Needle wounding methods for C. elegans were first developed by Nathalie Pujol and Jonathan Ewbank; we thank Nathalie Pujol for comments on the manuscript. Work in our laboratory on C. elegans epidermal wound repair is supported by a grant from the NIH to A.D.C. (R01 GM054657).

以下协议描述为C.详细过程线虫皮肤伤人和测定伤口响应。 1.针伤人3,4 <ol><li>健康成长unstarved蠕虫标准NGM（线虫生长培养基）板的E.大肠杆菌 OP50细菌作为食物，保持在20℃的培养箱中培养。 注:方法NGM琼脂平板和常规栽培的C.线虫可以在www.wormbook.org 6找到。 </li><li>在20°CO / N伤人及培养前挑选25 L4阶段蠕虫新鲜种子板块1天。 </li><li>前伤人，拉针使用针拉马毛细血管。在伤人使用的针是相同的用于C的显微注射线虫 。 </li><li>确保所有的蠕虫是在年轻的成年阶段。放置在冰虫的板约30分钟。冷却导致动物不振，促进针伤人。 <li>取出琼脂平板从冰到解剖体视显微镜。使用蠕虫挑，将25蠕虫到琼脂板的中心。 </li><li>将针头插入100微升枪头更容易持针。穿刺与针蠕虫的主体的前部或后部，避免了性腺。在一般情况下，试图将卷绕后咽和前性腺之间，或介于后部性腺和直肠。重复使用针管多次，直到他们分手。 注:确保伤口破洞的角质层和皮肤短暂的，温柔的刺，穿透〜5-10微米到动物。针头应该进入阿尼姆人在大约直角的皮肤。伤口不应该造成明显损害内部器官，或身体的即时断裂。观察到少量细胞质渗出的伤口后，几秒钟的;但是，如果细胞内容物泄漏出​​来过长时间的动物将无法生存。为获得最佳成像，卷绕在外侧表皮的合胞体（hyp7）或横向接缝。 注:此过程包括使用玻璃针。 </li><li>允许蠕虫恢复室温，然后在培养20℃。判断针伤人通过许多如下所述测定法的成功。后在年轻的成年阶段针伤人大于95％的野生型动物的存活24小时。伤人的幼虫或老年人的影响尚未得到广泛的分析。 </li></ol> 2.激光伤人3 用飞秒激光照射（800纳米），以进行更精确的伤人。 <ol><li>准备琼脂P广告上融化的2％琼脂7载玻片。确保垫类似于用于C的实时成像琼脂垫线虫 。 </li><li>转让10年轻的成虫与上挑的蠕虫琼脂糖垫，并增加12毫米左旋咪唑溶液2微升下降。涵盖的动物和用盖玻片液体。等待1-2分钟的蠕虫瘫痪。 </li><li>放置载玻片到一个旋转盘共聚焦显微镜。移动舞台，找到蠕虫和注重横向前或后合胞体表皮具有100X的目标（NA 1.4-1.46）。 </li><li>设置飞秒激光至140毫瓦的功率（目标之前测得）。使用飞秒激光以80兆赫的重复率。 注:两个脉冲的每个200毫秒（20毫秒分隔）足以伤人在我们手中。 </li><li>着眼于表皮细胞的顶端表面和伤口表皮。观察细胞质（气泡）的地方破裂，或局部脱色Ø˚F使用任何荧光标记。 注:按照适当的激光安全程序，使用飞秒激光的时候。使用飞秒激光器线或点扫描，如在双光子显微镜应该用于激光伤人提供足够的功率。 注意:尽管我们没有使用其它激光器的直接经验，原则伤人应使用常规的UV激光器（如用于线虫细胞消融）是可能的。