作者非常感谢，博士:瓦西里Gurchenkov（居里研究所）图像采集和处理在图3和PICT-IBISA的成像设备（居里研究所）。支持这项工作是由ANR-09-JCJC0023 01，ARC SFI20111203863和PIC 在体外细胞模型的3D -复杂。

1。 TAMRA-I型胶原贴标<ol><li>制备10毫克/毫升TAMRA的溶液，加入2.5毫升DMSO中提供的25毫克TAMRA粉末。溶解它通过涡旋，直到完全溶解。储存于-20ºC和避光。 </li><li>准备2 L的标记缓冲液（0.25 M，0.4 M氯化钠的NaHCO 3）。调节pH值至9.5，用10 M的NaOH溶液。保持在4℃从这一点来说，所有的操作都​​在4℃，除非另有说明外，荧光材料从光用铝箔保护。 </li><li>填充高度集中的大鼠尾I型胶原溶液用1毫升1毫升一次性注射器。高浓度的胶原蛋白的解决方案通常是设置在大约10毫克/毫升的浓度，是非常粘稠的。一定要慢慢地操纵它，以避免形成气泡。 </li><li>使用一个21 G注射器针头，注射胶原蛋白预浸3毫升透析盒10000截留分子量切断。谨慎使用，以避免大马动，由膜用针。删除所有空气透析盒拉动注射器的活塞。 1 L的标记缓冲液透析过夜。 </li><li>用900微升标记缓冲液，100微升10毫克/毫升TAMRA溶液混合。注:这应该是做TAMRA解决方案和贴标缓冲器，在室温下，因为DMSO冻结在4℃混合后，带来摊薄TAMRA解决方案4℃ </li><li>小心地取出胶原蛋白，用2毫升的一次性注射器用21 G皮下注射针头从透析盒。用1ml稀释的TAMRA溶液用移液管将1毫升的透析过的胶原蛋白溶液。 </li><li>到2毫升离心管中传输胶原/ TAMRA混合的旋转孵育过夜。 </li><li>成预浸3毫升透析盒TAMRA标记的胶原蛋白的2毫升转移和对1 L的标记缓冲液，以除去过量的游离的染料中透析过夜。 </li><li>要恢复TAMRA实验室ELED胶原蛋白，胶原蛋白原溶液，透析盒放置到1升0.2％（体积/体积）的乙酸溶液中，pH值为4，并透析过夜。 </li><li>测量TAMRA标记的胶原蛋白的最终体积，并计算它的最终浓度，考虑到所用的胶原蛋白溶液的初始体积和浓度。贮存在4℃ </li></ol> 2。 3D TAMRA-胶原嵌入式单细胞矩阵<ol><li>计算2 mg / ml的TAMRA-胶原蛋白混合的实验所需的体积。一定要准备20％以上，考虑到移液损耗，由于胶原蛋白的高粘度。 </li><li>准备股票10X PBS和1 N氢氧化钠溶液。过滤消毒，并保持在4℃从这点来说，所有的操作都​​在冰上进行，除非另有说明，并在无菌条件下。 </li><li>混合10倍的PBS和1N的NaOH在适当体积，以达到预期的胶原蛋白浓度和pH值7.4。例如，对于1毫升的最终体积TAMRA-胶原蛋白在pH 7.4混合，结合用5μl的1N的NaOH100μl的10倍PBS。拌匀。 </li><li>添加适当的两个TAMRA标记的胶原蛋白和未标记的胶原蛋白在1:6的比例，以实现最终的总胶原浓度为2毫克/毫升的体积。例如，对于2 mg / ml的TAMRA-胶原蛋白混合，最终体积为1毫升，添加90.48微升3.68毫克/毫升TAMRA标记的胶原蛋白和4.01毫克/毫升的未标记的胶原蛋白415.71微升。拌匀，慢慢移液，避免形成气泡。 </li><li>添加细胞悬浮在适当体积的不含FBS的冷冻细胞培养基的TAMRA-胶原蛋白混合以获得最终的细胞密度为10 5个细胞/ ml和一个最终的胶原蛋白的浓度为2毫克/毫升。例如，对于1毫升2毫克/毫升TAMRA-胶原蛋白混合，添加388.81微升含有10 5个细胞的培养基。拌匀，慢慢移液，避免形成气泡。确认pH值的pH值试验通过测试10微升混合之旅。 </li><li>移液管加入100μl滴TAMRA-胶原蛋白混合到玻璃底菜，并允许它在室温下放置30-45分钟，以聚合。将100μl的胶原蛋白滴有一个典型的直径为7mm，是2毫米高。