Name: quantification of reactive oxygen species using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe and flow-cytometry in m&#252;ller glial cells
Uploaded: 2022-05-13T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Reactive Oxygen Species Using 2Ã¢â‚¬Â²,7Ã¢â‚¬Â²-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in MÃƒÂ¼ller Glial Cells at JoVE.com

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。

注：有关缓冲液成分，请参见 表1。
1. 细胞培养制备
注意：这里描述的是MIO-M1细胞的培养制备，MIO-M1细胞是一种自发永生的人Müller神经胶质细胞系（Moorfield's/Institute of Ophthalmology-Müller 1）。始终使用适当的无菌技术，并在层流罩中工作。
<ol><li>准备Dulbecco的改良鹰培养基（DMEM）完整培养基。对于含有 4.5 g/L D-葡萄糖和 110 mg/L 丙...

<ol>
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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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2&#8242;,7&#8242;-DCFH-DA</td>
			<td>Sigma</td>
			<td>35845-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Gibco by life technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II flow cytometer</td>
			<td>BD Biosciences</td>
			<td>FACSCanto</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dishes 100x20 mm</td>
			<td>Cell Star- Greiner Bio-One</td>
			<td>664 160</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo</td>
			<td>Sorvall legend micro 17 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (15 ml)</td>
			<td>BIOFIL</td>
			<td>CFT011150</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>BIOFIL</td>
			<td>CFT011500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial</td>
			<td>CRYO.S - Greiner Bio-One</td>
			<td>126263</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>W387520-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium-hydrogen-phosphate heptahydrate</td>
			<td>Merck</td>
			<td>106575</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM without phenol red</td>
			<td>Gibco by life technologies</td>
			<td>31053-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Gibco by life technologies</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt, Dihydrate</td>
			<td>Merck</td>
			<td>324503</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Internegocios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo v10 Software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>108337</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer, Neubauer chamber</td>
			<td>BOECO,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>ESCO</td>
			<td>AC2-6E8</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine (GlutaMAX)</td>
			<td>Gibco by life technologies</td>
			<td>A12860-01</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoSOX Red</td>
			<td>Invitrogen</td>
			<td>&#160;M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco by life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium-dihydrogen phosphate</td>
			<td>Merck</td>
			<td>4878</td>
			<td></td>
		</tr>
		<tr>
			<td>Round polystyrene tubes 5 ml (flow cytometry tubes)</td>
			<td>Falcon - Corning</td>
			<td>BD-352008</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Merck</td>
			<td>822335</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck</td>
			<td>106462</td>
			<td></td>
		</tr>
		<tr>
			<td>SPINWIN Micro Centrifuge Tube 1.5 ml</td>
			<td>Tarson</td>
			<td>500010-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Plate 6 well</td>
			<td>BIOFIL</td>
			<td>TCP011006</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Merck</td>
			<td>111732</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.5% 10X</td>
			<td>Gibco by life technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Labnet International, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of reactive oxygen species using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe and flow-cytometry in m&#252;ller glial cells

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。

如方案部分所述，我们已经展示了具有代表性和定量的数据，这些数据证明了流式细胞术检测荧光探针DCFH-DA从用ROS诱导剂A或B刺激的MIO-M1细胞中产生ROS。正如预期的那样，我们观察到未受刺激细胞中FITC荧光的变化高于自发荧光水平（图1A，比较“基础对照”与“自发荧光对照”，点图）。这是由于MIO-M1细胞中ROS的基础产生而发生的。（图1B，比较“基?...

Watch this Scientific Journal Video about Quantification of Reactive Oxygen Species Using 2Ã¢â‚¬Â²,7Ã¢â‚¬Â²-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in MÃƒÂ¼ller Glial Cells at JoVE.com

Quantification of Reactive Oxygen Species Using 2ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â²,7ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â²-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in MÃƒÆ’Ã‚Â¼ller Glial Cells

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。

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

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

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探针是直接测量氧化还原状态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。这些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二氯荧光素。探针的氧化是多种类型ROS作用的产物。这可以通过流式细胞术或共聚焦显微镜检测。在本文中，我们使用二氯荧光素二乙酸探针通过流式细胞术测量和量化ROS。将6孔板从培养箱转移到层流罩中。吸出上清液。并用PBS洗涤细胞一次。每孔加入 2 mL 低血清 DMEM。并将6孔板在培养箱中孵育两小时。用抗氧化剂处理细胞六小时。用ROS诱导剂A或B处理细胞30分钟。吸出上清液。并用PBS清洗细胞三次，以去除所有刺激。关闭层流罩的灯，在黑暗中工作。在不含酚红的DMEM中加入1mL二氯荧光素二乙酸探针。向对照自发荧光对照细胞中加入1 mL不含酚红的DMEM。将6孔板在培养箱中孵育30分钟。将冰上的板转移到实验室。每孔用2mL冷PBS洗涤细胞三次。向每个孔中加入0.5 mL冷分离缓冲液。通过使用P1000移液器轻轻地上下移液来收获细胞。将它们收集在标记的1.5 mL管中。并在4°C下以600×g离心5分钟，小心地弃去上清液，轻轻地用敲击解离沉淀。加入1mL FACS缓冲液以将细胞洗涤到每个标记的试管中。如有必要，请使用最低强度的涡旋，以完全解离颗粒。在4°C下以600×g离心5分钟，重复此步骤两次。总共三次洗涤。将细胞沉淀重悬于200L FACS缓冲液中。使用涡旋轻轻地完全解离每个管中的沉淀。并转移到标记的5 mL用于流式细胞仪管。将试管保持在冰上，直到在流式细胞仪上进行分析。使用流式细胞术软件，创建一个新的实验。单击试样，将打开试管列表。双击并重命名它们。将自动荧光控制管放在吸气臂上，仅向左小心地移动底座。单击“采集数据”，然后单击“记录数据”，然后选择所需的停止条件。在感兴趣的细胞周围画一个门，排除死细胞和碎片。取下管子。单击“下一管”并运行基础对照样品。单击“采集数据”，然后在“记录数据”中打开软件。使用“添加示例”操作按钮添加示例。单击“添加样品”。选择“实验日期”文件夹，然后单击“选择”。双击工作区中的第一个样品，在本例中为自动荧光控制。绘制多边形门以分离感兴趣的细胞，排除死细胞和碎片。将绘图更改为直方图。在 M 中双击

Research

JoVE Journal

Neuroscience

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

活大鼠皮层神经元线粒体膜电位和活性氧的测定

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

神经母细胞含量：从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

胶质细胞和神经细胞的三维重建与虚拟现实分析方法

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...