或者，使用荧光漂白恢复（FRAP）模块上旋转盘共聚焦伤口表皮（N.普霍尔，个人通讯）之后。 </li></ol> 3.可视化的表皮钙响应伤人<ol><li>使用转基因C.线虫菌株表达的Ca 2+传感器（如那些GCaMP系列）在表皮，成人表皮特异性启动COL-19（ 图1A的控制之下;转基因列于<stroNG&gt;表1）。使用表达红色荧光蛋白的转基因，如tdTomato作为用于转基因表达水平的内部对照。 注意:我们已经使用tdTomato作为用于转基因表达的内部对照表皮tdTomato荧光是相对稳定的，并且不与GCaMP成像干涉。 </li><li>两个针和激光伤人触发钙升高表皮。然而，由于针伤人不能容易地在一个旋转盘共聚焦进行的，激光伤人是优选的钙离子反应（ 图1）的定量分析。 </li><li>收购使用旋转盘共聚焦与100X的目标（NA 1.4-1.46）和适当的过滤器设置多维采集模式（时间间隔2秒，114毫秒激发激光曝光）时间推移图像（GFP过滤器将工作GCaMP3）。 注:采集时间推移图像，每30秒约2小时检查钙 RESPONSE伤人在一个较长的时间过程。 </li><li>使用图像分析软件测量中的感兴趣区域（ROI）10等效区域，其中五个集中于在背景中的表皮细胞胞质溶胶和5的平均GCaMP荧光。 </li><li>通过平均荧光5投资回报在表皮，然后减去5投资回报平均在后台受伤前获得的基线荧光（F 0）。表达在荧光ΔF的变化的变化相对于所述基线[（F T -F 0）/ F 0]的比值。 </li></ol> 4.可视化F-肌动蛋白伤人后的<ol><li>注意:在响应于针伤人，观察肌动蛋白环在伤口部位伤口周围逐渐关闭。该协议部分检测通过测量肌动蛋白环直径伤口闭合。 </li><li>生成表达一种F-肌动蛋白标记物，例如稠合到果蝇膜突蛋白的肌动蛋白结合结构域的转基因蠕虫绿色荧光蛋白，表达表皮特异性启动子的控制下，如COL-19（ 图2A）（转基因juIs352）。 注意:我们已经获得使用其它的肌动蛋白结合结构域标记如Lifeact或F-tractin（未发表的结果）相似的结果。 </li><li>伤人的P COL-19- GFP-膜突蛋白的转基因蠕虫进行针。针伤人后，观察周围的伤口部位的GFP-膜突蛋白的由〜5分钟的环。肌动蛋白环变得在直径小，并最终关闭2-3小时交伤人，在野生型动物（ 图2A）。 </li><li>制备2％琼脂板在载玻片上，并在2微升12毫左旋咪唑溶液转移10蠕虫到垫。使用传统的共聚焦显微镜伤人后，图像的GFP-膜突环1小时。 注:使用共焦显微镜伤人后1小时获得肌动蛋白环Z-栈（13×0.5微米）。 </li><li>伤人后，测量肌动蛋白环的直径。定量表皮摹FP-膜突蛋白，首先使Z堆栈的最大强度投影，然后得出4线扫描（在45°至对方）通过GFP-膜突环。环直径被定义为平均峰至4行扫描（ 图2B）的峰值距离。对于已经关闭的环，该直径被定义为0微米。 注:F-肌动蛋白环方便地测定1小时后的伤人，因为这是通过在野生型的伤口闭合过程大约一半。 F-肌动蛋白环也可以在多个时间点测量来推导伤口闭合的时间过程。可以用左旋咪唑，固定蠕虫和旋转磁盘共聚焦显微镜获得的伤口闭合时间推移电影。 </li></ol>表皮抗菌肽5.分析感应注:伤人也触发了C.先天免疫反应线虫的皮肤。基因编码抗菌肽（安培）的转录INDuced在表皮。在NLP-29（神经肽样蛋白），AMP被打伤4强烈诱导。