低容量通气培养系统的初始开发部分得到了美国国家科学基金会（CRI-OA-1416690 给迪金森学院）的支持。Lauren Mullineaux 博士慷慨地为伍兹霍尔海洋研究所提供了实验室设施，在那里收集了该系统提供的数据（图 4）。

1. 建立和维持 C. fornicata 幼虫培养物的常规操作
注意：这种方法从一加仑（3.8 升）罐装海水开始，其中含有刚刚释放育雏的 Veliger 幼虫的成年 C. fornicata 。成虫可以实地采集或从 材料表中 给出的供应商处获得。成虫是原生雄性雌雄同体，生活在交配堆中，无柄育雏雌性生活在堆底。不要拆散成人堆栈。繁殖的季节性和调节成虫以在非季节产卵的方法之前已经描述过17。幼虫最好在释放后 2-3 小时内收集，此时它们具有很强的地理负性，并且会集中在罐子表面附近。
<ol><li>切下 400 mL 三角一次性塑料烧杯的底部，然后粘在 236 μm 尼龙网板上，制成筛子。为此，特别是对于 mesocosm 结构（如下），请使用对聚乙烯具有良好的粘合力的热熔胶。</li>
	<li>从成虫罐中取出曝气物，静置 15-20 分钟，以便碎屑沉淀，幼虫可以轻松游到水面。</li>
	<li>通过固定在 60-80 厘米长的软塑料水族箱管末端的短 （15-20 cm） 5 mm 玻璃管将幼虫虹吸到如上所述制备的筛子中，悬浮在 600 mL 玻璃烧杯中，使多余的海水溢出玻璃烧杯，而幼虫保留在筛子上。将筛子底部保持在水下，避免搁浅幼虫。</li>
	<li>将筛子短暂地从烧杯中取出，并使用过滤海水 （FSW） 的喷瓶将幼虫冲洗到一个玻璃碗中，该玻璃碗的大小便于在低倍解剖显微镜下进行操作。 注意：C. fornicata 的幼虫在盐度接近 30 ppt 的过滤天然海水或 20-25 °C17,18 的盐度高达 32 ppt. 的 Instant Ocean 人工海水中茁壮成长。< 1 μm 的过滤足以消除可能在实验室培养中引起问题的污染流行性原生生物。</li>
	<li>使用巴斯德移液管手动将所需数量的幼虫转移到培养罐中并计数。为获得最佳结果，请使用 1 只幼虫/5 mL 培养体积的 C. fornicata。数一数幼虫，避免估计数量的诱惑。</li>
	<li>当培养开始时，用所需口粮的微藻喂养幼虫，当更换培养水时，每隔一天更换一次微藻。 注意：1 x 105 个细胞/mL 等黄藻 （菌株 T-ISO）的饮食是一种很好的标准饮食，可产生接近最高的实验室生长速率。食物配给和 T-ISO 的培养方法在早期出版物中提供17。</li>
	<li>如上面的步骤 1.3 所示，将培养物的内容物倒入烧杯内的筛子中，每隔一天更换一次培养水。取出筛子，用 FSW 喷瓶将幼虫冲洗到装有新食物的新鲜 FSW 培养罐中。 注意：最佳做法是将用于幼虫培养的任何玻璃器皿分开，并避免该玻璃器皿与固定剂、可溶性重金属盐或清洁剂接触。日常清洁最好使用小苏打糊 （NaHCO3） 和尼龙擦洗垫进行，而矿物质沉积物可以用白醋或稀释的 HCl 进行温和的酸洗去除。</li>
</ol>2. C. fornicata 幼虫通风培养物的构建
注意：推荐的玻璃罐（材料表）有一个聚丙烯盖，该盖对海水呈惰性，并且具有适合固定管道倒钩入口的厚度，用于通风气流。