胶原蛋白的聚合，变成白色上下的凝胶。注意:允许胶原蛋白的聚合，但不关闭烹调避免形成水膜周围的胶原蛋白的下降，降低玻璃的脱离。为了提高依从性，玻璃菜可以涂有聚-L-赖氨酸在100微克/毫升。 </li><li>小心地将足够的培养基，以弥补胶原蛋白/细胞下降。胶原滴避免高通量媒体或突然运动，因为可以很容易地分离。请在37℃在10％CO 2的加湿空气中足够长的时间（通常为1-3天），在基体中用于细胞迁移。 </li></ol> 3。 3D TAMRA-胶原嵌入式细胞球体的矩阵<ol><li>计算体积的2毫克/毫升TAMRA-胶原蛋白中号㈨实验所必需的。一定要准备20％以上，考虑到移液损耗，由于胶原蛋白的高粘度。 </li><li>准备股票10X PBS和1 N氢氧化钠溶液。过滤消毒，并保持在4℃从这点来说，所有的操作都​​在冰上进行，除非另有说明，并在无菌条件下。 </li><li>混合10倍的PBS，1 N NaOH和在适当的卷不含FBS的细胞培养基，以实现所需的胶原蛋白的浓度和pH值7.4。例如，对于在pH 7.4，最终体积为1毫升的TAMRA-胶原蛋白混合，结合100μl的10倍PBS，5微升的1 N NaOH和388.81微升介质。拌匀。 </li><li>添加适当的两个TAMRA标记的胶原蛋白和未标记的胶原蛋白在1:6的比例，以实现最终的总胶原浓度为2毫克/毫升的体积。例如，对于2 mg / ml的TAMRA-胶原蛋白混合，最终体积为1毫升，加入90.48微升3.68毫克/毫升TAMRA标记的胶原蛋白和415.71微升4.01毫克/毫升UNLA贝莱德胶原蛋白。拌匀，慢慢移液，避免形成气泡。确认测试的pH试纸条上的混合10μl的pH值。 </li><li>吸取100微升滴TAMRA骨胶原混合到玻璃底菜，并允许2-5分钟，引发聚合。这将有助于防止下沉的球体凝胶粘度略有增加。将100μl的胶原蛋白滴有一个典型的直径为7mm，是2毫米高。 </li><li>收集的细胞球体，并把它放在一个干净的培养皿。删除任何多余的液体，悬浮在10微升TAMRA-胶原混合。这是重要的，以防止细胞培养基中的胶原蛋白稀释。 </li><li>使用一个P20吸管，收集胶原蛋白悬浮球体，并把它放在中心顶部的100微升TAMRA-胶原混合降。注意:不要放置多个球体每胶原蛋白的下降，因为它们可以相互影响的入侵和/或迁移能力。 </li><li>允许TAMRA-胶原混合聚merize在室温下放置30-45分钟。胶原蛋白的聚合，变成白色上下的凝胶。注意:允许胶原蛋白的聚合，但不关闭烹调避免形成水膜周围的胶原蛋白的下降，降低玻璃的脱离。为了提高依从性，玻璃菜可以涂有聚-L-赖氨酸在100微克/毫升。 </li><li>小心地将足够的培养基，以弥补胶原/球体滴。胶原滴避免高通量媒体或突然运动，因为可以很容易地分离。 </li><li>细胞入侵/迁移在基体中（通常为1-3天）足够长的时间保持在37℃在10％CO 2的加湿空气。 </li></ol> 4。三维免疫荧光染色<ol><li>小心取出细胞培养基及胶原基质含有细胞/球体用PBS冲洗。 </li><li>同时固定和提取与提取/固定缓冲液（4％PFA，0.3％的Triton X-100，5％蔗糖的PB孵育细胞S）5分钟。补充2μM鬼笔环肽和2μM紫杉醇骨架或细胞骨架相关蛋白可视化的缓冲区。 </li><li>进一步固定细胞，用4％PFA，5％蔗糖磷酸缓冲液30分钟。用0.05％Tween-20的PBS冲洗。 </li><li>准备在PBS的主要抗体解决方案。在室温下至少1小时或4℃过夜孵育初级抗体细胞请一定要补充足够的解决方案，覆盖整个胶原蛋白降。对于一个35毫米的玻璃底菜，长1.5-2毫升的解决方案将是必要的。注意:为了减少体积的抗体溶液，不溶于水的屏障，如圆线的硅脂或硅橡胶环，可放置周围胶原下拉。 </li><li>用0.05％Tween-20的PBS洗3次30分钟。 </li><li>准备Alexa的标记的二抗，Alexa的共轭毒伞和DAPI在PBS的解决方案。