为了测定表皮如何控制伤人后AMP表达，可以使用转基因记者或实时PCR来检测个体的AMP的转录水平。 <ol><li>对于伤人4先天免疫反应的转基因记者分析。 </li><li>执行针伤人（如在方案1）在成年动物表达转基因报告frIs7，其含有P NLP-29- GFP 和P COL -12- DsRed的作为内部对照。观察未受伤的成年动物的GFP长通滤波器为暗红色或橙色。 注:伤人野生型虫会诱发P NLP-29-GFP表达，但不会影响P COL-12 -dsRed，导致下的GFP长传照明出现橙，黄，绿或蠕虫。基因在P38 MAPK通路，如PMK-1或功能丧失NSY-1将阻止NLP-29-GFP伤人后，4诱导。 P NLP-29- GFP高表达水平在幼虫阶段</li><li>在年轻的成年动物进行AMP诱导测定。确保未受伤的控制动物有GFP的低水平。 注:P NLP-29- GFP表达也诱导渗透胁迫8，并可能对其他环境胁迫敏感。 </li><li>伤人后6小时，得分具有P NLP-29-GFP诱导（ 图3）9使用荧光解剖范围用GFP长通滤波器的蠕虫的数量。 注:在1小时后伤人P NLP-29- GFP有明显诱导，达到了约6个小时后伤人峰，然后剩余升高至24小时后的创伤。通过量化打分的视觉表达4,9。可替代地，量化使用荧光激活蜗杆分拣4表达水平。对于蠕虫分拣机，至少50动物需要被打伤。 </li><li>对于QUA实时PCR检测ntitating的各个AMP的基因的转录水平。测定的转录水平为几个基因:NLP-29，NLP-30，CNC-1，和CNC-5。 </li><li>执行针头伤人的野生型或突变蠕虫（见协议1） </li><li>收集50受伤蠕虫和50未受伤的蠕虫在所需时间（ 如 3，6，24小时）后伤人。 </li><li>使用商业试剂盒按照制造商的说明提取RNA。 </li><li>执行反转录使用商业逆转录酶试剂盒获得总共20微升的cDNA。 </li><li>用于RT-PCR中，使用0.2微升的cDNA（1/40）从每个样品中组合，带Green超用荧光素和0.2μM的引物。比较RNA的质量，使用AMA-1或SNB-1的RT-PCR作为参考;通过规范化的内部控制（AMA-1或SNB-1）比较AMP相对表达水平，或（在受伤的蠕虫/未受伤的蠕虫AMP感应）通过比较规范的诱导倍数后伤人alizing内部控制。 注:CNC-1和CNC-5 caenacin家庭抗菌肽的转录被炸伤10激活。典型地，针伤人诱导表达（〜500倍用于CNC-1）经4小时10的时间过程。在公开的工作中使用的引物列于表2中 。 </li></ol> 6.试验后生存伤人<ol><li>注:伤人活化至少两个独立的信号传导途径在表皮:所述的Ca 2+依赖的伤口修复途径和先天免疫应答的途径。两个途径都需要无菌伤人的充分的存活。为了测试突变或RNAi治疗是否会影响伤口愈合，测量生存在24-48小时后伤人。任选地，使用常规寿命测定法来确定伤人对长寿4,9的效果。 </li><li>年轻的成人（L4 + 24小时）蠕虫执行针头伤人（50蠕虫，重复塞弗拉尔次）按照上述方案1。维持未受伤蠕虫作为对照的相同数目。 </li><li>检查成活率，每24小时（转移伤员蠕虫到一个新的琼脂平板上，每24小时）。伤人的突变体有缺陷的创面修复，如GTL-2或TIR-1，归时，未受伤的对照组动物后观察〜50％存活24小时。死亡被定义为缺乏运动反应由蠕虫挑去触摸。 </li></ol>

