The work was funded by the Fund for Medical Research and Education, Wayne State University, Detroit, MI.

1.细胞培养<ol><li>种子3×10 5的HUVEC细胞在10ml完全生长培养基10的一个75厘米烧瓶中。 </li><li> 的 CO 2孵育细胞在37℃，5％至70-80％汇合。 </li></ol> 2.肾小管形成分析<ol><li>在4℃下解冻要么生长因子降低（GFR）的胞外基质或正常细胞外基质过夜冰上。 注意:为简便起见&#39;细胞外基质&#39;的缘故将从此用于引用两GFR和正常细胞外基质。让他们做好准备的过程是相同的。 </li><li>保持在冰上的96孔培养板，每使用预冷的技巧以及添加50微升冷却的细胞外基质。制备仅含正常细胞外基质5井一式三份。准备只含GFR细胞外基质的5口井的另一个一式三份。 </li><li>孵育96孔板在37℃下30分钟。 </li><li>洗内皮细胞一旦与没有钙和镁（PBS）的磷酸盐缓冲的溶液，并加入1 ml 0.05％胰蛋白酶-EDTA。孵育在37℃下的菜1-5分钟，检查每分钟细胞直至大多数细胞围捕。通过点击一次瓶打跑细胞。 <ol><li>加入5 ml基础培养基（ 即，内皮细胞生长介质中无任何内容（ 例如 ，补充剂）加）有1％胎牛血清（FBS）。收集在烧瓶中的细胞，通过移液并转移到15毫升管。离心细胞，在200×g离心5分钟并弃上清。 </li><li>重悬用2ml基础培养基，使用血球计数细胞，并调整细胞浓度为2-3×10 5个细胞/ ml。 </li></ol></li><li>制备100微升以下的10倍浓度CXCR2抑制剂SB225002的在基础培养基:11μM，5.6μM，1.1μM，0.56μM和0μM（对照）。 </li><li>吸管300微升的HUVEC悬浮液到每5 MICRocentrifuge管。添加抑制剂浓度的33微升（11μM，5.6μM，1.1μM，0.56μM和0μM）成一个管各自含有内皮细胞，和旋涡。 </li><li>吸移管将50μl各细胞悬浮液（1-1.5×10 4个细胞）的细胞外基质的各个孔中的。板各悬浮液一式三份，并在37℃，5％的CO 2孵育4-16小时。 </li><li> 4-16小时后，取4张照片每使用倒置显微镜相机11（原来的放大倍数×100）以及形成的管子。 </li></ol> 3.肾小管破坏试验<ol><li>根据相同的规格制备细胞外基质和板作为管道形成试验（步骤2.1至2.3）。 </li><li>如在步骤中管道形成试验和等分2.4.2以每孔50微升所述2.4-到含有细胞外基质的96孔板制备的HUVEC悬浮液。 注:板足够井小号Ø有每个药物治疗3口井。 </li><li>在37℃下生长的细胞外基质4小时，5％ 的 CO 2。药物处理开始之前拍摄图像。 </li><li>如在管道形成试验中的步骤2.5所述制备CXCR2抑制剂SB225002的2倍的浓度，并添加50微升每含有内皮细胞的96孔板的各浓度。板一式三份每个浓度。 </li><li>于37℃孵育另外2-12小时的板，5％ 的 CO 2。以形成在每个使用以及倒置显微镜照相机（原放大倍数×100）11管的4张图片。 </li></ol> 4.量化数据<ol><li>通过使用显微镜照相机软件测量4个随机显微镜视野管的总管长度评估所述图像中捕获管形成。 </li><li>打开显微镜摄像头软件图像，然后单击"注释和测量9 ;.根据"长度"部分中，选择"简单的线"工具。点击图片，然后绘制沿管长度的线，然后用鼠标点击。 注意:软件会自动计算在像素线的长度，并把数据转换成一个文件。 </li><li>用同样的方法通过选择"简单的线"工具在图像中的所有管系。点击图片，然后绘制沿管长度的线，然后用鼠标点击。 注意:该软件将记录每个线的长度并在屏幕上显示的数据。 </li><li>在图像测量每个管后，点击"导出"，然后选择"长到电子表格"。 注:长度数据将被导出到电子表格。 </li><li>添加所有的管长度从电子表格得到总的管长度。计算和记录管的平均长度。 注:四个重复推荐使用均值和确定的标准偏差。 </li></ol> 5.从细胞外基质培养细胞中恢复<ol><li>培养，并执行一个管形成试验在更大的范围（见部分2），为96孔板将不会产生足够的细胞数。 12孔板中，使用细胞外基质500微升和500微升的HUVEC细胞悬浮液（1.5×10 5个细胞）。 </li><li>孵育4-16小时的细胞，以便形成管。然后，吸出细胞培养基。冲洗细胞用1ml冷的1×PBS中的无钙和镁。 </li><li>加入1ml冰冷PBS-2.5毫摩尔EDTA缓冲液到培养，并保持在冰上10分钟。 </li><li>逐出使用1000微升枪头与尖端切断菜细胞和细胞外基质混合物，然后转移到冷15ml试管，洗井用4ml冰冷的PBS-2.5 1mM EDTA和放入管。 </li><li>雪藏管1-4小时，几次翻转试管，直到所有的外基质溶解。 </li><li>在1,620 xg离心离心10分钟在0℃下</li><li>重悬用1ml冷的PBS中的细胞沉淀在冰上一个1.ml管。 </li><li>离心再次在3000 xg离心在0℃下5分钟，然后丢弃上清液。 </li><li>存放在-80℃的细胞。 </li></ol>