孵育细胞与适当的二抗在室温下搅拌2小时。 </li><li>洗3X 30分钟用0.05％Tween-20的PBS。 </li><li>删除多余的液体，补充足够的安装媒体，填补了玻璃底菜底（约500微升），顶部放置一个24毫米盖玻片密封。请勿按压盖玻片，避免压缩胶原蛋白的下降和损坏的三维组织。 </li></ol> 5。样品成​​像 Classic或纺丝盘共聚焦显微镜都可以使用。一个倒置的系统应优先被用来确定从玻璃底部的成像单元的距离。该系统用于这项工作是一个倒立共轭焦配备一个40X/1.3NA和60X/1.4NA的油浸物镜（工作距离200微米和130微米，分别），光度CoolSNAP HQ2摄像头，1392 x 1040的旋转盘成像阵列，6.45 x 6.45微米像素的MetaMorph成像软件和控制。 405纳米，491纳米，561纳米和633纳米激光器通常用于在30-50％的功率，获得1和2的分级×2，以减少曝光时间。该显微镜还配备了一个汞灯泡和过滤器立方体（EXC 400-418纳米; EM 450-465）DAPI，FITC（EXC 478-495 510-555 EM）和德克萨斯红（EXC 560-580; EM 600 -650），用眼片。 <ol><li>使用的荧光灯中，将40倍物镜上面的中间的胶原下降。获得的样品，在xy轴和z轴​​的概述。确保不偏离超过100μm，从凝胶中的xy轴的边缘时，获取的图像，以避免边缘效应。当成像球状体，确保他们是根据客观的工作距离。更改目标（如适用）。 </li><li>切换到聚焦实时成像模式。通过使用在561纳米的激光，以可视化的TAMRA标记的胶原蛋白，从玻璃底部开始确定的z值，当胶原纤维开始出现。这是在基板的底部和z = 0处。 </li><li>使用对焦旋钮，100微米Z = 0。只有在z = 100微米的细胞或以上应进行成像，避免紧张的影响，从硬质玻璃底部。 </li><li>选择单元格图像。优化相机的曝光时间和激光功率为每个波长。典型的DAPI和Alexa-488鬼笔环肽的曝光时间是100-200毫秒之间。抗体染色的曝光时间可能会发生变化。 </li><li>在使用适当的激光共聚焦活模式，以可视化的鬼笔环肽染色，并使用聚焦旋钮，定义z系列间隔设置顶部和底部z值电流。 </li><li>定义的z步长。 40X的目标，用1微米。 60X，使用0.5微米。开始收购。 </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+blind+spot+in+confocal+reflection+microscopy:+the+dependence+of+fiber+brightness+on+fiber+orientation+in+imaging+biopolymer+networks.">A blind spot in confocal reflection microscopy: the dependence of fiber brightness on fiber orientation in imaging biopolymer networks.</a> Biophys. J. 98 (3), L1-L3 (2010).</li><li>Cicchi, R., Vogler, N., Kapsokalyvas, D., Dietzek, B., Popp, J., Pavone, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+molecular+structure+to+tissue+architecture:+collagen+organization+probed+by+SHG+microscopy.">From molecular structure to tissue architecture: collagen organization probed by SHG microscopy.</a> J. Biophotonics. 6 (2), 129-142 (2013).</li></ol>

