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 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

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

16.0K 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

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

科研

生物学

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

电子自旋共振氧测绘活体物种显微成像

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

侵入细胞DISC1蛋白种类的产生，纯化及表征

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

在型号蓝藻种类标记和无标记突变体的产生

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

灭鼠工作心脏模型心肌的性能和氧耗的研究

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

可视化吞噬的早期阶段

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

通过可切换原卟啉IX依赖性原位活性氧物种光生成刺激小鼠皮肤中的干细胞壁龛和组织再生

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...