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	<li>Sharma, B., Nawandar, D. M., Nannuru, K. C., Varney, M. L., Singh, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+CXCR2+enhances+chemotherapeutic+response,+inhibits+mammary+tumor+growth,+angiogenesis,+and+lung+metastasis.">Targeting CXCR2 enhances chemotherapeutic response, inhibits mammary tumor growth, angiogenesis, and lung metastasis.</a> Mol Cancer Ther. 12 (5), 799-808 (2013).</li><li>Birbrair, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type-2+pericytes+participate+in+normal+and+tumoral+angiogenesis.">Type-2 pericytes participate in normal and tumoral angiogenesis.</a> Am J Physiol Cell Physiol. 307 (1), 25-38 (2014).</li><li>Ferrara, N., Kerbel, R. 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The authors declare that they have no competing financial interests.

当进行该测定，必须在任何时候都保持在冰上或4℃下的细胞外基质，除非另有规定。如果细胞外基质温热以上4℃，将聚合，并测定将被破坏。同样重要的是保证任何与之接触（ 例如，枪头，板）与胞外基质是预冷却用于上述原因。在每个孔中接种细胞的数量是关键的，过少的细胞不会产生预期的纸幅的对照样品中，过多的细胞会形成大的细胞团或单层和测定将是无效的。在该测定中的成功的另一个重要因素是血管内皮细胞的细胞传代次数。该通道数量始终应为十下，否则可能不会出现强劲的管形成。此外，应该确保所用的细胞培养基还没有过期，或者细胞将是不可行的。 Alternatively，可以分装在介质及其组件，并将它们存储在-20℃保存用于在到期日之后的一段时间。当然，它仍然是优选事先使用介质的有效期限像所有的程序，也有一些缺点，在进行内皮细胞管形成测定。一个主要的问题是，由于有不同类型的内皮细胞和支持矩阵的，测定的结果可能会因该细胞与基质的类型用于上差异很大。所用的内皮细胞（HUVECs，HAECs或HMVECs）是原代细胞，它们是昂贵相比永生化细胞获得，并有可变性。主细胞具有使用有限的通道，并且因此不适合于长期的血管生成的实验14。对于可靠的数据，每个人都应该使用相同类型的分析。与体外测定其它，一个管形成测定的结果应该是CON坚挺体内 ，因为从二维组织培养的控制并人工条件的结果可能不总是在生物体的复杂生物系统反映。同样重要的是要记住，这种类型的测定法可以仅用于演示内皮细胞管的形成，并且不应当被用来测试其它非内皮，管形成细胞。 该测定是量化的化合物的血管生成潜力的比较简单而快速的方法。它也形成了进一步的实验平台，与​​单独，它不会产生关于由该化合物实际上影响了容器形成过程的特定机制的信息。然而，使用不同的细胞外基质的是如何进一步创造研究问题的框架是不常用的一个例子。有研究的化合物的特定的生化机制，包括靶向组分特异性抑制剂的方法血管生成途径。另一方法可以是从血管内皮细胞中提取的RNA，并使用RT-PCR（实时PCR）12来分析基因表达的改变可能导致在测定中观察到的生长行为。这种方法的未来应用包括转染的内皮细胞，以创建用于血管生成途径的具体步骤击倒测定。因此有可能将这种方法用于研究淋巴管途径。通过改变细胞外基质成分，基质和细胞过程中癌症支承血管发生的相互作用，可以进一步阐明。 RNA和蛋白质从这些特定条件下的内皮细胞中提取附加量化的数据对应于成像数据提供。