<ol><li>在每个培养瓶（包括盖子的内衬）的盖子上钻两个 5 毫米的孔，每个孔距边缘约 1.5 厘米，并且彼此相对。（13/64 英寸英制或 #7 ASME 标准机械师的钻头也将制作合适的间隙孔）。</li>
	<li>使用 10-32 尼龙螺母在每个孔中安装一个 1/8 英寸 x 10-32 螺纹尼龙管适配器，管子倒钩位于盖子的外表面。</li>
	<li>在其中一个管道适配器的螺纹内部的孔上涂抹少许硅橡胶水族密封剂，然后从内部将 20 厘米长的 2 毫米（外径）聚乙烯管推入孔中，使其突出几毫米超出管道倒钩的外孔。</li>
	<li>现在，管材的末端将有一小块水族密封剂塞，从管材倒钩中伸出，但在倒钩末端之外可以看到透明管材。让密封剂固化，然后修剪聚乙烯管的突出端，使其与管倒钩的末端齐平。</li>
	<li>用 FSW 填充培养瓶，使其距离罐顶部肩部 1 cm 以内，拧下盖子，然后将通风空气或实验气体混合物供应连接到固定聚乙烯通气管的管道倒钩上。修剪通风管的长度，使其末端整齐地位于罐子的底部。</li>
	<li>调整通风空气或气体混合物的流量，以产生缓慢的气泡流（图 1）。使用带有普通水族箱联阀的水族箱气泵为环境空气供应通风，或将质量流量控制器用于其他实验性大气气体混合物14。</li>
</ol>3. 构建 C. fornicata 幼虫的现场可部署中宇宙培养物
<ol><li>在标准 7 加仑聚乙烯桶的侧面切 4 个均匀分布的矩形开口，每个开口 25 厘米 x 14.5 厘米。将每个开口的底部放置在距离桶内底部约 3.5 厘米的位置（图 2A）。带有细齿刀片的手持式电刀锯是实现此目的的理想选择。保留切出的废料，用台锯将它们纵向切成 2 厘米宽的条状。</li>
	<li>剪下 4 块 236 μm 尼龙网，每块至少 30 cm x 20 cm，以便舒适地重叠（至少 2 cm）桶中的切口开口。使用小弹簧夹或衣夹将网面板的一个边缘暂时固定在其切口的长边上。</li>
	<li>用专为聚乙烯配制的热熔胶将网板的边缘粘到桶口上。固定第一个边缘后，在保持张力的同时粘合其他边缘，以在网格面板上获得拉紧的表面。</li>
	<li>修剪步骤 3.1 中桶废料条的长度，使加强条整齐地覆盖网板与桶开口重叠的胶合区域。用大量的热熔胶将每个覆盖条压入到位，在胶水硬化之前快速将每个覆盖条压到位。</li>
	<li>用从桶外部安装的尼龙盲铆钉固定每个条带的末端。用实用剃须刀从加固条的外边缘修剪掉多余的胶水和网眼。 注意：每个铆钉都需要一个 15/64 英寸的导向孔。在 图 2B 中，铆钉的内部（盲）部分在成品 mesocosm 的内部可见。</li>
	<li>将 mesocosm 部署在浮动机架中，该浮动机架将桶的顶部保持在水面以上（图 2C）。所示的浮动机架由 30 厘米的 2 英寸 Schedule 40 PVC 管段制成，与弯头和三通接头粘合在一起，形成一个气密结构，可以容纳一排 4 个复制中宇宙。</li>
	<li>根据当地情况，网板可能会在 1-2 天内结垢，从而阻碍海水通过中层的交换。在这种情况下，通过将污损的中胚层从水中拉出 2/3rd ，并用 2 L 水罐反复从其底部捞入新鲜的中胚层，将幼虫转移到新鲜的中层中。</li>
	<li>执行 20-25 次迭代以转移大部分幼虫，或直到在被污染的中宇宙中不再观察到幼虫。用装有小苏打糊 （NaHCO3） 的软海绵轻轻擦洗网板，然后用自来水冲洗，以清洁被污染的 mesocosm。</li>
</ol>