作者宣称，他们有没有竞争经济利益。

这里描述的协议，我使用TAMRA提供了一个很好的方法，让简单的​​可视化的胶原网络组织，通过使用标准的配备了一个561 nm激光共聚焦显微镜，荧光标记胶原蛋白。这种技术的一个优点，相对于反射率的共聚焦显微镜图像的胶原纤维深入到3D矩阵的能力。纤维反射的强度和对比度显着衰退的深度，由于激光的光吸收和散射。此外，胶原纤维的方向可以是一个主要的障碍时使用反射共焦显微镜中，由于纤维周围50°对齐从成像平面的完全未被发现。检测到荧光标记的纤维相似的亮度，独立于它们的方向20。另一种方法，已越来越多地用于可视化，无论是在体外和体内的胶原纤维束，是由二次谐波根忧思（SHG）。这个过程是基于非中心对称的结构，如胶原蛋白21的二次谐波信号的发射。但是中，SHG需要多光子显微镜，这是不的标准实验室成像设备的一部分。 作为荧光基团TAMRA使用不是唯一的。其他的荧光基团，如CY2，Cy5标记的Alexa Fluor系列，市售的蛋白质标记试剂盒，可用于为用户以最方便的颜色标签的胶原蛋白。使用荧光胶原蛋白的另一个优点是潜在的结合细胞转染与时间推移成像，荧光标记的蛋白质，要密切跟踪细胞 - 基质相互作用或细胞诱导矩阵变形。它也可以被用于在2D实验的薄胶原蛋白涂层的盖玻片。 嵌入细胞在3D矩阵是一个微妙的过程，特别是当使用大球体，因为他们有一种倾向，由于重力下沉。另外，细胞通常吸引到更严厉的环境中，在嵌入区域的三维矩阵​​足够接近的感觉张力增加引起的玻璃的玻璃盖玻片，移动朝刚性2D基板。一个技术诀窍，以帮助防止下沉的大球体是让胶原蛋白的简单聚合（2-5分钟）球体的矩阵下降之前。这将略微增加的凝胶的粘度，增加下沉的时间。然而，这是很好的做法，准备多次重复，以增加成功的可能性。另一方面，重要的是根据客观的工作距离，以保持细胞。较低倍物镜通常有更大的工作距离和可用于整体场图像，例如，比较不同类型的细胞的入侵，从旋转椭球体。当需要更高分辨率的图像，水浸泡的目标，更大的工作距离，大大推荐。 <p c小姑娘=的"jove_content"&gt;在三维基质中的细胞的细胞内蛋白的出版工作已完成由过度表达荧光标记的蛋白质，可能是由于缺乏可靠的免疫荧光染色协议兼容的3D特性在底层。如抗体的无障碍设施的深深嵌入细胞或矩阵（因为许多非特异性抗体可以装点胶原纤维）的高背景染色的问题是有原因的。此协议还介绍了在三维胶原基质的免疫组化。虽然这个协议已经优化骨架或骨架相关蛋白，它也被成功地用于标签粘着斑的标志物，如paxilin，纽zyxin的，在不同的细胞系10。我们不要试图创建一个通用的3D染色协议。在2D中，每种抗体可能需要不同的固定方法和各抗体可能会要求设计一个特定的协议四。该协议是作为出发点，并且它被建议，用户包括鬼笔环肽荧光染色，在基质中的细胞的形状和本地化提供一种思路。 厚胶原基质在体外系统研究细胞迁移的生理ECM是一个很好的简化。虽然它缺乏的生物体组织的化学和物理的复杂性，该系统允许特定的ECM的性能，如孔径大小，弹性和交联的操纵。我们希望这些协议将有助于更好地描述细胞结构的分子组成，定位和功能的多年分析2D和3D细胞行为的改善我们的知识。

细胞迁移的领域已经进入全新的第三维世界冒着。它是最近似于生理的一个，因此，三维（3D）的环境中，直观地研究细胞迁移。然而，由于技术的限制，大多数细胞迁移的研究已被被分析细胞的运动刚性的两维（2D）基板之间，未经处理或涂以适当的细胞外基质（ECM）蛋白。 第一个致力于研究细胞迁移的三维胶原蛋白晶格回去1-3超过20年。然而，仅在过去的5年中，它已成为游走细胞实质上的不同，其形态和模式的迁移取决于基材的维。在2D中，细胞只接触基底腹面使用焦距粘连，从而形成了宽阔平坦的突起（lamellipodia上）与嵌入式的指状突起（丝状伪足），他们的领先优势。这些结构中，一起连接的单元的从前至后缘的应力纤维，被认为是在2D细胞运动的关键。