为了获得养分生存所必需的，恶性肿瘤需要访问病人的血液流。为了得到该访问，肿瘤细胞释放，刺激的新血管的生长的肿瘤的化学信号，从而劫持称为血管2的正常的生理过程。它也可通过经由血管发生该转移（ 即，癌症到其他器官的扩散），可能会发生在血管。因为血管生成的过程是这样的多种癌症的进展至关重要，它是在抗癌治疗的研究3有吸引力的目标。 用来量化血管生成的一种方法是当放置在细胞外基质以测量内皮祖细胞的形成管的能力。由于这些形成管是关键的一步早在血管生成，试验环境条件（ 例如，一个给定的共同存在或缺乏mpound），可以刺激或抑制管道形成提供了深入了解，可以有针对性地抑制血管生成的具体步骤。内皮细胞管形成测定是测量细胞的形成管的能力最广泛使用4 体外方法之一。这是一个简单的分析，需要相对较少的部件和一个短培养周期5。或许最重要的是，然而，就是从这种类型的测定法中获得的数据是量化。 对使用管形成测定法的实例包括从生长在细胞外基质与生长因子降低（GFR）的胞外基质的血管内皮细胞进行比较管的发展。细胞外基质是从肉瘤细胞和其主要部件分离的基底膜样材料是层粘连蛋白，IV型胶原蛋白，生长因子和蛋白多糖。一些化合物对细胞的能力不同的效果的时候，以形成管具有降低的生长因子和降低的硫酸乙酰肝素蛋白多糖（聚糖）的环境。聚糖是细胞外基质的组分其在GFR外基质显著降低。作为一个例子，如果假设是血管生成抑制剂，如SB225002，其阻断CXCR2受体，具有相当弱的中断管形成时聚糖丰富能力，然后在细胞外基质和GFR外基质管生成活性之间的比较是在探索通过聚糖的控制抑制血管生成的可能性重要。 通常用于确定对血管生成化合物的作用的其他测定法体内方法。这种显着的例子是鸡胚尿囊膜（CAM）测定6使用鸡胚，并在体内基底膜利用老鼠堵塞血管生成测定7。尽管体内方法测量在T血管生成HREE尺寸和较有代表性人体相比， 体外管形成测定中，它们遭受需要显著更多时间的缺陷，并且相当多的困难来执行。两者CAM分析和细胞外插头测定至少需要8周做，而在比较，该管形成测定可以在一天完成，它不需要动物的使用。 要注意，本报告中所使用的血管内皮细胞是人脐静脉内皮细胞（HUVEC）是重要的。这些细胞在血管萌芽和血管生长的关键作用，并有足够的类似于内皮细胞中的癌症也可以应用于在体外和体内实验9以评价抗血管生成活性。也可以使用其它细胞如初级微血管内皮细胞。 同样重要的是要注意，除了具有低聚糖的水平，比较正常的细胞外基质时GFR细胞外基质也降低了许多组分的水平。这包括，但不限于:表皮生长因子，IGF-1，PDGF，和TGF-β。那些进行实验研究的血管生成的关系这些化合物的意义可能有兴趣使用GFR细胞外基质。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EGM-2 complete growth medium (EGM-2 bullet kit CC-3162 )</td>
			<td>Fisher</td>
			<td>NC9525043</td>
			<td>HUVEC complete growth medium: EGM-2 base medium mixed with BBE (Bovine Brain Extract), hEGF (Human Epidermal Growth Factor), Hydrocortisone, GA-1000 (Gentamicin, Amphotericin-B), 2% FBS (Fetal Bovine Serum), VEGF (Vascular Endothelial Growth Factor), hFGF-&#946; (Human Fibroblast Growth Factor Beta), R3-IGF-1 (Long R Insulin-like Growth Factor One), Ascorbic Acid, and Heparin.&#160; Aliquot the medium and its components and store them at -20 oC, warm&#160; in 37 oC&#160; before use.</td>
		</tr>
		<tr>
			<td>MATRIGEL MATRIX 10ML Growth Factor Reduced (GFR); 10mL; LDEV-Free</td>
			<td>Fisher</td>
			<td>CB-40230&#160;</td>
			<td>Growth Factor Reduced (GFR) extracellular matrix, keep in&#160;&#160; -20 oC , warm in 4 &#176;C&#160; before use.</td>
		</tr>
		<tr>
			<td>MATRIGEL MATRIX 10ML ; 10mL; LDEV-Free</td>
			<td>Fisher</td>
			<td>CB-40234&#160;</td>
			<td>extracellular matrix, keep in&#160;&#160; -20 oC, warm in 4 &#176;C&#160; before use.</td>
		</tr>
		<tr>
			<td>SB225002</td>
			<td>Fisher</td>
			<td>559405</td>
			<td>CXCR2 inhibitor, keep in&#160;&#160; -20 oC, warm in room temperature&#160; before use.</td>
		</tr>
		<tr>
			<td>Primary human umbilical vein endothelial cells (HUVEC)&#160;</td>
			<td>ScienCell Research Laboratories&#160;</td>
			<td>8000</td>
			<td>culture it according reference&#160; 10.</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%-EDTA phenol red</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>keep in 4 oC, warm&#160; in 37 oC&#160; before use.</td>
		</tr>
		<tr>
			<td>Nikon ECLIPSE Ti</td>
			<td>Nikon</td>
			<td></td>
			<td>Inverted Research Microscope.&#160;</td>
		</tr>
		<tr>
			<td>NIS-Elements AR 4.20.02 64 bit</td>
			<td>Nikon</td>
			<td></td>
			<td>Inverted Research Microscope software</td>
		</tr>
		<tr>
			<td>Graph-pad Prism 5</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td>scientific 2D graphing and&#160;statistics software, to calculate IC50.</td>
		</tr>
	</tbody>
</table>

