这项工作是由美国国立卫生研究院资助R37 AI34867和R01 GM064625为NWA我们感谢R. Twete​​n博士从俄克拉何马大学的SLO表达质粒的支持。

1。 ASM的转录沉默<ol><li>种子1.5×10 5个HeLa细胞于2ml DMEM生长培养基（DMEM高葡萄糖，10％胎牛血清（FBS），2mM的L-谷氨酰胺，1％青霉素-链霉素）在35 mm的玻璃底培养皿（MatTek）和在5％CO2 培养箱孵育过夜，在37℃。 </li><li>在第二天，吸出物离开的生长培养基，并添加siRNA转染混合物之前，用2ml的DMEM低血清培养基（DMEM，用4％FBS）中，至少1小时更换。 </li><li>准备4管的寡核苷酸的siRNA转染混合物。在管A和C，加入250μl的Optimem降低血清和4μL脂质体RNAiMax，每35mm培养皿进行转染。管B中，加入250μl的Optimem减少控制媒介GC含量寡（对照siRNA）血清和8μL（160皮摩尔），每35mm培养皿进行转染。在管D，加入250μl的Optimem减少SMPD1寡（热情）的血清和8μL（160皮摩尔），每35mm培养皿吨Ø转染。孵育所述管在室温下放置5分钟。 </li><li>结合管A和B，形成转染复合物为对照siRNA和管C和D为ASM的siRNA。孵育反应在室温下搅拌20分钟。 </li><li>添加对照siRNA和ASM siRNA转染反应混合物慢慢滴，到各自的35毫米的玻璃底菜和孵化在37℃/ 5％的CO 2。在24小时转染后，轻轻吸去培养基并更换新鲜的DMEM培养基。为了有效ASM击倒，该吃的食物应进一步前实时成像在37℃/ 5％的CO 2约31小时（共55小时）。 </li></ol> 2。试剂现场显微镜的研制<ol><li>在100纳克/微升冷的1X PBS的浓度准备重组的成孔毒素链球菌溶血素为O（SLO）溶液中不含Ca 2 +，Mg 2 +的。在这些测定中所使用的SLO是constitutivelY有功功率半胱氨酸突变体，不需要减少对活性剂，由Rod Twete​​n，奥克拉荷马大学博士惠赠。该毒素在大肠杆菌中表达大肠杆菌 BL21 DE3和天然条件下纯化，如先前描述的16。 </li><li>准备FM1-43原液:在DMSO中重悬FM1-43冻干粉来创建一个25毫米的解决方案。 </li><li>准备溶液A:与Ca 2 +的稀释FM1-43原液在寒冷的DMEM至4微米（需要每MatTek菜1.5ml）中的终浓度，直到需要在冰上保存。 </li><li>制备溶液B:稀释FM1-43储备溶液在冷的DMEM不含Ca 2 +和10mM EGTA（Ca 2 +的无DMEM）中于4微米（每MatTek菜需要1.5ml）中的终浓度，并在冰上储存直至需要的。 </li><li>预温（37°C）1毫升分装解决方案A和方案B的Eppendorf管。 </li><li>不断的DMEM冰解决方案的Ca 2 +和C一个2 +的DMEM。 </li></ol> 3。的FM1-43涌入SLO处理和未处理的细胞活细胞成像<ol><li>填充一个中等大小的浅的金属容器用碎冰，颠倒成冰的大玻璃容器中，并添加额外的冰留下露出的金属容器的唯一的底面。放置在暴露的金属底部湿纸巾以允许均匀的热传递。 </li><li>采取一对照siRNA处理MatTek菜（ 样品1），并将其放置在冰冷的湿纸巾。轻轻吸了所有媒体，并与冷的Ca 2 +的DMEM洗3次。最后一次洗涤后，吸过的菜所有媒体。 </li><li>对细胞图像进行相关的FM1-43细胞中没有SLO伤人（ 样品1）中的Ca 2 +的存在，加入180微升冰冷溶液B到35毫米的玻璃底培养皿的中心玻璃盖玻片，静置5分钟在冰上（ 表1）。 </li&gt; <li>放置MatTek盘上的共焦显微镜下加 ​​热至37℃，并配有环境室（即保持温度，湿度和CO 2水平）的阶段。发现细胞将成像期待通过40X 1.3 NA物镜（尼康）的领域。如果有的话，打开PerfectFocus或类似装置，以校正热漂移。 </li><li>加入1毫升预热的解决方案A轻轻的35毫米的菜一边，更换环境室（ 表1）的封面。 </li><li> （中的Ultraview Vox的珀金埃尔默旋转盘共聚焦显微系统Volocity）采用适当的图像处理软件在一帧图像采集4分钟，每3秒。 FM1-43的激励是通过使用二向色滤光片的488nm的带通488nm的激光线，并且发射鉴别是通过使用一个527（W55）发射滤波器的实现。激光功率水平和相机感光度设置达到100毫秒的形象曝光。 </li><li>重复步骤3.2至3.6，以检测FM1-43的对照siRNA处理的细胞与无SLO伤人（ 样品2）在不存在的Ca 2 +的，不同之处在于，在步骤3.5 B液应加入（ 表1）。 </li><li>测量FM1-43流入对照siRNA处理的细胞与SLO受伤或者在存在（ 样品3）或不存在的Ca 2 +的（ 样品4）中，添加180微升冰冷溶液B含有SLO到的中心玻璃盖玻片35毫米的玻璃底菜和孵化5分钟，冰如步骤3.3。重复步骤3.2至3.6;调整加入溶液A或溶液B在步骤3.5（ 表1）。 </li><li>来确定ASM在质膜修复中的作用，重复使用ASM siRNA处理的细胞中的所有条件下（ 样品5-8）（表1）步骤3.2-3.6。 </li><li>为了确定细胞外重组鞘磷脂（SM）上的作用细胞膜修复动力学（由抑制在FM1-43潮反映）（ 样品9-10），SM加（0-50μU）到溶液A或溶液B在步骤3.5，然后在活细胞加入细胞成像在1毫升（ 表1）的总体积。 </li></ol> 4。的FM1-43定量潮进入细胞<ol><li>电影已被收购后，使用图像分析软件（Volocity套房，珀金埃尔默）测量FM1-43的细胞内荧光强度。绘制的感兴趣区域中的字段中的每个小区的胞质区域和应用的软件工具，以确定在整个视频的所有帧的均值FM1-43的荧光强度。 </li><li>来调整变化的初始FM1-43染色，对于每个视频数据被表示为在一段时间内荧光强度倍的增加，并绘制为每个不同的条件。折叠增加每各个时间点的荧光细胞被计算为:F T / F 0，其中F T是平均荧光在特定的时间点，和F 0是平均荧光在t = 0（ 图1-2）。 </li></ol>

