该研究得到了Bambino Gesù基金会和Ricerca Corrente（意大利卫生部）对C.C.的资助。 我们要感谢Enrico Bertini博士（神经科学系，神经肌肉和神经退行性疾病部门，分子医学实验室，Bambino Gesù儿童研究医院），Stefania Petrini博士（共聚焦显微镜核心设施，研究实验室，Bambino Gesù儿童研究医院），Giulia Pericoli（肿瘤血液学，基因和细胞治疗科，儿童研究医院Bambino Gesù） Roberta Ferretti（Bambino Gesù儿童研究医院肿瘤血液学、基因和细胞治疗科）进行科学讨论和技术帮助。玛丽亚·芬奇是"英国癌症儿童奖学金"的获得者。

1. 涂覆 96 孔板
注意:在同一板中测试不同的涂层，但不同的孔（见 补充文件）。
<ol><li>在DMEM中以1:100稀释基质胶。向96孔板中每孔加入100μL，并在室温下孵育1小时。之后，除去溶液并用 100 μL DMEM 洗涤孔两次。</li>
	<li>在PBS（含钙和镁）中稀释层粘连蛋白（20μg/ mL，LN-521）。向孔中加入100μL并在4°C下孵育过夜。第二天在接种细胞之前用DMEM进行两次洗涤。</li>
	<li>在稀释缓冲液中稀释玻连蛋白（10μg/mL）。向 96 孔板中每孔加入 100 μL，并在室温下孵育 1 小时。在电镀细胞之前，用PBS（不含钙和镁）清洗孔。</li>
	<li>在ddH2O中稀释HU-纤连蛋白（30μg/ mL），向孔中加入100μL并在室温下孵育45分钟。之后，在接种细胞之前用培养基清洗孔。</li>
</ol>2. 培养中iPSCs的维持
注意:iPSC 是商业购买的。iPSCs来源于健康的人成纤维细胞，并使用游离体技术重新编程。
<ol><li>从-80°C冰箱或液氮中，在37°C水浴中解冻冷冻保存的iPSC。在将其移入生物安全柜之前，用70%乙醇清洁装有细胞的小瓶。</li>
	<li>用 1000 μL 移液器在 15 mL 无菌锥形管中逐滴将细胞悬液加入 5 mL 预热的细胞培养基（例如 mTeSR1）中。</li>
	<li>在室温（RT）下以304× g 离心细胞5分钟。</li>
	<li>取出培养基并将细胞沉淀重悬于 4 mL 细胞培养基中。</li>
	<li>将细胞悬液接种到 6 孔板的两个孔中（每个 6 孔细胞培养皿 105 个 iPSC），其中小鼠胚胎成纤维细胞 （MEF） 先前已接种。在DMEM中以2.4 x 104 / cm2 的密度接种iPSCs前两天播种MEF（含有10%胎牛血清，1%L-谷氨酰胺和1%青霉素 - 链霉素）。</li>
	<li>接种后，用10μM的ROCK抑制剂Y-27632补充细胞培养基。</li>
	<li>在 MEF 上培养 iPSC 前 4-5 周，然后在无饲养层条件下（无 MEF 条件），使用 mTeSR1 中感兴趣的包衣之一（参见步骤 1 和 表 1）。</li>
	<li>当 iPSC 汇合 70-80% 时，使用 0.5 mM EDTA 处理在室温下 1:4 传代 3-5 分钟。 为 6 孔板添加 1 mL 的 0.5 mM EDTA（或其他类型的板按比例）。在无饲养器条件下转移到新孔中，并在37°C，5%CO 2，20%O2下孵育。</li>
	<li>每天用新鲜的mTeSR1更换培养基，每2天分裂一次细胞。</li>
</ol>3. 细胞汇合的表征
<ol><li>使用96孔板进行实验。</li>
	<li>在无饲养层条件下培养至少 1 个月后，每孔接种 10，000 个细胞，以确保 MEF 未传代。使用一次性计数玻片用光学显微镜对细胞进行计数。</li>
	<li>一式三份进行实验。因此，在三个孔中测试每个播种条件。</li>
	<li>从接种后的第1天开始，在明场模式下使用细胞仪进行自动图像采集。每24小时进行一次自动图像采集，持续5天。有关实验参数的详细信息，请参阅 补充文件。</li>
	<li>使用自动对比和自动曝光来更好地可视化细胞。</li>
	<li>设置汇合分析的分析设置（参见 补充文件），以应用每孔60%、80%和100%的掩模，以评估由于孔边界的光折射引起的焦点变化。使用上面提到的不同掩模分析设置来分析每个时间点的细胞汇合度。</li>
</ol>4. 统计分析
<ol><li>将定量结果报告为平均值的标准误差 （SEM） ±平均值。</li>
	<li>为了比较不同涂层条件的总体差异，使用相同的样本获取数据并执行学生配对样本t检验。小于 0.05 的 P 值被认为具有统计显著性，并且所有报告的 p 值都是双侧的。</li>
</ol>5. 细胞骨架微丝的表征
<ol><li>用4%多聚甲醛（4%PFA）在PBS中固定细胞在室温下10分钟，然后在PBS中洗涤两次（总共10分钟）。</li>
	<li>在室温下向每个孔中加入 100 μL 封闭溶液（由 5% BSA、0.1% Triton 在 PBS 中组成）1 小时。</li>
	<li>除去封闭溶液并用PBS洗涤样品两次10分钟。</li>
	<li>每个样品加入 100 μL 鬼笔环肽偶联工作溶液，并在室温下孵育 1 小时。</li>
	<li>用PBS洗涤细胞两次（室温下10分钟）。</li>
	<li>用Hoechst 33342染色细胞核，在PBS中稀释1:10000，在室温下10分钟。</li>
	<li>取出Hoechst溶液，用PBS洗涤细胞两次，每次10分钟。</li>
	<li>用H2O洗涤样品，并在化学罩下干燥。</li>
	<li>加入 100 μL 封片剂（即 PBS:甘油，1:1）以覆盖细胞并保留样品荧光。</li>
	<li>在配备白光激光 （WLL） 源和 405 nm 二极管激光器的激光扫描共聚焦显微镜上观察 Ex/Em 493/517 nm 处的细胞。使用HC PLAPO 40x油浸物镜采集连续共聚焦图像。对所有检查样品使用相同的激光功率、分束器、滤光片设置、针孔直径和扫描模式。</li>
</ol>

