笔者想感谢6月米德尔顿供给E.粪肠球菌细菌培养和指导。C.线虫提供了CGC，这是由美国国立卫生研究院办公室研究基础设施建设项目（P40 OD010440）。

1）制备线虫生长培养基（NGM） <ol><li>为了使50板，加1.5克氯化钠，8.5克超纯琼脂和1.25克蛋白胨1 Lflask的。 </li><li> DH 2 O添加487.5毫升的烧瓶。轻轻摇晃混合覆盖开幕烧瓶用一块铝箔。 </li><li>消毒的解决方案，通过在标准条件下高压灭菌（121磅，120℃，20分钟）。 </li><li>让溶液冷却至45℃的水浴中。 </li><li>要解决方案，添加下面的顺序：500微升胆固醇5毫克/毫升原液（溶解在乙醇中），500微升1 2 M的氯化钙，500微升1 M 硫酸镁和12.5毫升KPO 4缓冲液（54.15克KH 2 PO 4和17.8克K 2 HPO 4 500毫升蒸馏水2 O）中所描述的18。 </li><li>使用无菌移液器，加10毫升琼脂溶液各60毫米的聚苯乙烯培养皿。允许介质在室温下固化overn洞察力。 </li></ol> 2）制备的B肉汤E.大肠杆菌媒体<ol><li>五个100毫升文化，加2.5克酵母提取物，蛋白胨，5.0克氯化钠5.0克到烧杯中。 </li><li>添加无菌蒸馏水至终体积为500ml。在热板上加热溶液，边搅拌，直至溶解溶质。 </li><li>分为五个玻璃瓶（至少200毫升）倒入100毫升的B肉汤。 </li><li>在标准条件下高压灭菌（121磅，120°C，20分钟）消毒解决方案。 </li><li>允许乙肉汤冷却至室温后再使用。 </li></ol> 3）播种C.线虫维护板<ol><li> NGM板在室温下干燥至少一个星期后，一个100毫升乙肉汤培养物接种的大肠杆菌菌落有几大肠杆菌 （OP50）。 </li><li> E.成长菌在37℃过夜。 </li><li>吸取1滴（约10081，L）OP50文化到每个NGM板的中心。片通常叠盖侧。移液时（播种），开始在堆栈的底部吹打文化上板。您的工作方式，每个堆栈。尽量不要移动的板，因为它是重要的文化太靠近板的边缘后播种。 </li><li>允许种子板在室温下干燥，在使用前至少一个星期。 </li><li>种子板块在密封的塑料容器中，在室温下保存（倒）。 </li></ol> 4）维护C.线虫菌株<ol><li>为了维持精心喂养C.线虫股票，使用蠕虫挑L4S或妊娠成人移动到一个新的种子NGM板每3-4天。 </li><li>商店板（倒）里面最好的孵化器在15 - 22°C。所描述的测定中的培养物储存在20℃下下线虫的发展是温度相关的。由于maintenaNCE温度的升高，发育阶段之间的时间减少。 </li></ol> 5）胰酶大豆琼脂（TSA）E.粪文化传媒<ol><li> 50板，测量一瓶500毫升的无菌蒸馏水，把煮沸，一边搅拌，在电炉上。 </li><li>粉状TSA琼脂20克加入烧瓶，并允许溶液溶解。 </li><li>在标准条件下高压灭菌（121磅，120°C，20分钟）消毒解决方案。 </li><li>让溶液冷却至45℃的水浴中。 </li><li>使用无菌移液器，加10毫升琼脂溶液各60毫米的聚苯乙烯培养皿。允许介质在室温下固化过夜。 </li></ol> 6）胰酶大豆肉汤（TSB）E.粪文化传媒<ol><li>为了使250个培养管，测量到一个烧瓶和温暖的烤盘上，而500毫升的无菌蒸馏水搅拌。 </lI&gt; <li> TSB琼脂粉15克加入到烧瓶中，使粉末溶解。 </li><li>在标准条件下高压灭菌（121磅，120°C，20分钟）消毒解决方案。 </li><li>让溶液冷却至室温，然后移液管2毫升，放入灭菌的5毫升试管。 </li></ol> 7）脑心浸液（BHI）媒体，链霉素，E.粪文化<ol><li>测量无菌蒸馏水500毫升的烧瓶中，并把煮沸，一边搅拌，在电炉上。 </li><li> 26克BHI培养基加入到烧瓶中，使该溶液溶解。 </li><li>在标准条件下高压灭菌（121磅，120°C，20分钟）消毒解决方案。 </li><li>让溶液冷却至45℃的水浴中。 </li><li>使用无菌技术，加入0.5毫升10毫克/毫升链霉素的原液和涡流混合。 </li><li>加入10毫升的溶液，以每60毫米的聚苯乙烯培养皿中。 </li><l&gt;允许媒体在室温下固化过夜。商店板（倒）在4℃，直到几个小时后再使用。 </li></ol> 8）维护E.粪肠球菌 <ol><li>为了维持E。粪股市连胜单独的胰酶大豆琼脂（TSA）平板上的单个菌落，用无菌环。 </li><li>文化生长在37℃过夜。店板（倒）了几个星期在室温下在密封的袋子或盒子中。 </li></ol> 9）准备E.粪板EIW含量的<ol><li>接种2毫升TSB文化的殖民地E.粪肠球菌和孵育过夜，在37°C。 </li><li>移液管加入20μl 大肠杆菌粪到准备好的BHI链霉素板的中心文化，孵育过夜，在37°C。 </li></ol> 10）准备E.大肠杆菌板EIW含量（控制板） <ol><li>接种了100毫升的B-BRO次文化的殖民地E.大肠杆菌 OP50在37℃孵育过夜</li><li>吸取20μl的的OP50培养的到非种子选手NGM板的中心和允许种子板在室温下干燥过夜。 </li></ol> 11）蛋蠕虫含量<ol><li>挑选15 - 20上演L4 C.线虫 （ 图1）上制备的BHI-链球菌， 大肠杆菌到草坪的细菌的中心粪板。要小心不转让了很多OP50的BHI板的。接送L4S相同数量的到控制NGM-OP50板。 </li><li> 40小时，在20°C孵育板</li><li>准备20％的漂白粉溶液。的商业漂白剂（6.0％次氯酸钠）混合，用适当体积的蒸馏水。 </li><li>加入10微升滴漂白剂溶液，以10个不同的位置上的塑料盖（一个蠕虫病毒或细菌板）。 </li><li>使用蠕虫挑，一个蠕虫传输到每个漂白下降。轻轻旋转的尖端挑中的漂白剂液滴，，确保蠕虫洗掉挑年底（确认在解剖显微镜下观察液滴）。 </li><li>使角质层溶解约10分钟或直到蠕虫爆裂开来，驱逐鸡蛋（ 图2）。要小心，不要将鸡蛋从板块。 </li><li>使用解剖显微镜每一滴漂白剂，算上鸡蛋。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