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作者宣称他们没有竞争的经济利益。

机械损伤和细胞膜修复早期的研究依赖于细胞损伤的机制多种多样，从掉落的玻璃珠，micropipetting，剪切，划伤或者刮伤。所有这些测定的读数是定性而非定量的，并没有给于修复机制的动力学精确的信息。细胞的这些形式的损伤之后重新密封其质膜的能力被加入到在细胞质中，渗透膜不透染料，细胞内的酶，ATP水平，或长期的损失外液示踪物的存在或不存在下测定细胞存活。虽然几个这些测定?...

人们已经知道了几十年了机械性损伤后质膜修复是的Ca 2 +依赖性过程1,2。后来的研究表明，通过伤口的Ca 2 +内流触发细胞内囊泡的胞吐一场轰轰烈烈的过程在损伤部位，这是需要重新密封3,4。加载到细胞的细胞质中的荧光染料的损失率被用来评估修复的速度，并得出结论，被重新密封于&lt;30秒5伤后完成。两款车型最初提出来解释在质膜修为胞吐作用的要求:1）"补丁"的模式，这表明，Ca 2 +的内流，通过病灶触发细胞内囊泡最初同型融合，形成了一个庞大的"补丁"，将然后可以应用到质膜重新密封伤口6和2）的张力减小模型，其中提出，膜ADDEð通过在伤口附近的Ca 2 +依赖性胞吐作用将降低细胞膜张力，促进双层7的重新密封。 钙在细胞膜修复+触发胞吐作用通过研究表明，溶酶体含有Ca 2 +的传感器分子，突触七，这有利于它们的胞外分泌和质膜修复受损细胞，进一步加强8,9,10,11 。 然而，更多的证据表明，单纯胞吐作用不足以促进细胞膜修复。除了 ​​在质膜上的机械眼泪，细胞损伤的常见形式是透由细菌12,13或免疫细胞14,15产生的成孔毒素。不像机械眼泪，成孔蛋白插入本身对质膜形成稳定，蛋白质内衬的孔隙无法通过应用膜"补丁"或由红眼简单地重新密封ING膜的张力。有趣的是，研究发现，哺乳动物细胞具有与透孔形成蛋白后，以修理其质膜一个有效的机制，而这个过程也需要胞外Ca 2 +的12的存在。这一发现提出了从细胞表面跨膜孔隙去除是否也是一个快速的过程的问题，作为具有机械伤口5观察到。出人意料的是，我们最近的研究表明，细胞透化与成孔毒素的再密封具有非常相似的性质，以机械创伤的修复:这个过程需要细胞外Ca 2 +的，并且在约30秒完成。更详细地调查这个过程中，我们最近了解到，除了Ca 2 +的调节溶酶体的胞吐，质膜修复涉及内吞作用的快速形式，它是由溶酶体的释放引发酶的酸性鞘磷脂酶（ASM），是必需的不仅为日ë去除跨膜孔的，而且对机械创伤16的维修。 为了确定通透与成孔蛋白后细胞再密封的动力学，在我们的实验室，我们改编以前已经用于评估激光隔离受伤的肌肉纤维17的重新密封的实时成像方法。该测定依赖于亲脂性染料FM1-43，它稳定地可嵌入到脂质双层中的荧光强度增加的外小叶的属性。当细胞膜双分子层被破坏的细胞外染料得以进入细胞内的膜，提供了一个敏感的检测，以检测细胞膜损伤和修复18,16,19,20。要通透与成孔蛋白后细胞再密封的评估适应这个实验中，我们预孵育细胞在4℃与细菌毒素链球菌溶血素为O（SLO），其中结合细胞膜胆固醇21。同步单元透然后可以通过移动从冰细胞温热介质中加热的显微镜载物台，其激活低聚和变化中的构象，导致跨膜孔形成很容易地实现。这种方法的一个优点，通过使用激光伤人先前公布的测定中，是细胞中的更大的数目可以在一个显微镜视野同时进行分析，提供上述细胞群的较好采样。给定的机械损伤和透化与成孔毒素后见到的细胞重新密封过程之间的机械的相似性，我们在这里描述的测定法提供了解剖参与质膜修复的基本过程的因素非常灵活和强大的方法。作为一个例子，我们表明，它有可能使用该测定，以确定在修复过程中的Ca 2 +依赖性和独立的步骤。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>HeLa 229 cell line</td>
			<td>ATCC</td>
			<td>CCL2-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose </td>
			<td>Invitrogen</td>
			<td>11965</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose No Calcium</td>
			<td>Invitrogen</td>
			<td>20168</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Optimem Reduced Serum</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Control medium GC content RNAi oligo</td>
			<td>Invitrogen</td>
			<td>12935300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>SMPD1 RNAi oligo</td>
			<td>Invitrogen</td>
			<td>HSS143988</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum Heat-inactivated</td>
			<td>Gemini-Bioproducts</td>
			<td>100-106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sphingomyelinase from Bacillus cereus</td>
			<td>Sigma</td>
			<td>S7651</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 mm-glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-14</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material</td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C9100-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spinning disk confocal microscope</td>
			<td>PerkinElmer</td>
			<td>UltraViewVoX</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software analysis software</td>
			<td>PerkinElmer</td>
			<td>Volocity Suite</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Environmental chamber</td>
			<td>Pathology Devices</td>
			<td>LiveCell System Chamber</td>
		 </tr>
		</tbody>
 </table>