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The authors are not aware of any competing interests.

这里介绍的针和激光伤人的方法提供互补的方法来评估的表皮上皮细胞修复损伤的能力。激光伤人是相对局部和（取决于激光构造）可以被限制在表皮，而针伤人破坏表皮，角质层，并有可能内部基底膜。针头伤人可能更准确地类似于伤口病原体或机械损伤自然环境造成的。作为一项规则，针伤人需要更多的实践来实现精确的伤口与动物存活兼容。该细胞对针和激光伤人显示许多相似之处。然而?...

皮肤伤口愈合机制是基本生物学的兴趣和相关的人体健康。在脊椎动物和哺乳动物伤口愈合包括一系列复杂的多种组织和信令1协调反应。许多简单的遗传模型有机体也能愈合皮肤伤口2。因此，感兴趣的分析的皮肤伤口修复基因易于处理的模型。我们和其他人已经开始使用线虫作为皮肤创伤新型治疗3,4。该协议的目的是使一组研究人员使用C的更广泛的线虫作为工具来调查表皮伤口愈合的分子和细胞机制。 C.将线虫皮肤包括表皮（也称为皮下组织）和细胞外角质层5。成人表皮是从少数多核合胞体，其中最大的是在合胞体形成的ķnown为hyp7。表皮是一个简单的上皮细胞分泌在其顶端表面的角质层。皮肤能够积极抵御皮肤穿透病原体和修复小伤口4。 C.在创伤修复线虫的皮肤是强大的，因为几乎所有的野生型动物生存才能生存造成的针，或因激光照射局部皮肤损伤小刺伤。C.线虫皮肤伤人引发一系列的反应，包括表皮先天免疫反应，伤口愈合和瘢痕形成4。成人表皮是有丝分裂后，和伤口愈合涉及相对于表皮增殖或细胞迁移的地方的细胞反应。我们已经表明，皮肤创伤触发大量持续增加表皮的Ca 2+，要求该膜TRPM通道GTL-2和内部的Ca 2+商店3。表皮钙离子信号所需的形成和封闭F-肌动蛋白环的WOUND网站。伤人也诱导了激活抗菌肽如NLP-29的转录先天免疫反应。抗菌肽的伤人诱导的转录依赖于TIR-1 / PMK-1的p38 MAP激酶级联在表皮4作主。在任一所述的Ca 2+信号传导途径，或在先天免疫应答缺陷将导致较低的生存后伤人。在C的相对简单的结构线虫表皮，其遗传易处理性和优点用于体内成像使其成为出色的系统来研究的伤口修复的多个方面。 在这里，我们提出的协议伤人的两种常用方法:针伤人和伤人的激光。针伤人不需要专门的设备（比针拔出器等），并与经验，可以在数百每天蠕虫来执行。针伤人的是，动物在琼脂平板上生长进行。与此相反，激光伤人的是ANES执行thetized动物装在琼脂焊盘下盖玻片，并适合于所述细胞反应的损害实时成像。

<table><tbody><tr><td>Agarose</td><td>Denville Scientific</td><td>CA3510-8</td><td></td></tr><tr><td>Plastic Petri plate</td><td>Tritech Research</td><td>T3308</td><td>60 mm</td></tr><tr><td>Levamisole</td><td>Sigma</td><td>L9756</td><td>12 mM</td></tr><tr><td>Borosilicate glass capillary</td><td>World Precision Instruments</td><td>1B100F-4</td><td></td></tr><tr><td>Needle puller</td><td>Sutter Instrument&amp;nbsp;</td><td>P-2000</td><td></td></tr><tr><td>Worm pick (platinum wire)</td><td>Tritech Research</td><td>PT-9010</td><td></td></tr><tr><td>M9 solution</td><td>Home-made</td><td></td><td>3 g KH&lt;sub&gt;2&lt;/sub&gt;PO4, 6 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 5 g NaCl, 1 ml 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;, H&lt;sub&gt;2&lt;/sub&gt;O to 1 liter. Sterilize by autoclaving</td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596026</td><td>Use 500 ml for 50 worms</td></tr><tr><td>iQ SYBR Green</td><td>Bio-Rad</td><td>1708882</td><td></td></tr><tr><td>SuperScript III</td><td>Invitrogen</td><td>18080-051</td><td></td></tr><tr><td>PCR machine</td><td>Bio-Rad</td><td>C1000/CFX96</td><td></td></tr><tr><td>96-well PCR tubes</td><td>Bio-Rad</td><td>MLL9601</td><td></td></tr><tr><td>Image analysis software</td><td>Molecular Devices</td><td>Metamorph</td><td></td></tr></tbody></table>

