我们感谢博士卡尔 - 海因茨·克劳斯和Vincent的Jaquet寻求帮助和建议设立这些协议，同时也感谢克里斯托夫·鲍威尔和杰罗姆Bosset从NCCR的生物成像平台和纳文Gopaldass博士为他们提供技术支持。这项研究是由瑞士国家科学基金会的PRODOC资助。

1。可视化和ROS的产生在吞噬体的定性测量该OBG包被的磁珠应该提前做好准备。珠涂布过程和琼脂覆盖技术是改编自出版的参考文献5,14。 <ol><li>加入1毫升悬浮液中3.0微米羧化硅珠（约1.8×10 9个珠）到1.5mL管中，洗涤珠粒通过快速旋转和涡旋3倍，用1ml的PBS中。 </li><li>重悬珠在1ml的PBS中含25毫克​​/毫升氨腈（激活的羧化的二氧化硅珠共价结合预先标记的BSA）孵育在车轮15分钟。 </li><li>洗涤3次用1ml偶联缓冲液（0.1M硼酸钠，pH 8.0）中通过快速离心（离心机全速在台式微型离心机，持续5秒或替代离心机在2000×g下在台式离心机1分钟），并涡旋去除多余的氰胺。 </li><li>混洗过的小珠用500μl的coupling含有1毫克的OxyBurst绿色（OBG）H2HFF（二氢-2&#39;，4,5,6,7,7&#39; - hexafluorofluorescein）-BSA缓冲液，填补该管，用氮气从标准气体罐压盖之前，并孵化在车轮14小时（过夜）在室温在黑暗中。 </li><li>洗珠2倍1毫升缓冲液淬火（250 mM的甘氨酸的PBS），以除去未反应的OBG，并两次偶联缓冲液通过快速旋转和涡旋消除淬火缓冲区。 </li><li>加入1 ml偶联缓冲液含50微克的Alexa Fluor 594琥珀酰亚胺酯共轭对BSA。用氮气填充管压盖之前，并孵育在车轮为1.5小时，在室温下在黑暗中。 </li><li>洗珠3倍，用1ml通过快速旋转和涡旋停止反应淬火缓冲区，然后洗珠通过快速旋转和涡旋3倍用1毫升的PBS。 </li><li>最后，重悬珠在1ml的PBS中与2微升10％w / v的叠氮化物对于长期贮存。测量concentration个珠子的悬浮液用血细胞计数器（通常大约1-2×10 9个珠/毫升）中，填充在管与氮封端之前，并在4℃储存在暗处。 </li><li>该OBG荧光素的涂布效率可以通过使用激发波长为500nm的检查的发射光谱进行测试。当通过H 2 O 2中的辣根过氧化物酶（HRP）的存在下被氧化，被氧化的珠的荧光强度，在发射峰（538纳米），是11〜12倍的比未氧化涂覆的珠粒更高。 </li><li>收获指数从10厘米的培养皿中生长粘菌细胞，细胞板在3厘米菜肴的不同密度用光学透明的塑料或玻璃底部，并且它们生长过夜。选择大约80％汇合用于实验的菜肴。培养盘基网柄细胞的详细协议已经出版15。 </li><li>与LoFlo培养基（LF介质）更换培养基，孵育在实验中，为了降低HL5C培养基中的细胞外和内体内的自体荧光前2小时。 </li><li>并联，熔体10毫升1.5％细菌用琼脂的LF介质中，以形成一个琼脂层约1mm厚，等待10-15倒入琼脂在一个平面上（10厘米×10厘米，即在玻璃板）分钟固化。切琼脂层成2厘米×2厘米的正方形并将其放置在LF媒体以备后用。 </li><li>同时，制备纺丝盘或宽视场显微镜中，将环境室的温度保持在22℃，并调节该实验设置。 （见讨论补充意见）。 </li><li>经过2小时温育后，吸自3 cm培养皿中的LF介质，但细胞单层仍应被覆盖的介质薄膜。稀释包被的珠子，以1.5×10 7个珠/毫升，并加入10微升到细胞层。 </li><li>就拿一平方琼脂板，排出多余液体，但保持湿润。轻轻的把日ë琼脂平上的细胞层的顶部。不要移动琼脂广场时，躺在细胞。该琼脂覆盖是用来增加珠和细胞之间的接触，从而改善吸收，并且它也略微压缩的细胞，更好保持它们在物镜的焦平面上。 </li><li>将盖子上的菜，把它放在显微镜载物台，并自动拍照的红色，绿色和相位通道间隔1分钟，2小时或更长的时间。 </li><li>选择并专注于含有吞噬的整个过程的细胞活动。合并优化的3个通道，并使用专业图像处理软件组装的图片拍成电影。量化的红色和绿色通道，每个通道选择的珠的荧光强度，和绿色/红色的比例将反映动态的吞噬体ROS产生的细胞。 </li></ol> 2。细胞内超氧化物歧化酶生产的定量和中等通量的测量<ol><li>征收o盘基网柄的10ml HL5C介质的NE 80％汇合的菜。离心机粘菌细胞在850×g离心5分钟，仔细，彻底吸出多余的介质。重悬细胞SS6.4缓冲液[0.12 1M山梨醇中索伦森缓冲液（14.69毫KH 2 PO 4和6.27毫米的Na 2 HPO 4），pH值6.4]，计数细胞并稀释至6×10 6细胞/ ml的最终密度（见讨论补充意见）。 </li><li>加50微升细胞悬浮液变成白色不透明的96孔板的各孔中（见讨论额外注释）。 </li><li>稀释DHE股票（30毫米DMSO）500倍的SS6.4，移液管加入50μl稀释DHE到每​​一个使用多道移液器如果有必要很好。 DHE的最终浓度为30μM。反应立即开始。 </li><li>刺激剂或抑制剂可以在这一点上，根据具体需要可以添加，或者在其它的时间点。 </li><li>使用结束点"顶部阅读＆＃34，荧光酶标仪，荧光激发/发射在522 nm/605 nm处的模式，阅读每2分钟1小时，中摇5秒/分，在22°C </li></ol> 3。定量和细胞外的高通量测量H 2 O 2生产<ol><li>跟随在2.1和2.2中所述的相同步骤。 </li><li>加入5微升的HRP的股票（100 U / ml的蒸馏水中）为10毫升SS6.4，旋涡准备稀释的HRP或颠倒离心管拌匀并保持在冰上，供以后使用。稀释的HRP解决方案是在0.05单位/毫升。 </li><li>通过混合AUR库存中（10mM AUR在DMSO中）和稀释的HRP溶液注入SS6.4缓冲到最终浓度为6.25μM，0.005 U / ml的，准备AUR反应混合物分别。作为一个例子，对40的反应制备反应混合物中，混合1600微升SS6.4用400微升稀释的HRP和2.5微升AUR原液。 </li><li>加入50微升AUR组合TURE到每个使用多道移液器如果需要良好。现在，反应开始。 </li><li>刺激剂或抑制剂可以在这一点上，根据具体需要可以添加，或者在其它的时间点。 </li><li>使用结束点"顶部读数"模式中的荧光微板读数器，具有荧光的激发/发射在530 nm/590 nm处，读取每2分钟1小时后，培养基中摇动5秒/分钟，在22℃下</li></ol>