live imaging assay for assessing the roles of ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds

细胞膜损伤是一种常见的事件，伤口必须迅速修复，以确保细胞存活。的Ca 2 +的内流，触发机械创伤的修复上内〜30秒质膜中的关键信号传导事件。最近的研究表明，与透孔形成在一个的Ca 2 +依赖性的过程，涉及溶酶体酶酸性鞘磷脂酶随后孔隙的内吞作用的胞外分泌的毒素后的哺乳动物细胞也重新密封的质膜。在这里，我们描述了用于证明细胞毒素链球菌溶血素Ô透的再密封也是快速和依赖的Ca 2 +内流的方法。该法的设计允许伤害事件的同步和细胞的成像，恢复细胞膜的完整性，并通过量化其liphophilic染料FM1-43达到细胞内膜的程度的能力的精确动力学测量。这活法阿尔斯O允许的外源能力的评估敏感添加可溶性因子如鞘磷脂酶抑制FM1-43大量涌入，反映细胞的修复他们的质膜的能力。该测定法允许我们表明:第一次，鞘磷脂酶的作用的Ca 2 +的依赖性胞吐作用的下游，由于胞外加入酶促进细胞透化在不存在的Ca 2 +的再密封。

低浓度的细菌鞘磷脂（SM）的救援细胞膜修复细胞贫溶酶体酸性鞘磷脂酶。 用FM1-43的成像方法，我们以前表明，细胞缺乏的溶酶体酶ASM和暴露于SLO伤人中的Ca 2 +的存在下具有质膜缺陷是由细胞外加入的重组人酶19的获救。由于人溶酶体ASM具有低的pH最佳值，我们研究是否重新密封也可以恢复增加的SM从蜡状芽孢杆菌 （Sigma公司），它是在中性pH下完全有活性...

Watch this Scientific Journal Video about Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds at JoVE.com

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

细胞暴露于所述亲脂性染料FM1-43的实时成像可以精确测定由该成孔毒素从质膜除去动力学。这是一个敏感的测定法可以用于评估要求的Ca 2 +，鞘磷脂和其它因素对质膜修复。

实时成像检测方法的评估钙的作用<sup&gt; 2 +</sup&gt;和鞘磷脂酶在成孔毒素伤口的修复

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

live-imaging-assay-for-assessing-roles-ca2-sphingomyelinase-repair

Research

JoVE Journal

Biology

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

11.6K 次观看.  University of Maryland. 细胞暴露于所述亲脂性染料FM1-43的实时成像可以精确测定由该成孔毒素从质膜除去动力学。这是一个敏感的测定法可以用于评估要求的Ca 2 +，鞘磷脂和其它因素对质膜修复。 

视频: Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

科研

生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

在体内的Ca 2 + -蜜蜂的嗅觉学习期间的蘑菇体神经元的成像

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

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 ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量:贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动:Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

实时成像检测方法的评估钙的作用

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克隆形成含量:贴壁细胞

为外植体含量的颅间充质细胞行为评定

凋亡细胞清除过程中实时成像

活细胞成像早期自噬活动:Omegasomes与超越

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像