澳大利亚核科学与工程学院的支持，是公认的事实。 TCK的是AINSE奖项的收件人。表观医学实验室是支持由国家卫生和澳大利亚的医学研究理事会（566559）。人力资源是由澳大利亚的研究生和BakerIDI明亮的火花奖。生物医学成像发展有限公司（CRC投标），建立和澳大利亚政府的合作研究中心计划下支持，这项工作是由"儿童权利公约"。 CO是收件人澳大利亚研究生奖和一个CRC投标补充奖学金。

1。细胞培养和实验装置<ol><li>人角质形成细胞保持在75厘米的2 - SFM角质形成细胞含有15毫升（K - SFM）的组织培养烧瓶单层培养基（GIBCO公司，无血清培养基）补充L -谷氨酰胺（2毫米），表皮生长因子（5纳克/毫升），牛垂体提取物（40微克/毫升）和20毫克/毫升的庆大霉素。细胞是生长在37℃饱和湿度5％的CO 2环境</li><li>细胞接种到12 × 25厘米2组织培养的K - SFM培养基中含有5毫升烧瓶。 </li></ol>单细胞悬液准备胰蛋白酶。细胞洗净，用磷酸盐缓冲液和0​​.05％胰蛋白酶/ EDTA 5-10分钟的解决方案孵育。当细胞开始变得圆润，3〜30％是分离的贝科的修改鹰含10％胎牛血清的介质卷被添加到中的胰蛋白酶。这些细胞被分离移液器向上和向下（20倍）。使用血球细胞计数。 接种相应的单元格的数字是根据细胞株人类FEP - 1811角质形成细胞（约20小时）的倍增时间。我们的目标是达到实验的日子〜90％汇合（〜10 6细胞每烧瓶） 。 12烧瓶组成的一个实验是为一个单一的克隆形成实验（6个未辐照的控制和六个照射烧瓶），它可以在大约四个小时完成的最佳选择。 2。治疗与辐射<ol><li>一个适当的时候与有关辐射修改复合治疗的细胞和揭露细胞无论是γ-辐射或X射线电离辐射。 </li></ol>通常情况下，六个烧瓶作为电镀效率（治疗）和药物只控制。其他六个烧瓶照射。 在这个例子中，被视为人类角化细胞与不同浓度Cinnulin公积金（CPF;诚信保健品国际，春山，TN，美国为20μg/ mL的下面是有代表性的数据），一种水溶性天然抗氧化剂配方1小时，在37 ° C细胞使用的137 Cs源（1000 Gammacell精英辐照; Nordion公司国际上，加拿大1.6 GY /分钟）与4 Gy的照射。 3。电镀<ol><li>治疗后，获得单细胞悬液，如前面所述。 </li><li>每个样本中的细胞数量是使用血球计数仔细稀释等适当的手机号码接种到培养皿（五个每个复制15毫米的菜肴）。 电镀效率和/或存活分率决定细胞的数量，以每盘种子时，应当预见。目的是实现范围内有20 - 150菌落。 </li>培养皿中被安排在一个加湿塑料克隆盒和5％的CO 2环境在37 ° C的集落形成。 </ol>集落形成的孵化时间，少则1-3周不同的细胞系，它被接受，必须相当于至少6个细胞分裂的时间。在这个例子中，对人类角质形成细胞的控制菜肴需要八天，以形成足够大的克隆，50个或更多的细胞组成。 4。固定和染色殖民地在通风柜完​​成下列步骤。 <ol><li>吸轻轻地从每个板块中删除媒体。 </li><li>用5 mL 0.9％生理盐水清洗每块板。 </li><li>修复与5毫升10％中性缓冲15-30分钟的福尔马林溶液菌落。 </li><li>染色5毫升0.01％（W / V）的水晶紫卫生署2 O为30-60分钟。 </li><li> DH 2 O洗净多余的水晶紫，让菜干。 </li></ol> 5。菌落计数体视显微镜<ol><li>包含超过50个单个细胞的菌落计数使用立体显微镜。 </li></ol>数码成像和点票的使用成像软件<ol><li>殖民地的数字图像获得使用照相机或扫描装置</li><li>菌落计数使用图像分析软件如下所述的软件包。 </li></ol>细胞计数使用ImageJ（斐济版本1.44a） <ol><li>在斐济打开图像文件，转到文件 - &gt;打开。 </li><li>如果需要将图像转换为8位格式，图像 - &gt;调整...-&gt;阈值。 </li><li>调整阈值，以减少非特异性背景水平，使被检测到，只有殖民地。 </li><li>计数菌落使用以下过程 - &gt;二进制 - &gt;查找最大值。 </li></ol>对于这种图像格式，可以设置噪声容限为0。确保浅色背景"选项被勾选预览检测的最大值，以检查所有细胞克隆已正确注册。

