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，应当注意的是，形成的肌动蛋白环的不再现地飞秒激光伤人后观察。 针头穿刺的一个限制是，它不是一个精确的伤口:针也损害内部组织的贡献蠕虫生存都没有受到调查。这个协议的一个可能的修改是结合微流控为基础的显微注射系统11和荧光记者进行更精确的物理伤人。 这里详述针和激光伤人方法简单而相当密集的劳动，并在最佳媒介吞吐量，使其具有挑战性的使用基因组或生化途径伤口响应特性。角质层穿透病原体如真菌或原生动物12,13可以被传递到更大的动物种群，并可能引发与该响应伤人重叠的反应，但引入额外的病原体特异性应答的复杂性。进一步的工作将需要探索是否其他非生物方法如弹道轰击14或微机械刺穿结构15的阵列可适于大规模伤人。

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 border="0" cellpadding="0" cellspacing="0" width="908">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Name</td>
			<td style="width:227px;">Company</td>
			<td style="width:227px;">Catalog Number</td>
			<td style="width:227px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Agarose</td>
			<td style="width:227px;">Denville Scientific</td>
			<td style="width:227px;">CA3510-8</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Plastic Petri plate</td>
			<td style="width:227px;">Tritech Research</td>
			<td style="width:227px;">T3308</td>
			<td style="width:227px;">60 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Levamisole</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:227px;">L9756</td>
			<td style="width:227px;">12 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;"><a name="RANGE!A5">Borosilicate Glass Capillary&#160;</a></td>
			<td style="width:227px;">World Precision Instruments</td>
			<td style="width:227px;">1B100F-4</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Needle puller</td>
			<td style="width:227px;">Sutter Instrument&#160;</td>
			<td style="width:227px;">P-2000</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Worm pick (Platinum wire)</td>
			<td style="width:227px;">Tritech Research</td>
			<td style="width:227px;">PT-9010</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="86">
			<td height="86" style="height:87px;width:227px;">M9 solution</td>
			<td style="width:227px;">Home-made</td>
			<td colspan="2" style="width:453px;">3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. Sterilize by autoclaving</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Trizol</td>
			<td style="width:227px;">Invitrogen</td>
			<td align="right" style="width:227px;">15596026</td>
			<td style="width:227px;">Use 500 ml for 50 worms</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">iQ SYBR Green</td>
			<td style="width:227px;">Bio-Rad</td>
			<td align="right" style="width:227px;">1708882</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">SuperScript III</td>
			<td style="width:227px;">Invitrogen</td>
			<td style="width:227px;">18080-051</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">PCR machine</td>
			<td style="width:227px;">Bio-Rad</td>
			<td style="width:227px;">C1000/CFX96</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">96-well PCR tubes</td>
			<td style="width:227px;">Bio-Rad</td>
			<td style="width:227px;">MLL9601</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Image analysis software</td>
			<td style="width:227px;">Molecular Devices</td>
			<td style="width:227px;">Metamorph</td>
			<td style="width:227px;"></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（ 图3）检测出的表达。 AMP感应很明显在2-4小时后伤人，持续约24小时。野生型的针伤人不应显著影响存活率（在24小时后伤人测量）或寿命。 <img alt="图1" src="/files/ftp_upload/51959/51959fig1highres.jpg" /> 图1.激光伤人引发的C.一个钙反应线虫表皮。 （A）表皮GCaMP3荧光（P COL-19 -GCaMP3（juIs319））飞秒激光伤人后的水平。中前体表皮的侧视图。 X，激光创面; N，表皮细胞核。旋转盘共聚焦图像，强度代码。 UW:未受伤，W:GCaMP3ΔF受伤（B）定量/ f 0的20，40和60微米的伤口部位;代表性的痕迹。 <a href="https://www.jove.com/files/ftp_upload/51959/51959fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/51959/51959fig2highres.jpg" /> 图2.针伤人引发各地伤口部位的肌动蛋白聚合。 （A） </s仲&gt;表皮伤口针触发形成肌动蛋白环的创缘，可视化与P COL-19- GFP-膜突蛋白（juIs352）的。规模:10微米。 UW:未受伤，W:从线扫描肌动蛋白环直径受伤（B）定量，每个动物4行扫描（点缀在面板上红色的线条（A），WT） <a href="https://www.jove.com/files/ftp_upload/51959/51959fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/51959/51959fig3highres.jpg" /> 图3.表皮伤人激活先天免疫反应。代表图像（WT未受伤，受伤的WT + 3小时）的P NLP-29-GFP（frIs7）感应针伤人后。规模:200微米<a href="https://www.jove.com/files/ftp_upload/51959/51959fig3large.jpg" target="_blank">请点击这里查看</a>更大的版本这个数字。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 记者 </td><td> 转基因 </td><td> 荧光 </td><td> 等位基因 </td></tr><tr><td>钙</td><td> PCOL -19- GCaMP3; PCOL -19- tdTomato </td><td> GFP / tdTomato </td><td> juIs319 X </td></tr><tr><td>肌动蛋白</td><td> PCOL-19-GFP ::膜突蛋白</td><td> GFP </td><td> juIs352我</td></tr><tr><td> NLP-29 </td><td>子Pnlp-29-GFP / PCOL-12，红色荧光蛋白</td><td> GFP /红色荧光蛋白</td><td> frIs7 IV </td></tr></tbody></table> 表1.转基因创伤反应实验中使用 。下表列出了染色体整合在秀丽隐杆线虫遗传中心（http://www.cbs.umn.edu/research/resources/cgc）可转基因。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 基因名称 </td><td> 序列 </td></tr><tr><td> NLP-29 </td><td> F:cttctcgcctgcttcatggc </td></tr><tr><td> R:gtccgtatccaccatatcctcc </td></tr><tr><td> NLP-30 </td><td> F:TTCTTCTCGCCTGCTTCATGG </td></tr><tr><td> R:CATAACCTCTACCATATCCACCG </td></tr><tr><td> CNC-1 </td><td> F:gccattgtcgccatttcctc </td></tr><tr><td> R:cctccatacattggatatcctc </td></tr><tr><td> CNC-5 </td><td> F:CTTCTTCTAGCAATGCTCGCTC </td></tr><tr><td> R:GTATCCTCCACCATACCCTCC </td></tr><tr><td> AMA-1 </td><td> F:ACTCAGATGACACTCAACAC </td></tr><tr><td> R:GAATACAGTCAACGACGGAG </td></tr><tr><td> SNB-1 </td><td> F:tccagcagacacaagctcagg </td></tr><tr><td> R:gagacaacttctgatcacgctc </td></tr></tbody></table> 表2.引物用于AMP成绩单RT-PCR。工作浓度为0.2微米。SNB-1用作内部对照。

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 ...

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

Nanyang Technological University

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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

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

为线虫突触传递的突变体的药理特性的范式

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

科研

生物学

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit检测癫痫发作癫痫突变果蝇</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法:同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

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 ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...