Name: detecting, visualizing and quantitating the generation of reactive oxygen species in an amoeba model system
Uploaded: 2013-11-05T18:30:00
Description: Watch this Scientific Journal Video about Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System at JoVE.com

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）?...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+superoxide+and+hydrogen+peroxide+detection+in+biological+assays.">Enhanced superoxide and hydrogen peroxide detection in biological assays.</a> Free Radic. Biol. Med. 49, 61-66 (2010).</li><li>Chen, J., Rogers, S. C., Kavdia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Kinetics+of+Dihydroethidium+Fluorescence+with+Superoxide+Using+Xanthine+Oxidase+and+Hypoxanthine+Assay.">Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay.</a> Ann. Biomed. Eng. , (2012).</li><li>Lee, C. W., Chen, Y. C., Ostafin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+accuracy+of+Amplex+Red+assay+for+hydrogen+peroxide+in+the+presence+of+nanoparticles.">The accuracy of Amplex Red assay for hydrogen peroxide in the presence of nanoparticles.</a> J. Biomed. Nanotechnol. 5, 477-485 (2009).</li><li>Guimaraes-Ferreira, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+creatine+supplementation+decreases+reactive+oxygen+species+content+with+no+changes+in+expression+and+activity+of+antioxidant+enzymes+in+skeletal+muscle.">Short-term creatine supplementation decreases reactive oxygen species content with no changes in expression and activity of antioxidant enzymes in skeletal muscle.</a> Eur. J. Appl. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agar-overlay+immunofluorescence:+high-resolution+studies+of+cytoskeletal+components+and+their+changes+during+chemotaxis.">Agar-overlay immunofluorescence: high-resolution studies of cytoskeletal components and their changes during chemotaxis.</a> Methods Cell Biol. 28, 347-356 (1987).</li><li>Fey, P., Kowal, A. S., Gaudet, P., Pilcher, K. E., Chisholm, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+growth+and+development+of+Dictyostelium+discoideum.">Protocols for growth and development of Dictyostelium discoideum.</a> Nat. Protoc. 2, 1307-1316 (2007).</li><li>Bloomfield, G., Pears, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+signalling+required+for+multicellular+development+of+Dictyostelium.">Superoxide signalling required for multicellular development of Dictyostelium.</a> J. Cell. Sci. 116, 3387-3397 (2003).</li><li>Dieckmann, R., Gopaldass, N., Escalera, C., Soldati, T., Deretic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagosomes+and+Phagosomes.">Autophagosomes and Phagosomes.</a> Methods in Molecular Biology. 445, 317-328 (2008).</li><li>Amir, Y., Edward, O. -. 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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 border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</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></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>ibidi &#956;-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

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