在实验室和田间条件下培养 Crepidula fornicata 的幼虫

<ol>
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没有需要报告的利益冲突。

尽管与其他浮游营养海洋幼虫相比，C. fornicata 的幼虫相对容易养殖，但关注良好养殖规范的基本原则仍然是必不可少的17,19。健康的幼虫应在孵化后立即开始摄食。这在孵化后的第二天通过使用具有透射功能的解剖显微镜观察它们充满藻类细胞的完整肠道，很容易验证。健康幼虫的壳应保持清洁、明亮，并且没有茎纤毛原生生物或无柄羽状硅藻?...

拖鞋帽贝 Crepidula fornicata （腹足纲：Calyptraeidae） 在当前和历史研究文献中得到了很好的体现，因为它作为一种发展模式的实用性以及它作为入侵物种的广泛影响。它是实验胚胎学经典时代螺旋发育的基础例子1，并且随着现代成像和基因组工具的应用来剖析 Lophotrochozoan 早期发育的机制，它经历了兴趣的重生 2,3。在其生命周期历史的另一端，其他研究集中在这种生态系统工程师的成年种群对温带沿海海洋环境的影响，这些海洋环境远非其在北美东部的原始分布 4,5。在胚胎和成虫之间，该物种的绒毛幼虫一直是幼虫发育和生态学的大量研究的主题，特别是影响生长和获得能力的因素，介导幼虫定居的内部和外部线索，以及幼虫经历对幼虫性能的影响 6,7,8,9,10,11 .最近的研究揭示了 C. fornicata 的幼虫和幼虫对海洋酸化的适应能力，这是这种动物的另一种生产性研究利用途径 12,13,14,15,16。
C. fornicata 用于海洋幼虫生物学研究的一个优点是，它在实验室中相对容易在天然或人工海水中以鞭毛状 Isochrysis galbana 的单一饮食生长。作者在早期以方法为重点的印刷出版物中详细介绍了培养方法17。目前做出贡献的原因有两个。首先，建立和照顾培养物所涉及的日常物理操作在概念上非常简单，但如果没有动手或视频演示，很难正确执行。其次，描述了先前描述的培养方法的两种变体，它们特别适用于对环境压力源（如海洋酸化、富营养化和氧气消耗）的反应的实验室和现场研究。其中第一个是小容量 （800 mL） 培养系统，适用于通过少量气泡气体控制海水中的 pH 值和溶解氧，第二个是较大容量 （15 L） 的中层系统，可以放置在田间并允许自由交换环境海水。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bucket, Polyethylene, 7 gallon</td>
			<td>US Plastic</td>
			<td>16916</td>
			<td>for mesocosm</td>
		</tr>
		<tr>
			<td>Crepidula fornicata</td>
			<td>Marine Biological Laboratory, Marine Resources Center</td>
			<td>760</td>
			<td>adult broodstock</td>
		</tr>
		<tr>
			<td>Hotmelt glue, Infinity Supertac 500</td>
			<td>Hotmelt.com</td>
			<td>INFINITY IM-SUPERTAC-500-12-1LB</td>
			<td>good for bonding polyethylene</td>
		</tr>
		<tr>
			<td>Jar, glass, 32 oz, with polypropylene lid</td>
			<td>Uline</td>
			<td>S-19316P-W</td>
			<td>for 800 mL ventilated cultures</td>
		</tr>
		<tr>
			<td>Nitex mesh, 236 &#181;m</td>
			<td>Dynamic Aqua Supply Ltd.</td>
			<td>NTX236-136</td>
			<td>for mesocosm</td>
		</tr>
		<tr>
			<td>Nut, hex, nylon, 10-32 thread</td>
			<td>Home Depot</td>
			<td>1004554441</td>
			<td>for fastening tubing barbs</td>
		</tr>
		<tr>
			<td>Rivets, nylon, blind, 15/64&#34; diameter, 5/32&#34;-5/16&#34; grip range, pack of 8</td>
			<td>NAPA auto parts</td>
			<td>BK 6652844</td>
			<td>4 packs needed per mesocosm</td>
		</tr>
		<tr>
			<td>Tubing barb 1/8&#34; x 10-32 thread</td>
			<td>US Plastic</td>
			<td>65593</td>
			<td>2 needed per culture jar</td>
		</tr>
		<tr>
			<td>Tubing, polyethylene, 2.08 mm OD</td>
			<td>Fisher Scientific</td>
			<td>14-170-11G</td>
			<td>for ventilating gas stream inside culture jar</td>
		</tr>
		<tr>
			<td>Tubing, Tygon, 1/8&#34;x3/16&#34;x1/32&#34;</td>
			<td>US Plastic</td>
			<td>57810</td>
			<td>fits barbs for ventilating cultures</td>
		</tr>
	</tbody>
</table>

laboratory and field culture of larvae of the slipper limpet, crepidula fornicata