strategic endothelial cell tube formation assay: comparing extracellular matrix and growth factor reduced extracellular matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

相当多的，健康的内皮管形成可以很容易地进行对比，以抑制血管形成的显微​​图像。健康管形成显示为毛细管状结构（ 图1）的一个有组织的纤维网。相比较而言，抑制血管形成本身表现为散射的细胞（ 图2）。管形成测定数据是通过测量毛细管（ 表1）的总管长度定量。除了总管长度，平均管长度，管的总数，或分支点总数可以被测量。结构管形成产生更大的净管长度不是分散抑制花粉管生长。在图3中 ，剂量反应曲线允许内皮细胞对GFR外基质和CXCR2抑制剂SB225002的实验条件下的IC 50的计算。 对细胞外基质和生长因子降低（GFR）的细胞外基质 图1. 内皮吨UBE形成两个图像显示在细胞外基质（A）和GFR细胞外基质（B）的管形成成功的。管的互连网络清楚地表明，管的生长是在这些内皮健康。比例尺= 10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54074/54074fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54074/54074fig2.jpg" /> 图2. 抑制对细胞外和生长因子降低（GFR）由CXCR2抑制剂SB225002（5.6μM）细胞外基质。管形成两个图像显示管形成已经由一个CXCR2抑制剂SB225002（5.6μM）对细胞外基质（A）和GFR细胞外基质（B）的抑制。在该图像显示看到HUVEC细胞的分离的团块，这些细胞无法以形成必要的相互连接的管中。比例尺= 10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54074/54074fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54074/54074fig3.jpg" /> 上生长因子降低（GFR）的细胞外基质 图3 的剂量反应曲线 ，X轴示出了管的长度与Y轴表示CXCR2抑制剂的浓度。剂量反应曲线表明，对GFR外基质的CXCR2抑制剂的HUVEC反应是剂量依赖性的。的IC 50（半最大抑制ÿ浓度）用软件13和可与细胞外基质的剂量响应曲线进行比较，例如计算。集成电路50是下降到50％的响应的抑制剂的浓度。误差棒代表标准偏差。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 生长因子降低（GFR）的细胞外基质和抑制剂 （4个重复的每个） </td><td> 总管长度平均（像素）（1 像素= 0.34微米） </td><td> 标准偏差 </td><td> 中P值 </td></tr><tr><td> GFR细胞外基质控制</td><td> 2447 </td><td> 168.174 </td><td></td></tr><tr><td> GFR细胞外基质+ 0.56μMSB225002 </td><td> 1422.5 </td><td> 185.4218 </td><td> 0.000395 </td></tr><tr><td>GFR细胞外基质+ 1.1μMSB225002 </td><td> 1004 </td><td> 126.1784 </td><td> 2.14×10 -5 </td></tr><tr><td> GFR细胞外基质+ 5.6μMSB225002 </td><td> 519.25 </td><td> 129.6368 </td><td> 4.19×10 -6 </td></tr><tr><td> GFR细胞外基质+ 11μMSB225002 </td><td> 393.75 </td><td> 212.6857 </td><td> 1.21×10 -5 </td></tr></tbody></table> 表1在不同条件下的量化生长因子降低（GFR）的细胞外基质总管的长度。利用记录在生长于GFR的细胞外基质与不同浓度的CXCR2抑制剂的样品总导管段的表的一部分。数据表明CXCR2抑制剂不抑制管道形成。包括的是例如平均的四次重复，标准偏差，和P值相关联的值。测量像素，显示要导出的数据。集成电路50可以通过使用统计软件13来计算。 （1个像素= 0.34微米）

Watch this Scientific Journal Video about Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix at JoVE.com

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

strategic-endothelial-cell-tube-formation-assay-comparing

Nanyang Technological University

Research

JoVE Journal

Medicine

13.3K 次观看.  Wayne State University School of Medicine. This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

视频: Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

科研

医学

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