<ol>
	<li>Puck, T. T., Marcus, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+x-rays+on+mammalian+cells.">Action of x-rays on mammalian cells.</a> J Exp Med. 103, 653-666 (1956).</li><li>Hurlin, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+human+papillomavirus+type+18-immortalized+human+keratinocytes+to+a+malignant+phenotype.">Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.</a> Proc Natl Acad Sci U S A. 88, 570-574 (1991).</li></ol>

在这个例子中，人类的FEP - 1811角质形成细胞处理不同浓度高达100微克/毫升的中央公积金;数据显示为20微克/毫升中央公积金为1小时37 ° C。治疗后细胞使用的137 Cs源（1000 Gammacell精英辐照; Nordion公司国际上，加拿大1.6 GY /分钟）与4 Gy的照射。对于控制未经处理和药物治疗只有100个细胞接种在每个培养皿中，细胞每盘1000辐照样品镀。正如上述表格中所指出的，使用立体显微镜或数码图像（图1）菌落计数计数使用ImageJ。五菜的平均菌落数来计算电镀效率和存活率。数据表明辐射与天然抗氧化剂配方的保护作用（保护因子〜3）。 代表性的成果<table border="1"><tbody><tr><td> 治疗 </td><td> 计数方法 </td><td> 细胞镀 </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 电镀Efficiency1 </td><td> 尚存 Fraction2 </td></tr><tr><td>未经处理的细胞</td><td>手册 （立体显微镜） </td><td> 100 </td><td> 19 </td><td> 22 </td><td> 38 </td><td> 14 </td><td> 20 </td><td> 0.23 </td><td> 1 </td></tr><tr><td></td><td>手册 （数字图像） </td><td> 100 </td><td> 22 </td><td> 23 </td><td> 34 </td><td> 15 </td><td> 22 </td><td> 0.23 </td><td> 1 </td></tr><tr><td></td><td>寻找千里马 （斐济） </td><td> 100 </td><td> 23 </td><td> 21 </td><td> 33 </td><td> 14 </td><td> 21 </td><td> 0.22 </td><td> 1 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 20 mM的中央公积金</td><td>手册 （立体显微镜） </td><td> 100 </td><td> 18 </td><td> 11 </td><td> 21 </td><td> 15 </td><td> 11 </td><td> 0.15 </td><td> 0.66 </td></tr><tr><td></td><td>手册 （数字图像） </td><td> 100 </td><td> 19 </td><td> 11 </td><td> 23 </td><td> 16 </td><td> 8 </td><td> 0.15 </td><td> 0.67 </td></tr><tr><td></td><td>寻找千里马 （斐济） </td><td> 100 </td><td> 17 </td><td> 11 </td><td> 22 </td><td> 17 </td><td> 8 </td><td> 0.15 </td><td> 0.65 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 4戈瑞</td><td>手册 （立体显微镜） </td><td> 1000 </td><td> 39 </td><td> 27 </td><td> 28 </td><td> 44 </td><td> 28 </td><td> 0.03 </td><td> 0.14 </td></tr><tr><td></td><td>手册 （数字图像） </td><td> 1000 </td><td> 42 </td><td> 33 </td><td> 31 </td><td> 42 </td><td> 31 </td><td> 0.04 </td><td> 0.16 </td></tr><tr><td></td><td>寻找千里马 （斐济） </td><td> 1000 </td><td> 42 </td><td> 35 </td><td> 30 </td><td> 43 </td><td> 31 </td><td> 0.04 </td><td> 0.16 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 20毫米的中央公积金+ 4戈瑞</td><td>手册 （立体显微镜） </td><td> 1000 </td><td> 115 </td><td> 105 </td><td> 98 </td><td> 108 </td><td> 101 </td><td> 0.11 </td><td> 0.47 </td></tr><tr><td></td><td>手册 （数字图像） </td><td> 1000 </td><td> 117 </td><td> 102 </td><td> 104 </td><td> 103 </td><td> 99 </td><td> 0.11 </td><td> 0.45 </td></tr><tr><td></td><td>寻找千里马 （斐济） </td><td> 1000 </td><td> 121 </td><td> 102 </td><td> 104 </td><td> 102 </td><td> 97 </td><td> 0.11 </td><td> 0.47 </td></tr></tbody></table> 1电镀效率=菌落数计算/镀细胞的数量 2存活分率=（计算/细胞镀金数量的殖民地数）/电镀效率电镀效率和生存和菌落计数的比较，使用各种方法计算见表 1。 <img src="/files/ftp_upload/2573/2573fig1.jpg" alt="图1" /> 图1。数字图像显示人类，FEP - 1811，电镀1000细胞的角质形成细胞和8天的潜伏期后产生的殖民地。 （一）细胞与4 Gy的照射和（二）细胞经1小时20微克/毫升中央公积金在37 ° C前4 Gy的照射。天然抗氧化剂配方与辐射保护作用是显而易见的。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Keratinocyte-serum free medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>17005042</td>
<td>Supplemented with 2mM L-glutamine, 20&#x03BC;g/ml gentamicin, epidermal growth factor, bovine pituitary extract.
</td>
</tr>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15400-054</td>
<td>0.05% trypsin / EDTA to detach adherent cells; 10 x stock diluted in PBS w/o Ca2+/Mg2+.</td>
</tr>
<tr>
<td>Dulbecco&#x2019;s modified essential medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>Supplemented with 10% FBS and 20&mu;g/ml gentamicin; used to neutralise trypsin/EDTA.</td>
</tr>
<tr>
<td>0.09% Saline</td>
<td>Solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td> Used to wash colonies.</td>
</tr>
<tr>
<td>10% Neutral buffered formalin solution</td>
<td>Solution</td>
<td>Sigma-Aldrich</td>
<td>HT501128</td>
<td>~4% formaldehyde; used to fix colonies.</td>
</tr>
<tr>
<td>Crystal violet</td>
<td>Powder</td>
<td>SPI-Chem</td>
<td>02577-MB</td>
<td>0.01% crystal violet solution, prepared in dH2O, used to stain colonies.</td>
</tr>
<tr>
<td>Gammacell 1000 elite irradiator</td>
<td>&nbsp;</td>
<td>Nordion International Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes</td>
<td>60 x 15 mm</td>
<td>Falcon</td>
<td>353002</td>
<td>&nbsp;</td>
<tr>
<td>Haemocytometer</td>
<td>&nbsp;</td>
<td>Hawksley; Medical and Laboratory Equipment</td>
<td>AC1000</td>
<td>&nbsp;</td>
<tr>
<td>Cloning box</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Plastic box with holes punched at opposite sides of lid; for storing petri dishes during prolonged incubation for colony formation.</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Type 102</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>Used to count colonies consisting of &gt;50 cells.</td>
</tr>		</tbody>
		</table>
		</div>