在三维基质中，细胞通常是更细长，与整个细胞表面接触的ECM，许多这些结构的形成和功能的相关性造成了相当大的变化。相反，其他细胞的功能，获得相关的三维偏移，如参与ECM重塑4核变形和结构。 尽管这些已知的形态学改变，以及迁移模式5-7，可取决于对ECM和类型的细胞，嵌入在三维基质中的细胞的结构和功能的分析的差异仍然不寻常的。又浓又密的3D矩阵进行技术困难，如高分辨率的显微成像和不兼容性与大多数站优化的ndard协议为2D的文化，如内源性蛋白免疫荧光标记。此外，因为使用的3D矩阵是一个相对较新的方法，研究人员仍在调查酷似具体在体内的情况，如不同的组织器官或ECM肿瘤组织周围的正常基质结构的最佳条件。在结果中关于不同群体的差异，例如，肿瘤细胞迁移模式或粘着斑的存在，产生了一些争议8。很多努力，最近一直致力于ECM化学性质，孔径，纤维厚度和刚度矩阵方面达成了共识。三维的ECM的许多不同类型的当前使用，不同，从细胞衍生的矩阵市售基质胶，pepsinized牛胶原I或nonpepsinized鼠尾胶原一，这些矩阵中的每一个都具有特定的物理和化学性质，并需要相对德选择矩阵的生理过程正在研究中。此外，孔径和纤维厚度可以依赖于聚合条件下，如pH值和温度9,10。的绑定和距离，可从硬质基材，例如玻璃，也可以改变基体10,11的弹性性能。 本文介绍了编制和成像3D细胞培养的方法，无论是作为单细胞或球体。使肿瘤细胞球粒的方法前面已经描述的，最流行 ​​的是悬滴法12,13和琼脂糖涂层板的方法14。肿瘤细胞迁移作为一个适当的细胞外基质底物，nonpepsinized大鼠尾I型胶原聚合在室温，用2毫克/毫升。 Nonpepsinized酸提取胶原蛋白我从鼠尾保留N和C-末端的端肽，的​​nonhelical部分负责天然胶原intermolec的胶原蛋白分子ular的交联和fibrilar的的稳定性15。总之，这些条件允许的情况下形成的胶原网络10 在体内观察到的那些最相似的。一个详细的协议，以允许可视化的胶原纤维，无论是在固定和活的文化，提供荧光标记胶原蛋白在体外 10，使用5 - （6） -羧基四甲基罗丹明（TAMRA），琥珀酰亚胺基酯。该协议是改编自拜辞等。16,17，用来标记异硫氰酸荧光素可溶性胶原蛋白分子。 ，TAMRA荧光素反应，质子化脂族氨基基团的蛋白质，如上面的N-末端的自由氨基基团，而更重要的是，侧氨基的赖氨酸的氨基的反应性荧光染料。该反应只发生在碱性pH值下，当赖氨酸的氨基质子化的形式。除了TAMRA更稳定，比荧光时间的推移，其发射光谱落在范围橙/红（EX / EM = 555/518纳米），它可以有效地活细胞成像GFP标记的蛋白质相结合。使用与氨基反应性染料的水溶性胶原蛋白标记的分子不影响聚合反应的过程中，还是的密度，微孔尺寸和胶原基质的交联状态10,16,18,19。 此协议还包括一个三维的内源性蛋白，其已被进一步优化标记细胞骨架或细胞骨架相关蛋白的免疫荧光标记的方法。此协议的最终焦点是获得高分辨率图像，利用共聚焦显微镜的3D文化与刚性盖玻片贡献减少胶原基质张力的方法。

<table border="0" cellpadding="0" cellspacing="0" width="603">
	<colgroup>
		<col span="2" />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">TAMRA, SE</td>
			<td style="width:195px;">Invitrogen</td>
			<td style="width:136px;">C-1171</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat tail Collagen High Concentration</td>
			<td>BD Biosciences</td>