成功地执行这个实验中最关键的步骤是：1）使用精心喂养C.股票线虫 ，2）单一类型的细菌培养测定板，3）准确吞吐量上演L4蠕虫暴露于E。粪 ，4）保持曝光时间E.粪一致的，所有试验和5），漂白时间不应超过10分钟，以防止鸡蛋解体。 这个实验，重要的是要挑选健康，良好的喂养蠕虫。产妇饥饿会影响后代的繁殖力和生长19,20，因此重要的...

一个微小的，自由生活的蛔虫， 线虫 ，是一种模式生物，传统上用来研究人类发育和细胞的信令流程，因为透明解剖特征的发展，全基因组测序，短代时，基因的同源性。最近，C.线虫已经成为一种模式生物在该领域的环境毒理学和先天免疫10，11。 这些自我施肥雌雄同体蠕虫从卵孵化后的两到三天内性成熟。在它的生命周期，C.线虫通过四个幼虫阶段（L1-L4），达到成年之前。可以产生一个孤立的雌雄同体，平均300个后代在三天之内的峰值繁殖力。在生殖成熟C.线虫雌雄同体，受精卵在子宫内保留几个小时前被解雇。该存储在子宫中的蛋在任何一个时间的正常数量的（在繁忙繁殖力）是在10和15之间12。鸡蛋在子宫产蛋率和产蛋率的函数。受精卵从子宫排出，外阴13日开幕的周围布置的16个外阴部肌肉的收缩。的雌雄同体特定运动神经元（HSN）和VC影响肌肉收缩阴部肌肉的运动神经元突触上，从而鸡蛋产蛋行为5,7,13,14。驱逐鸡蛋从子宫的发生是由于神经元和肌肉的协调活动。 C.实验室培养线虫通常提出非致病性大肠杆菌 OP50的饮食。在自然环境中，C。线虫接触到的各种食物来源，如致病性细菌，也可以有潜在危害。当暴露于有害物质的的环境中，C。线虫保留鸡蛋，直到环境变得更加有利。据推测，这鸡蛋保留是为了保护他们的后代。 在这个蛋虫（EIW）测定，C.线虫暴露于潜在的致病细菌， 粪肠球菌 ，这是存在于环境中。 大肠杆菌致病形式曝光粪可引起持续性肠道感染，甚至死亡，在C线虫 15。暴露于其他形式的致病性细菌已被证明影响蛋保留16,17，但是没有量 ​​化的效果。此外，轻度致病性菌株的大肠杆菌的效果肠球菌 ，菌株不会立即致命，产蛋行为还没有被研究。 EIW检测涉及计数鸡蛋的数量保留在子宫C.线虫 4。尽管C。线虫是透明的，积聚在子宫的卵可以是难以量化，在一个完整的动物。 EIW检测涉及漂白妊娠成人C.线虫暴露于细菌一段固定的时间。漂白粉溶液溶解身后留下的鸡蛋外被角质层。鸡蛋是折射的漂白剂，由于存在保护蛋壳的效果。漂白后，能够非常容易地发布从子宫的蠕虫后漂白的鸡蛋的数量来计算。 检测是一种简单，廉价和快速的方法来量化在子宫内一次鸡蛋的数量，从而量化的影响E.粪蛋保留。本试剂盒可用于其他类型的细菌，环境毒素或药物的效果进行量化的蛋性。本试剂盒也有可能被用作细菌致病性的屏幕。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Agar, ultrapure</td>
			<td style="width:191px;">Affymetrix</td>
			<td style="width:119px;">10906</td>
			<td style="width:1791px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bacto Peptone</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bacto Tryptone</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Brain Heart Infusion dehydrated medium</td>
			<td style="width:191px;">Carolina Biological Supply</td>
			<td style="width:119px;">781781</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:371px;">C. elegans, N2 strain</td>
			<td style="width:191px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:371px;">Cholesterol</td>
			<td style="width:191px;">Alfa Aesar</td>
			<td style="width:119px;">A11470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Culture plates for C. elegans</td>
			<td style="width:191px;">Tritech Research Inc.</td>
			<td style="width:119px;">T3308</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Culture plates for E. faecalis</td>
			<td style="width:191px;">Fisher Scientific-Fisherbrand</td>
			<td style="width:119px;">875713</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">E. coli (OP50)</td>
			<td style="width:191px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">E. faecalis strains</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>provided by J. Middleton. All isolates were confirmed as enterococci</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>by observing growth on enterococcosel agar (BBL) and in 6% NaCl broth;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>
			all strains grew at 44.5 &#186;C and were catalase negative and hydrolyzed esculin. A simplified&#160;dichotomous key based on pigmentation and fermentation reactions for six sugars&#160;(arabinose, mannitol, methyl-&#945;-D-glucopyranoside (MGP), ribose, sorbose and sorbitol) allowed&#160;presumptive identification of all E. faecalis strains (Efs lacks pigmentation and is arabinose, MGP&#160;and sorbose negative and sorbitol, mannitol and ribose positive). All presumptive Efs strains&#160;were confirmed using the API 20 STREP system (Biomerieux).
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microscope</td>
			<td style="width:191px;">Motic</td>
			<td style="width:119px;">SMZ 168B</td>
			<td>any microscope with transmitted illumination and 50X magnification should be sufficient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Streptomycin sulfate</td>
			<td style="width:191px;">Fisher BioReagents</td>
			<td style="width:119px;">BP910-50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Tryptic Soy Agar (Soybean-Casein Digest Agar Medium), Difco</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">236950</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Trypticase Soy Broth (Soybean-Casein Digest Medium), BBL</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211768</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Yeast extract</td>
			<td style="width:191px;">Acros</td>
			<td style="width:119px;">61180-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the effects of bacteria on c. elegans behavior using an egg retention assay