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没有利益冲突的声明。

相比于先前描述的方法中，OBG测定动态可视化，而不是从细胞群中测量的平均ROS信号吞噬体ROS的产生的过程中在单细胞水平。这样的人口平均的方法往往掩盖造成的非同步吞噬作用的关键信息。我们成功地适应DHE和AUR检测到粘菌和优化的协议。最重要的是，我们指定这两个实验测得的种类和活性氧的亚细胞定位。两者合计，整个集粘菌活性氧测量协议提供了一个有用和强大的系统来?...

活性氧（ROS）参与多种生物过程，如宿主防御，信号传导，组织发育和响应于损伤以及高血压和癌症。在充分研究的吞噬体NADPH氧化酶的机器是专门为快速ROS的产生，被称为氧化爆发，杀死细菌摄取中性粒细胞"吞噬体1。此外，电子作为线粒体呼吸链的副产品泄漏，以前被认为是只对活性氧的不受管制的来源负责。但最近，它被确定为促进小鼠巨噬细胞2 intraphagosomal杀灭细菌的重要机制。近年来，社会变形虫， 粘菌 ，已经成为一个强大的和流行的模型来研究先天免疫反应的细胞的内在机制。事实上， 粘菌和人分享的吞噬细胞节约molecula的一个令人惊讶的高度负责检测的细菌，吞噬和杀灭3,4Ř机器。蛋白和相关的活性氧的产生或解毒，如NADPH氧化酶，过氧化氢 ​​酶，超氧化物歧化酶的酶的同源物，可以在人类和粘菌中找到。作为最好的学习变形虫盘基网柄呈现在哺乳动物模型系统的一些独特的优势。它们生长于室温下，无需对CO 2，具有8-10个小时的倍增时间。他们可以很容易地保持为贴壁或悬浮培养。此外，由于他们的全部测序和注释的单倍体基因组，并易于遗传操作， 盘基网柄已经成为一个非常有吸引力的实验模式生物。 在以前的研究中，荧光素（统称为OxyBurst绿，OBG），该氧化的ROS后发出荧光的各种氯化和氟化衍生物，已被用来作为一个ROS记者。细胞穿透物ESTerified衍生工具是用来衡量细胞质活性氧，而细胞透性葡聚糖或蛋白偶联衍生物用于测量细胞外活性氧。特别是，OBG BSA包被的磁珠已经被用来检测吞噬体ROS产生在哺乳动物细胞5。然而，酶标仪为基础的方法只能给从细胞群体的平均ROS生成曲线。本协议中，通过使用粘菌 ，专业的吞噬细胞，优化实验条件下，我们获得稳定，高效的吞噬功能无任何偶联上调理素珠。 DHE已被应用于各种模型系统中，如哺乳动物嗜中性粒细胞和巨噬细胞，以检测ROS产生6-9。同时，也有一些争议的方法8,10的特异性和灵敏度。如Amplex Red试，AUR的是pH值，这使得它更适合于测量较不敏感的荧光强度的改进版本活性氧在非中性或弱酸性milieus。 AUR已在几种哺乳动物系统11,12最近被应用，但其在非哺乳动物模型，这往往需要弱酸性生长介质，使用了尚未见报道。此外，也没有公布协议的定量和动态测量ROS的产生和本地化的社会变形虫盘基网柄 。 所提出的协议集的目标是提供一个简单而灵活的解决方案来监控各种活性氧和他们的定位，并进一步给出见解活性氧有关的细胞机制。对于OBG实验中，我们用显微镜实时监测吞噬体ROS生成的OBG包被的磁珠由盘基网柄细胞摄取后的全过程，并提供了一种新的方法来研究intraphagosomal杀灭细菌的机制。我们在盘基网柄优化培养基通量DHE和AUR分析，测量细胞内superoxIDE和细胞外H 2 O 2生产，分别通过使用LPS作为一种有效的ROS刺激物。需要注意的是脂多糖，最近证实会增加对细菌的吞噬13 粘菌的杀菌活性。此外，随着DEDTC和过氧化氢 ​​酶处理明确地证实了这两种方法特异性地测量不同类型和ROS在盘基网柄亚细胞定位。最后，OBG测定可适应任何粘附的吞噬细胞，通过进一步opsonizing珠粒与配体为动物细胞的吞噬受体。原则上，DHE和AUR分析也可以进行微调，根据合适的实验条件下在任何附着或不依从小区的测量。

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>OxyBURST Green H2HFF BSA</td><td>Invitrogen</td><td>O-13291</td><td></td></tr><tr><td>Alexa Fluor 594</td><td>Invitrogen</td><td>A-20004</td><td></td></tr><tr><td>Carboxylated silica beads</td><td>Kisker Biotech</td><td>PSi-3.0COOH</td><td></td></tr><tr><td>HL5C medium</td><td>ForMedium</td><td>HLC0102</td><td></td></tr><tr><td>LoFlo medium</td><td>ForMedium</td><td>LF1001</td><td></td></tr><tr><td>Bacto agar</td><td>BD</td><td>204010</td><td></td></tr><tr><td>Dihydroethidium</td><td>Sigma</td><td>37291-25MG</td><td></td></tr><tr><td>Amplex UltraRed</td><td>Invitrogen</td><td>A36006</td><td></td></tr><tr><td>Horseradish peroxidase</td><td>Roche</td><td>10108090001</td><td></td></tr><tr><td>Diethyldithiocarbamate (DEDTC)</td><td>Sigma</td><td>D3506-100G</td><td></td></tr><tr><td>Catalase</td><td>Sigma</td><td>C9322-1G</td><td></td></tr><tr><td>Cyanamide</td><td>Sigma</td><td>187364-25G</td><td></td></tr><tr><td>Lipopolysaccharides</td><td>Sigma</td><td>L2630-25G</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>ibidi &amp;mu;-Dish 35 mm, high</td><td>ibidi</td><td>81156</td><td>Bottom made of optically clear plastic</td></tr><tr><td>MatTek dish 35 mm</td><td>MatTek corporation</td><td>P35G-1.5-14-C</td><td>Bottom made of a glass coverslip</td></tr><tr><td>Scepter Cell Counter</td><td>Merck Millipore</td><td>PHCC00000</td><td></td></tr><tr><td>White 96 well plate</td><td>Nunc</td><td>236108</td><td></td></tr><tr><td>Centrifuge</td><td>SORVALL</td><td>Legend RT</td><td></td></tr><tr><td>Microplate reader</td><td>BioTek</td><td>Synergy Mx</td><td></td></tr></tbody></table>