methods for skin wounding and assays for wound responses in c. elegans

C.将线虫表皮角质层，并形成一个既简单又复杂的皮肤层，它可以修复由伤人造成局部损坏。伤口反应和修复中该模型的研究已经照亮了细胞骨架和基因组响应组织损伤的理解。两种最常用的方法，以卷绕在C.线虫成人皮肤都刺有注射针头，和当地的激光照射。针伤人局部破坏角质层，表皮，和相关的细胞外基质，并且还可能会损坏内部组织。激光照射导致更多的局部损坏。伤人触发容易测定应答，包括升高的表皮的Ca 2+（秒分钟），形成与含有肌动蛋白环在伤口部位（1-2小时），升高的转录的抗微生物肽基因的闭合连续（2-24小时），和瘢痕形成。基本上所有的野生型成年动物生存伤人，在那里作为突变体缺陷的创面修复或其他答复表明存活率降低。对针和激光伤人检测的定量和伤口反应和修复过程（钙动力学，肌动蛋白，抗菌肽的诱导和生存）的可视化的详细协议，并介绍了。

激光或针伤人将触发快速和持续升高表皮钙水平，作为可视化与钙传感器，诸如GCaMPs（ 图1）。钙高程发生在几秒钟内，并持续升高几十分钟。针伤人再现地导致形成F-肌动蛋白环中的伤口位点（ 图2）;这些出现在几分钟之内，并逐渐接近过1-2小时后伤人。肌动蛋白环更精确的激光伤人后较少形成。两个针和激光伤人诱导表皮抗菌肽（AMPS）如NLP-29，与转录记者（ <st...

Watch this Scientific Journal Video about Methods for Skin Wounding and Assays for Wound Responses in C. elegans at JoVE.com

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

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Research

JoVE Journal

Biology

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

10.3K 次观看.  University of California, San Diego. The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity. 

视频: Methods for Skin Wounding and Assays for Wound Responses in C. elegans

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

在 C 在体内激光干切断线虫

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

科研

神经科学

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

创伤显微注射和分析<em&gt;在体内</em&gt;表皮的伤口本地化响应记者在<em&gt;果蝇</em&gt;胚胎。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

生物工程

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

基于线虫人群的化学感应优选测定的自动分析

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

行为学

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

一种在96孔板表演细胞迁移/伤口愈合设备

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

发育生物学

A Drosophila Model to Study Wound-induced Polyploidization

Polyploidy is a frequent phenomenon whose impact on organismal health and disease is still poorly understood. A cell is defined as polyploid if it contains more than the diploid copy of its chromosomes, which is a result of endoreplication or cell fusion. In tissue repair, wound-induced polyploidization (WIP) has been found to be a conserved healing strategy from fruit flies to vertebrates. WIP has several advantages over cell proliferation, including resistance to oncogenic growth and genotoxic stress. The challenge has been to identify why polyploid cells arise and how these unique cells function. Provided is a detailed protocol to study WIP in the adult fruit fly epithelium where polyploid cells are generated within 2 days after a puncture wound. Taking advantage of D. melanogaster&#39;s extensive genetic tool kit, the genes required to initiate and regulate WIP, including Myc, have begun to be identified. Continued studies using this method can reveal how other genetic and physiological variables including sex, diet, and age regulate and influence WIP&#39;s function.

A Drosophila Model to Study Wound-induced Polyploidization

Wound-induced polyploidization is a conserved tissue repair strategy where cells grow in size instead of dividing to compensate for cell loss. Here is a detailed protocol on how to use the fruit fly as a model to measure ploidy and its genetic regulation in epithelial wound repair.

研究伤口引起的多倍体化的果蝇模型

Polyploidy is a frequent phenomenon whose impact on organismal health and disease is still poorly understood. A cell is defined as polyploid if it ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

利用微流体装置进行机械刺激和高分辨率成像的研究C.线虫

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

使用多盐皮下聚乙烯醇海绵植入和外向尾皮肤伤口模型进行急性伤口愈合评估。

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

免疫与感染

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

划痕迁移分析和背皮折叠室的体外和在Vivo分析伤口愈合

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

医学

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

生物学

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

对于皮肤伤人方法和测定的伤口响应

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