基质辅助重塑和修复软骨缺损的自体软骨细胞移植在兔模型

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

注重形成:一个基于Cell的检测，以确定一个基因的致癌性

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

人掌腱膜挛缩的<em&gt;离体</em&gt;文化为肌成纤维细胞的研究和细胞外基质相互作用

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

从人乳腺癌组织及细胞系微球体形成分析

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

在Ciliopathy鼠标突变耳蜗平面 - 细胞极性表型的评价

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage and that carries factor VIII in the circulation. VWF is synthesized by endothelial cells and is either released constitutively into the plasma or is stored in specialized organelles, called Weibel-Palade bodies (WPBs), for on-demand release in response to hemostatic challenge. Procoagulant and proinflammatory stimuli can rapidly induce WPB exocytosis and VWF release. The majority of VWF released by endothelial cells circulates in the plasma; however, a proportion of VWF is anchored to the endothelial cell surface. Under conditions of physiological shear, endothelial-anchored VWF can bind to platelets, forming a VWF-platelet string that may represent the nidus of thrombus formation. A flow chamber system can be used to visually observe the release of VWF from endothelial cells and the subsequent platelet capture in a manner that is reproducible and relevant to the pathophysiology of VWF-mediated thrombus formation. Using this methodology, endothelial cells are cultured in a flow chamber and are subsequently stimulated with secretagogues to induce WPB exocytosis. Washed platelets are then perfused over the activated endothelium. The platelets are activated and subsequently bind to elongated VWF strings in the direction of fluid flow. Using extracellular histones as a procoagulant and proinflammatory stimulus, we observed increased VWF-platelet string formation on histone-treated endothelial cells compared to untreated endothelial cells. This protocol describes a quantitative, visual, and real-time assessment of the activation of VWF-platelet interactions in models of thrombosis and hemostasis.

In this paper, we describe a method to assess endothelial von Willebrand factor release and the subsequent platelet capture under fluid shear stress in response to inflammatory stimuli using an in vitro flow chamber system.

血管性血友病因子-血小板串形成的流室模型研究血管血友病因子的病理生理学

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

白念珠菌实时细胞外通量实时分析的生物能量学研究

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...