丹麦独立研究、技术和生产理事会, 赠款 #4005-00029

实验设置没有道德和法律上的限制, 并与罗斯基勒大学进行了协商。在图1和图 2中的光学摄像头的机制中可以看到实验装置的概述。
1. 准备
<ol><li>在实验开始前, 用70% 乙醇清洗工作台和所有设备, 在无菌层流工作台执行所有步骤。</li>
	<li>将光学相机放在传统的孵化器中, 37 摄氏度, 5% CO2 , 以确保细胞的最佳条件。</li>
	<li>在 DMEM 培养基中生长 HaCaT 细胞, 其中有10% 只血清和1x 抗生素 (青霉素和链霉素) 在 T75 瓶中。</li>
	<li>使用1毫升1x 胰蛋白酶分离细胞和分培养他们每3-4 天, 通过分散它在细胞层。在使用前先加热到室温。</li>
</ol>2. 在48井微量滴定板中生成一层 HaCaT 细胞
<ol><li>用巴斯德吸管从 T75 瓶中取出培养基。</li>
	<li>在 T75 瓶中加入5毫升1x 无菌 PBS, 洗涤烧瓶中的黏附细胞。 注:确保 PBS 没有钙和镁。</li>
	<li>分发 PBS 并用巴斯德吸管将其取出。</li>
	<li>重复步骤 2.2-2.3。</li>
	<li>添加1毫升1x 胰蛋白酶到烧瓶, 并分散在细胞层。</li>
	<li>在传统的孵化器中孵化出37摄氏度的烧瓶, 直到细胞分离 (5-8 分钟足够用于大多数细胞系)。</li>
	<li>在显微镜下查看细胞是否分离。一旦细胞分离, 添加9毫升的常规培养基和10% 的血清, 以使1x 胰蛋白酶失效。</li>
	<li>用 hemocytometer、库尔特计数器或类似的计数单元格。 注:台盼蓝可用于查看死细胞。然而, 在1x 胰蛋白酶 HaCaT 细胞是完全圆的, 当活着。</li>
	<li>转移 7.5 x 104细胞/井在250µL 10% 血清介质到一个圆底的48井组织培养板材 (标准板材)。 注: 还可以在6、12、24或96井板中设置化验, 所有这些都可以在光学显微镜下测定。</li>
	<li>轻轻地将盘子从一侧移到一边, 以确保细胞在井中均匀传播, 否则细胞将聚集在水井的中心。在光显微镜下检查以确保电池在介质中均匀传播。</li>
	<li>把盘子放在传统的 CO2孵化器中, 一夜之间37摄氏度, 或者直到细胞粘在井底。</li>
</ol>3. 增加对移徙影响的刺激
<ol><li>检查油井是否在光镜下90-95% 汇合。</li>
	<li>在适量的培养基中制备10µg/毫升丝裂霉素 C。 注:在本研究中, 丝裂霉素 C 在1毫克/毫升的库存溶液中保持在4摄氏度。丝裂霉素 C 在溶液中可以储存至多一个星期在黑暗中2-8 °c。</li>
	<li>用巴斯德吸管从每个井中取出所用的介质。</li>
	<li>添加250µL 5 µg/毫升丝裂霉素 C 的每一个井。</li>
	<li>在37摄氏度和 5% CO2上孵育2小时的板材。</li>
	<li>通过稀释适量的培养基来制备肽的刺激物。 注:在这项研究中, 人和牛乳铁蛋白和乳铁素的测试在25µg/毫升的数量250µL</li>
	<li>用德霉素 C 去除培养基, 用巴斯德吸管取出。</li>
	<li>用250µL 1x PBS (室温) 冲洗井 1x, 用巴斯德吸管去除洗涤液。</li>
	<li>添加250µL 1x PBS 和手动划伤井垂直与200µL 吸管提示。为每个井使用新的吸管尖端。</li>
	<li>取出 PBS 并添加250µL 肽刺激。</li>
	<li>通过调整板两侧的螺钉, 将板安装在光学摄像系统中。把盖子放在微量滴定板上。 注:如有必要, 相机可以在盖子打开的情况下操作。</li>
</ol>4. 为计算机上的光学摄像机设置程序
<ol><li>单击左侧任务栏中的 "获取"。</li>
	<li>选择左侧任务栏中的功能伤口愈合。</li>
	<li>通过点击板块插图, 选择板块类型。</li>
	<li>取消选择未使用的井, 标记它们并单击屏幕左上角的 "启用"。 注:默认情况下, 所有井都被选中。</li>
	<li>用右键单击其中一个井, 调整井上透镜的焦距, 以适应细胞的位置。 注:自动对焦也可以手动调整, 通过移动栏的右侧的板块插图。</li>
	<li>设置图片间隔(如(00:30:00)) 中图像的时间段。</li>
	<li>将重复次数设置为适合所需周期 (例如97 重复)。</li>
	<li>通过单击右下角的 "开始" 按钮开始运行。</li>
</ol>

