶点。使用探针测量ROS水平，如DCF…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢CIBIICI（CENTRE de Investigaciones en Bioquímica Clínica e Inmunología，CONICET-UNC，阿根廷科尔多瓦）的María Pilar Crespo和Paula Alejandra Abadie，以及Gabriela Furlan和Noelia Maldonado的细胞培养协助。我们还感谢Victor Diaz（FCQ机构传播副部长）的视频制作和编辑。
本文由2018-2021年科尔多瓦国立科学与技术大学（SECyT-UNC）Consolidar，科学与技术研究基金会（FONCyT）和科学与技术研究中心（PICT）2015 N° 1314（全部至M.C.S.）资助。
Materials
| 2′,7′-DCFH-DA | Sigma | 35845-1G | |
| 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | Gibco by life technologies | 15630-080 | |
| BD FACSCanto II flow cytometer | BD Biosciences | FACSCanto | |
| BD FACSDiva software | BD Biosciences | ||
| Cell Culture Dishes 100×20 mm | Cell Star- Greiner Bio-One | 664 160 | |
| Centrifuge | Thermo | Sorvall legend micro 17 R | |
| Centrifuge Tubes (15 ml) | BIOFIL | CFT011150 | |
| Centrifuge Tubes (50 ml) | BIOFIL | CFT011500 | |
| Cryovial | CRYO.S – Greiner Bio-One | 126263 | |
| Dimethyl Sulfoxide | Sigma-Aldrich | W387520-1KG | |
| Disodium-hydrogen-phosphate heptahydrate | Merck | 106575 | |
| DMEM without phenol red | Gibco by life technologies | 31053-028 | |
| Dulbecco’s modified Eagle’s medium (DMEM) | Gibco by life technologies | 11995065 | |
| Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt, Dihydrate | Merck | 324503 | |
| Fetal Bovine Serum | Internegocios | ||
| FlowJo v10 Software | BD Biosciences | ||
| Glucose | Merck | 108337 | |
| hemocytometer, Neubauer chamber | BOECO,Germany | ||
| Laminar flow hood | ESCO | AC2-6E8 | |
| L-glutamine (GlutaMAX) | Gibco by life technologies | A12860-01 | |
| MitoSOX Red | Invitrogen | M36008 | |
| Penicillin/Streptomycin | Gibco by life technologies | 15140-122 | |
| Potassium Chloride | Merck | 104936 | |
| Potassium-dihydrogen phosphate | Merck | 4878 | |
| Round polystyrene tubes 5 ml (flow cytometry tubes) | Falcon – Corning | BD-352008 | |
| Sodium Azide | Merck | 822335 | |
| Sodium Chloride | Merck | 106404 | |
| Sodium Hydroxide | Merck | 106462 | |
| SPINWIN Micro Centrifuge Tube 1.5 ml | Tarson | 500010-N | |
| Tissue Culture Plate 6 well | BIOFIL | TCP011006 | |
| Trypan Blue | Merck | 111732 | |
| Trypsin-EDTA 0.5% 10X | Gibco by life technologies | 15400-054 | |
| Vortex Mixer | Labnet International, Inc. |
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