clonogenic assay: adherent cells

克隆形成（或菌落形成）检测已建立了超过50年;，原文件，描述该技术是在1956年1出版。除了 ​​记录方法，初步具有里程碑意义的研究产生的X射线照射哺乳动物细胞（HeLa细胞）培养1的辐射剂量反应曲线。从根本上讲，克隆形成实验，使生殖可行性的差异（细胞产生的后代的能力，即一个单细胞，形成了50个或更多细胞的殖民地）的评估，经历了各种治疗方法，如曝光控制未经处理的细胞和细胞之间的电离辐射，各种化学化合物（如细胞毒性药物）或在其他情况下的遗传操纵。该试剂盒已成为辐射生物学中最广泛接受的技术，并已被广泛用于评估不同的细胞系的放射敏感性。此外，克隆形成实验是常用的监测辐射修改化合物的疗效，并为确定集落形成能力，在不同的细胞系，细胞毒性药物和其他抗癌疗法的影响。一个典型的形成群落的生存实验，使用贴壁细胞线涉及到三个不同的组件，1）单层细胞在组织培养瓶中治疗，2）制备单细胞悬液，电镀适当数量的细胞在培养皿中和3），固定和染色菌落相关的潜伏期之后，范围从1-3周，这取决于细胞系。在这里，我们展示了执行与使用同一个永生化人角质形成细胞线（FEP - 1811）2贴壁细胞系的克隆形成实验的一般程序。此外，我们的目的是描述克隆形成实验的共同特征，包括细胞对辐射的暴露后的电镀效率和生存分数的计算，并体现与使用的一种天然抗氧化剂配方辐射响应的修改。

Watch this Scientific Journal Video about Clonogenic Assay: Adherent Cells at JoVE.com

Clonogenic Assay: Adherent Cells

克隆形成生殖可行性的评估检测的适用性已成立超过50年。在这里，我们展示了贴壁细胞进行克隆形成实验的一般程序。

克隆形成含量:贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Nanyang Technological University

Research

JoVE Journal

Biology

47.9K 次观看.  The Alfred Medical Research and Education Precinct. 克隆形成生殖可行性的评估检测的适用性已成立超过50年。在这里，我们展示了贴壁细胞进行克隆形成实验的一般程序。

视频: Clonogenic Assay: Adherent Cells

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

为贴壁细胞培养的氧气插入的制备及操作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

科研

生物学

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

基于一个基质胶管形成实验，以评估对肿瘤细胞的血管生成活性

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

对缝隙连接检查功能测定;贴壁细胞的氧化铟锡电

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...