蛾蛾腹足类软体动物 Crepidula fornicata 已广泛用于幼虫发育生物学、生理学和生态学的研究。该物种的育雏绒幼虫在成虫自然释放后通过虹吸到筛子上收集，以 200/L 的密度分配到培养物中，并以 1 x 105 个细胞/mL 的密度喂食 Isochrysis galbana（菌株 T-ISO）。记录了在通风的 800 mL 培养物中饲养的兄弟幼虫的壳生长和能力的获得，这些培养物旨在平衡与环境空气或确定的大气气体混合物。与这些实验室培养条件形成对比;还收集了在位于繁殖成虫田间种群中的 15 L 流过环境海水中层饲养的幼虫的生长和能力数据。实验室培养物中能力的生长速率和时间与先前发表的研究中报道的相似。在田间中饲养的幼虫生长得比任何实验室研究报道的要快得多，也早得多。总之，这些方法适用于在实验室中预定的受控条件下以及在田间自然发生的条件下探索幼虫发育。

在 800 mL 通气培养物的 4 个同时重复中测量幼虫生长和能力的获得，每个重复包含 160 只幼虫，来自从单个卵块孵化的兄弟姐妹批次的幼虫，并以 1 x 105 个细胞/mL 的密度喂食 Isochrysis galbana。pH 值为 7.9-8.0，温度为 20-21 °C，盐度为 30-31 ppt。在美国马萨诸塞州秃鹰湾部署的 15 L 中层环境中，还用不同兄弟批次的幼虫测定了生长和，该试验中含有 600 只幼虫，在 pH 值、温度和盐度与实验?...

Read this Scientific Article Protocol about Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata at JoVE.com

作者聚焦：弥合幼虫生态学研究中现场观察和实验室操作之间的差距

本文介绍了在小容量实验室规模系统和可在现场部署的环境海水中宇宙系统中培养腹足类 Crepidula fornicata 幼虫的方法。

鹦鹉幼虫 Crepidula fornicata 幼虫的实验室和田间培养

The calyptraeid gastropod mollusk, Crepidula fornicata, has been widely used for studies of larval developmental biology, physiology, and ecology. Brooded ...

我对海洋幼虫的发育如何受到多种环境因素和压力源的影响以及幼虫神经系统如何整合来自环境的信息来调节幼虫的定居、为底栖幼虫以及招募到成年种群感兴趣。一些海洋物种的幼虫，如拖鞋帽贝 （Crepidula Fornicata），对海洋酸化具有很强的适应能力，但酸化与其他因素（如营养和盐度）之间存在重要的相互作用。我们对幼虫在自然界中的生长、发育和行为知之甚少。野外中宇宙观测可以填补其中一些空白，并提出假设以通过实验室操作进行测试。我们可以开始在神经行为、发育和转录层面上询问幼虫如何在特别容易受到人类影响的生产性沿海环境中处理短时间变化的压力源。

laboratory-field-culture-larvae-slipper-limpet-crepidula

Research

JoVE Journal

Biology

Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata

337 次观看.  Dickinson College. 我对海洋幼虫的发育如何受到多种环境因素和压力源的影响以及幼虫神经系统如何整合来自环境的信息来调节幼虫的定居、为底栖幼虫以及招募到成年种群感兴趣。一些海洋物种的幼虫，如拖鞋帽贝 （Crepidula Fornicata），对海洋酸化具有很强的适应能力，但酸化与其他因素（如营养和盐度）之间存在重要的相互作用。我们对幼虫在自然界中的生长、发育和行为知之甚少。

视频: 作者聚焦：弥合幼虫生态学研究中现场观察和实验室操作之间的差距

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

高分辨率测量的气味驱动在果蝇幼虫的行为

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

科研

生物学

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.

Microgavage斑马鱼幼虫

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

设计和适体 - 金纳米粒子基于比色法的开发在这领域的应用

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Establishment of an Extracellular Acidic pH Culture System

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

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

胞外酸性 pH 培养体系的建立

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

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

绿松石鳉鱼实验室外壳的协议 (Nothobranchiusfurzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

利用膜成熟腺细胞聚合培养（MAAC）的人类成熟腺细胞的分离与培养

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

原原小鼠肝细胞的分离和3D胶原蛋白三明治培养，研究细胞骨架在胆汁形成中的作用

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

果蝇幼虫的欧诺细胞的解剖和脂滴染色

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...