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作者没有什么可透露的。

移徙在发展、创伤愈合、免疫学和癌症研究中的重要性导致了研究迁移的几种不同方法。迁移可以是由单元格扩展或关闭间隙的表达式。最常见的抓痕的闭合是测试, 因为它是更容易种植细胞在单层, 然后做一个擦伤比生长的细胞在特定的形式8。我们的研究表明, 手工抓伤的优化方法, 因为该技术是易于掌握和低成本。我们介绍了 DNA 合成抑制剂丝裂霉素 C, 一个额外的清洗, 以优?...

迁移是影响几个生理方面的重要过程。在伤口愈合中, 迁移有利于皮肤的重新上皮化。在非愈合伤口, 如慢性伤口, 机会性细菌引起的伤口感染, 开放性伤口是理想的细菌生长和形成生物膜1,2。生物膜是耐药性细菌发展的主要原因之一, 是我国现代社会面临的最大威胁之一3。在伤口愈合过程中, 免疫细胞无法穿透生物膜。在癌症中, 癌细胞的迁移是转移的关键步骤, 这是4、5实体肿瘤患者死亡的主要原因。因此, 能够研究迁移是至关重要的。
擦伤检测通常用于测试细胞迁移, 因为它便宜, 易于执行黏附细胞线, 如成纤维细胞, 内皮, 和上皮细胞线6,7。今天, 有几个方法来执行划痕8, 并报告了不同的材料, 包括牙签9, 电池铲运机10, 激光11, 电流12。所有的方法都有不同的优缺点, 应该根据单元类型、预算和分析工具来确定方法。举例来说, 如果使用的细胞类型需要涂层, 手动减压,如牙签或吸管提示, 将影响在该地区的迁移, 应使用不同的方法。在本研究中, 我们将专注于手工刮擦。
手工刮擦的缺点是大小、划痕的形状和划痕边缘的单元格堆积的变化。许多协议存在, 并且一张高度被引述的纸劝告增加的速度和/或者彻底洗涤在划伤13以后。此外, 还使用了几种方法来最大限度地减少扩散, 因为细胞的增殖不能与移徙区分开来, 因此可能给移徙带来假阳性结果。一些报告的方法是血清饥饿前的14和减少的数量的血清15。然而, 这两种方法都不能确保没有扩散。因此, 我们报告一个前处理细胞丝裂霉素 C, 以确保真正的迁移结果。丝裂霉素 C 是一种抗生素, 它通过与 dna 形成共价键来抑制 dna 合成, 从而防止 dna 分离16。通过对丝裂霉素 C 的预处理, 在刮伤前用 PBS 洗涤, 检测到的间隙闭合显示了细胞的真正迁移 (图 1)。
所有方法的一个特点是需要一个系统来分析迁移17的过程。一个常见的和廉价的方法来分析的进展是使用光场显微镜拍摄的图像的划痕在预先确定的时间点, 然后分析关闭的差距在图像软件18,19。这种方法是费时的, 不可靠的关于拍照在同一地点和焦点, 和运动从孵化器到相机的力量周期的温度变化, 而拍摄。还开发了其他方法, 通过测量电流流量来检测偏移量。目前的变化, 因为细胞覆盖更多的空间在板块上, 这是读取作为增加阻抗20。通过测量阻抗, 细胞的环境不受影响, 因此该方法被认为是无侵入性的。这种方法依赖于专门的板, 金电极, 这是昂贵的, 但它们能够检测到许多过程, 如细胞黏附, 增殖, 迁移和细胞死亡。这种方法的一个很大的缺点是阻抗测量不能直接表示迁移, 因为温度等因素对阻抗有很大的影响。阻抗的变化可能是由软件没有审查的几个因素引起的, 使得对数据的分析更加困难。因此, 缺乏可视化需要常规的光显微镜来验证迁移。
为了克服现有技术的许多缺点, 我们使用实时自动光学相机。光学摄像头是一种检测系统, 具有高吞吐量的显微镜, 在一个小的平台上有透镜, 照明单元, 和数码相机。它有一个内置的软件, 它可以测试迁移和扩散等其他事情。为了检测迁移, 分别用两种算法对细胞的生长和迁移动力学进行了分析, 称为增殖和创面愈合分析。扩散分析是基于两步图像分割算法, 应用纹理分析方法, 通过高级直方图分析来检测细胞覆盖区域 (黄色) 和空背景区域之间的差异。伤口愈合分析是基于三步算法检测细胞单层, 定位伤口间隙, 确定间隙宽度。这些步骤是在用户确定的时间点进行的, 数据显示为覆盖区域、间隙宽度和间隙区域。这台机器的最大优点之一是, 它能够通过液体, 因为相机21的角度来成像粘附细胞。相机 lenstakes 倾斜的图像, 投影到 z 栈的单 z 平面生成, 以确保图片的焦点 (图 2)。z 栈是通过合并的一系列图片 (如40) 垂直于伤口边缘, 并贯穿整个伤口生成。z 栈能够捕捉到细胞层的深度, 以及贯穿伤口的聚焦平面。此外, 该系列图像可以放在一起的图像视频收集, 点击一个按钮。
我们演示了一个优化的协议, 以检测角质形成细胞线 HaCaT 的迁移。我们测试了乳铁蛋白及其 n-端肽, 乳铁素, 从人类和奶牛, 分析其对迁移的影响, 因为这些分子已经证明有许多应用作为抗菌和免疫调节分子22,23. 将四种化合物与未经处理的 HaCaT 细胞进行比较, 并将表皮生长因子 (EGF) 用作阳性对照。