线虫产卵行为是受环境因素如渗透压和振动2。在完全没有食物C.线虫也停止产蛋，并保留受精卵在子宫3。然而，不同来源的食品，特别是病原细菌，特别是粪肠球菌 ，对产卵行为的影响没有得到很好的特征。蛋在蜗杆（EIW）法是一种有用的工具，不同类型的细菌的影响量化，在这种情况下， 大肠杆菌粪，产蛋行为。 EIW检测涉及计数鸡蛋的数量保留在子宫C.线虫 4。 EIW检测涉及漂白上演，妊娠成人C.线虫去除角质层，从动物中分离保留的鸡蛋。在漂白之前，蠕虫暴露于细菌（或任何不同的环境线索）一段固定的时间。漂白后，一个是很容易能够指望鸡蛋保留子宫内蠕虫的数量。在这个实验中，一个量化的增长后，在鸡蛋保留E.粪曝光可以很容易地测量。的的EIW测定试验，可用于筛选潜在致病细菌或环境毒素的存在是一种行为。此外，EIW检测可能是一个工具，屏幕药物，影响神经递质的信号，因为产卵的行为是由神经递质，如5 -羟色胺和乙酰胆碱的5-9调制。

此法可以让一个量化C.内保留的鸡蛋数量线虫暴露于E.后粪 。 L4上演蠕虫（特点是透明开放的空间高于其外阴， 图1）暴露于E.粪到成年。经过40个小时的接触E.粪 ，漂白。由于角质层崩解在漂白剂液滴，蛋变得更加明显（ 图2）。保留鸡蛋的数量很容易量化计算，而解剖显微镜下观看。 存在的大肠杆菌中...

Watch this Scientific Journal Video about Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay at JoVE.com

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay | Biology | Video

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

一个鸡蛋在蠕虫（EIW）法是一种有用的方法来量化产蛋行为。产蛋的改变可以是一个模型有机体的行为反应<em&gt;线虫</em到潜在的有害环境的物质，例如由致病细菌产生的。

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

measuring-effects-bacteria-on-c-elegans-behavior-using-an-egg

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Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

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16.9K 次观看.  Fairleigh Dickinson University. 一个鸡蛋在蠕虫（EIW）法是一种有用的方法来量化产蛋行为。产蛋的改变可以是一个模型有机体的行为反应线虫

视频: Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

测量线虫固体介质的寿命

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

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 ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用“冻结开裂”

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

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 ...