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作者没有什么可透露的。

使用iPSCs进行疾病建模和未来的药物筛选以及它们在精准医学中的可能应用使其成为一项具有重要意义的技术，因此我们认为有必要清楚地了解更类似于胚胎干细胞生理状况的体外培养条件。在这种情况下，我们使用野生型iPSCs测试了不同的ECM涂层，以了解允许细胞保持健康和未分化状态的条件。除此之外，一个关键点是在MEF和基质胶的异种成分中培养iPSC，这可能解释了一式三份之间的实验变异?...

诱导多能干细胞（iPSC）是从体细胞中获得的，可以分化成不同的细胞类型。它们通常被用作模拟疾病发病机制或进行药物筛选的系统，并且还提供了用于个性化医疗的潜力。由于iPSCs具有巨大的潜力，因此充分表征它们以用作可靠的模型系统非常重要。我们之前展示了在缺氧环境中生长iPSCs的重要性，因为这些细胞依赖于糖酵解，而有氧环境会导致氧化还原失衡1。iPSCs也容易受到其他培养条件的影响，特别是细胞外环境。优化培养条件是保持其健康和增殖的关键问题。健康的iPSC培养将产生健康的分化细胞，这些分化细胞通常是用于了解特定人类疾病或细胞过程的分子，细胞和功能特征的模型的终点。
在这项研究中，已经使用一个简单的方案来测试在不同孔中使用不同包衣条件的iPSCs的汇合度。iPSCs需要鼠胚胎成纤维细胞（MEF）的饲养层才能正确附着，但是iPSCs和MEF的共存使得难以进行RNA或蛋白质提取等分析，因为存在两个细胞群。为了避免饲养层，属于细胞外基质（ECM）的不同蛋白质已被用于重建天然细胞生态位并具有无饲养层iPSC培养物。特别是，Matrigel是从Engelbreth-Holm-Swarm（EHS）小鼠肉瘤中提取的可溶基底膜制剂，其富含细胞外基质蛋白（即层粘连蛋白，胶原蛋白IV，硫酸乙酰肝素蛋白聚糖，entactin/nidogen和生长因子）2，3。其他使用的包被条件是纯化的蛋白质，这些蛋白质在构建ECM中具有已知的相关性:已知层粘连蛋白-521由胚胎内部细胞团中的人多能干细胞（hPSCs）分泌，它是出生后体内最常见的层粘连蛋白之一4，5，6，7，8，9， 10，11;玻连蛋白是一种无异种细胞培养基质，已知可支持hPSC12，13，14，15，16的生长和分化;纤连蛋白是一种ECM蛋白，对脊椎动物发育以及胚胎干细胞在多能状态下的附着和维持很重要17，18，19，20，21，22，23，24，25。由于存在不同的包被条件，我们根据它们对iPSC汇合的影响来比较它们。