<table border="0" cellpadding="0" cellspacing="0" width="712">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">oCelloScope</td>
			<td style="width:156px;">BioSense Solution ApS</td>
			<td style="width:119px;"></td>
			<td style="width:221px;">Optical camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UniExplorer software&#160;</td>
			<td style="width:156px;">BioSense Solution ApS</td>
			<td style="width:119px;"></td>
			<td>(8.0.0.7144 (RL3))</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HaCaT cell</td>
			<td style="width:156px;"></td>
			<td style="width:119px;"></td>
			<td>Donated from Bispebjerg Hospital</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM (1x) + GlutaMAX</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:119px;">31966-021</td>
			<td>Medium for HaCaT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:119px;">10270</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:119px;">D837</td>
			<td>Without Mg+ and Ca2+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pencillin-Streptomycin</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">P0781</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin (10x)</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">T3924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mitomycin C</td>
			<td style="width:156px;">Tocris</td>
			<td style="width:119px;">3258</td>
			<td>Inhibits proliferation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microtiter plate</td>
			<td style="width:156px;">Greiner Bio-one</td>
			<td style="width:119px;">677 180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator</td>
			<td style="width:156px;">Panasonic</td>
			<td style="width:119px;"></td>
			<td>37&#176;C, 5% CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:156px;">Assistent</td>
			<td style="width:119px;"></td>
			<td>T&#252;rk (0.1 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipet boy</td>
			<td style="width:156px;">Accu-Jet pro</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized scratch assay for in vitro testing of cell migration with an automated optical camera

细胞迁移是一个重要的过程, 影响健康的许多方面, 如伤口愈合和癌症, 因此, 这是至关重要的发展方法, 研究的迁移。由于其成本低、操作简单, 在体外检测化合物的抗性和亲迁移性方面一直是最常用的方法。然而, 一个经常报告的问题的检测是积累的细胞跨越边缘的划痕。此外, 为了从化验中获取数据, 不同曝光的图像必须在同一地点拍摄一段时间, 以比较迁移的运动。不同的分析程序可以用来描述的划痕闭合, 但他们是劳动密集型, 不准确, 和力量循环的温度变化。在这项研究中, 我们展示了一个优化的方法来测试的迁移效果,如自然发生的蛋白质人-和牛乳铁蛋白及其 n-末端肽乳铁素在上皮细胞系 HaCaT。一个关键的优化是清洗和刮在 PBS, 这消除了前面的细胞积累沿边缘。这可以通过去除阳离子来解释, 这已经被证明对角质形成细胞的连接有影响。为确保对迁移的真实检测, 在该协议中加入了丝裂霉素 C (DNA 合成抑制剂) 的预处理。最后, 我们演示了自动光学相机, 它消除了过度的温度循环, 手工劳动与划痕分析, 同时提高重现性和确保分析的相同部分的划痕随着时间的推移。

HaCaT 是角质细胞系, 是皮肤中最丰富的细胞类型之一。当伤口发生时, 皮肤细胞开始增殖并迁移到伤口床上以闭合伤口 (图 3)。因此, 这两个过程对正确愈合至关重要。
光学摄像头可以实时跟踪这两个过程。对于迁移, 系统提供三个数据集: 单元格覆盖区域、间隙宽度和间隙区域。当监测用乳铁蛋白、乳铁?...