detecting, visualizing and quantitating the generation of reactive oxygen species in an amoeba model system

活性氧簇（ROS）包括一系列反应性和短命的，含氧分子，这被动态地相互转化或消除或者催化或自发的。由于大多数ROS和它们的来源和亚细胞定位的多样性的生命周期短，一个完整的画面只能通过协议的组合仔细的测量来获得。这里，我们提出使用OxyBurst绿色（OBG）一组三个不同的协议包被的珠，或二氢乙（DHE）和的Amplex红外线的（AUR），并监控定性和定量在专业的吞噬细胞如粘菌各种活性氧。我们优化了小珠包衣方法和用过的OBG-包被的珠子和现场显微镜来动态可视化intraphagosomal ROS生成在单细胞水平。我们从大肠杆菌鉴定脂多糖（LPS） 大肠杆菌作为一种有效的刺激活性氧产生在盘基网柄 。此外，我们ðeveloped实时，用DHE和AUR定量测定细胞内超氧化物和细胞外H 2 O 2生产，分别介质通量测定。

ROS在吞噬体的生成，可以通过显微镜（ 文件S1）定性和动态可视化。红色荧光通过的Alexa Fluor 594发射的是pH值不敏感的，并保持在吞噬体环境恒定，而OBG的氧化增加其荧光的绿色通道。 在体外的发光光谱的氧化和未氧化的OBG-包被的珠子相比较， 如图1B所示 ，示出的氧化后的强度显著增加。为方便现场活动的可视化，这两个通道内摄取1分钟​​合并，由红吞噬过?...

Watch this Scientific Journal Video about Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System at JoVE.com

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

我们适于一组协议对活性氧簇（ROS）可以在不同的变形虫和哺乳动物细胞模型被应用于用于定性和定量研究的测量。

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

detecting-visualizing-quantitating-generation-reactive-oxygen-species

Research

JoVE Journal

Biology

16.1K 次观看.  University of Geneva. 我们适于一组协议对活性氧簇（ROS）可以在不同的变形虫和哺乳动物细胞模型被应用于用于定性和定量研究的测量。 

视频: Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

科研

癌症研究

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

生物化学

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

血管钙的可视化 2 +信号旁分泌源性活性氧引发

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

生物学

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

中性粒细胞中活性氧物种的实时定量 感染脑膜性 大肠杆菌

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...

免疫与感染

在肝癌细胞中使用二氢乙锭进行新型 ROS 定量

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

作者聚焦：肝细胞系中细胞内 ROS 水平的高通量测量

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...

生物工程

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

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

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 ...

神经科学

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

检测，可视化和定量活性氧的生成在变形虫模型系统

摘要

探索更多视频

此视频中的章节

用基因编码荧光传感器评估毒理学氧化应激的成像方法

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

血管钙的可视化^{2 +}信号旁分泌源性活性氧引发

中性粒细胞中活性氧物种的实时定量感染脑膜性大肠杆菌

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

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

使用活性氧敏感荧光探针分析小鼠肠道类器官中的氧化应激

检测，可视化和定量活性氧的生成在变形虫模型系统

摘要

探索更多视频

此视频中的章节

用基因编码荧光传感器评估毒理学氧化应激的成像方法

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

血管钙的可视化 2 +信号旁分泌源性活性氧引发

中性粒细胞中活性氧物种的实时定量 感染脑膜性 大肠杆菌

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

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

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中性粒细胞中活性氧物种的实时定量感染脑膜性大肠杆菌

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估