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td style="width:281px;">Corning</td>
			<td style="width:77px;">4488</td>
			<td style="width:176px;">Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Tool</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (With Ca2+; Mg2+)</td>
			<td>Thermofisher</td>
			<td>14040133</td>
			<td>Medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (without Ca2+; Mg2+)</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td>Corning</td>
			<td>4487</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture microplate, 96 WELL, PS, F-Bottom</td>
			<td>Greiner Bio One</td>
			<td>655090</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture plate, 6 well</td>
			<td>Costar</td>
			<td>3516</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium- high glucose)</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>ED4SS-500g</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epi Episomal iPSC Reprogramming Kit</td>
			<td>Invitrogen</td>
			<td>A15960</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FAST - READ 102</td>
			<td>Biosigma</td>
			<td>BVS100</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Merck</td>
			<td>FC010</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Reagent</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">H2O</td>
			<td></td>
			<td>MILLIQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Thermofisher</td>
			<td>33342</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin 521</td>
			<td>Stem Cell Technologies</td>
			<td>77003</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>LS25030081</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>Corning Matrigel hESC-Qualified Matrix</td>
			<td>354277</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">Mouse embryonic fibroblasts (MEF)</td>
			<td>Life Technologies</td>
			<td>A24903</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Medium</td>
			<td>Stem Cell Technologies</td>
			<td>85851</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>85852</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vitronectin</td>
			<td>Stem Cell Technologies</td>
			<td>7180</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

measuring the confluence of ipscs using an automated imaging system

本研究的重点是了解在不同ECM包衣底物上生长iPSC如何影响细胞汇合。已经建立了实时评估iPSC汇合的协议，而无需对单细胞悬液中的细胞进行计数，以避免任何生长扰动。使用高内涵图像分析系统以自动方式评估 4 种不同 ECM 上的 iPCS 汇合度。使用不同的分析设置来评估贴壁iPSC的细胞汇合度，并且仅观察到使用60%、80%或100%面膜的微小差异（在层粘连蛋白24和48小时）。我们还表明，与基质胶，玻连蛋白和纤连蛋白相比，层粘连蛋白导致最佳汇合。

在这项研究中，我们研究了在不同包衣条件下生长时的iPSCs汇合。使用细胞仪，我们能够在5天内获得一式三份的简单信息结果。由于iPSCs几乎不附着在塑料容器上，并且需要涂层来支持其增殖，因此我们决定监测人iPSC的汇合，因为它表明细胞培养物的健康状况，并可能反映其分化潜力。体外扩增后，我们将iPSCs接种在不同的ECM底物上，并通过观察在明场中获得的样品图像并使用鬼笔环肽染色（用?...

Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Measuring the Confluence of iPSCs Using an Automated Imaging System

该方案的目标是比较不同的细胞外基质（ECM）包被条件，以评估差异涂层如何影响诱导多能干细胞（iPSC）的生长速率。特别是，我们的目标是创造条件以获得iPSC培养物的最佳生长。

使用自动成像系统测量 iPSC 的汇合度。

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

measuring-the-confluence-of-ipscs-using-an-automated-imaging-system

Research

JoVE Journal

Biology

2.0K 次观看.  Ospedale Pediatrico Bambino Gesù, IRCCS. 该方案的目标是比较不同的细胞外基质（ECM）包被条件，以评估差异涂层如何影响诱导多能干细胞（iPSC）的生长速率。特别是，我们的目标是创造条件以获得iPSC培养物的最佳生长。

视频: Measuring the Confluence of iPSCs Using an Automated Imaging System

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

被截肢者的智能植入种植体设计系统的生物电分析

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

科研

生物学

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

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

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 egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

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

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

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 ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

研究蛋白质的质量控制体系<em&gt;Ð。菌</em&gt;使用温控活细胞成像

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

利用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 对生物发光记者进行灵活测量

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

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 ...

使用自动成像系统测量 iPSC 的汇合度。

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