Watch this Scientific Journal Video about Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera at JoVE.com

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

在这里, 我们描述了一个程序, 以测试的细胞迁移体外与自动光学相机显微镜。刮伤试验已被广泛使用, 在结束后的划痕是一个设定的周期。光学显微镜能够可靠、廉价地检测细胞的迁移。

用自动光学相机进行细胞迁移的体外试验优化划痕试验

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

optimized-scratch-assay-for-vitro-testing-cell-migration-with-an

Research

JoVE Journal

Biology

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

22.8K 次观看.  Roskilde University. 在这里, 我们描述了一个程序, 以测试的细胞迁移体外与自动光学相机显微镜。刮伤试验已被广泛使用, 在结束后的划痕是一个设定的周期。光学显微镜能够可靠、廉价地检测细胞的迁移。

视频: Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

科研

生物学

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

一个<em在体外 FluoroBlok肿瘤侵袭实验

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

对于侧线再生成年斑马鱼的含量

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

2维直接随机光学重建显微成像中间丝和微管

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill

The ease of genetic manipulation and the strong evolutionary conservation of eukaryotic cellular machinery in the budding yeast Saccharomyces cerevisiae has made it a pre-eminent genetic model organism. However, since efficient protein isolation depends upon optimal disruption of cells, the use of yeast for biochemical analysis of cellular proteins is hampered by its cell wall which is expensive to digest enzymatically (using lyticase or zymolyase), and difficult to disrupt mechanically (using a traditional bead beater, a French press or a coffee grinder) without causing heating of samples, which in turn causes protein denaturation and degradation. Although manual grinding of yeast cells under liquid nitrogen (LN2) using a mortar and pestle avoids overheating of samples, it is labor intensive and subject to variability in cell lysis between operators. For many years, we have been successfully preparing high quality yeast extracts using cryogrinding of cells in an automated freezer mill. The temperature of -196 &#176;C achieved with the use of LN2 protects the biological material from degradation by proteases and nucleases, allowing the retrieval of intact proteins, nucleic acids and other macromolecules. Here we describe this technique in detail for budding yeast cells which involves first freezing a suspension of cells in a lysis buffer through its dropwise addition into LN2 to generate frozen droplets of cells known as "popcorn". This popcorn is then pulverized under LN2 in a freezer mill to generate a frozen "powdered" extract which is thawed slowly and clarified by centrifugation to remove insoluble debris. The resulting extracts are ready for downstream applications, such as protein or nucleic acid purification, proteomic analyses, or co-immunoprecipitation studies. This technique is widely applicable for cell extract preparation from a variety of microorganisms, plant and animal tissues, marine specimens including corals, as well as isolating DNA/RNA from forensic and permafrost fossil specimens.

We describe a reliable method for the preparation of whole cell extracts from yeast or other cells using a cryogenic freezer mill that minimizes degradation and denaturation of proteins. The cell extracts are suitable for purification of functional protein complexes, proteomic analyses, co-immunoprecipitation studies and detection of labile protein modifications.

在自动冷冻机中冷冻处理细胞提取物的制备

The ease of genetic manipulation and the strong evolutionary conservation of eukaryotic cellular machinery in the budding yeast Saccharomyces cerevisiae ...

A Cost Effective and Adaptable Scratch Migration Assay

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as development and mounting an immune response. When a defect or alteration occurs with the cell migration process, it can have detrimental outcomes (i.e., cancer metastasis, wound healing, and scar formation). Due to the importance of cell migration, it is necessary to have access to a cell migration assay that is affordable, adaptable, and repeatable. Utilizing the common scratch migration assay, we have developed a new approach to analyzing cell migration that uses general laboratory equipment. The method described uses visual markers that allow for recapturing specific areas of interest without the use of time-lapse microscopy. In addition, it provides flexibility in the experimental design, ranging from altering the migration matrix substrate to the addition of pharmacological modifiers. Furthermore, this protocol outlines a way to account for the area of cell migration, which is not considered by several methods when examining cell migration. This new approach offers a scratch migration assay to a larger audience and will provide greater opportunity for researchers to examine the physiological and pathophysiological impact of cell migration.

We present a cost-effective method to the scratch migration assay that provides a new approach for determining cell migration without the use of equipment-intensive methods. While fibroblasts were used in this protocol, it can be adapted and utilized to study additional cell types and influences on cell migration.

经济高效且适应性强的划痕迁移检测

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.