			<td>354249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat tail Collagen</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P2141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taxol (Paxitel)</td>
			<td>Sigma</td>
			<td>T7402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-alpha Tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 633 Goat anti-Mouse antibody</td>
			<td>Invitrogen</td>
			<td>A21052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting media</td>
			<td>Fisher Scientific</td>
			<td>106 226 89 </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name of Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis Cassette</td>
			<td>Pierce</td>
			<td>66380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom dishes</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MetaMorph Microscopy Automation &#38; Image Analysis Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris 7.2.3</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

revealing the cytoskeletal organization of invasive cancer cells in 3d

在传统上一直在2D基板研究细胞迁移。然而，它已成为越来越明显的是，需要研究更合适的3D环境，更好地类似于维度的生理过程中细胞迁移。游走细胞实质上的不同，其形态和模式的迁移取决于他们是否正在2D或3D基板上。由于与大多数标准协议，细胞内嵌入3D矩阵的结构和功能分析的技术困难和不兼容性仍然屡见不鲜。本文介绍了编制和成像3D细胞培养的方法，无论是作为单细胞或球体。肿瘤细胞迁移作为一个适当的细胞外基质底物，我们使用nonpepsinized的大鼠尾I型胶原在室温下聚合，荧光标记，以方便使用标准的共聚焦显微镜的可视化。这项工作还包括一个协议传输醇三维​​免疫荧光标记的内源性细胞骨架。使用这些协议，我们希望能有助于更好地描述的分子组成，定位，细胞结构和功能在3D。

TAMRA标记鼠尾I型胶原，使一个简单的编制和可视化三维胶原网络。在室温下缓慢聚合的查询结果发现在体内 10的具有可比性的组织中形成的胶原蛋白网络。 在这里，我们提出了一个协议CT26癌症细胞骨架的免疫荧光染色，球体和单细胞。为了更好地保护细胞骨架，缓冲器增加了未标记的鬼笔环肽和紫杉醇，已知F-肌动蛋白和微管稳定的药物，分别。此外，细胞被同时固定的，提取以除去非聚合的细胞内微管蛋白池可能会干扰单个微管的可视化。这种技术同样可以用于可视化细胞骨架相关的蛋白质。当在3D，CT26细胞提出了一种典型的骨髓间充质的形态，其特征在于由一个细长的细胞体放倒，与F-肌动蛋白细胞突起丰富，类似于丝状伪足和伪足（ 图1和图2）。这个狭长的形态更是明显在试图逃避细胞球体入侵胶原基质（ 图2）细胞。微管使用抗微管蛋白抗体染色显示一个保存完好的组织的微管网络（ 图1和2）。此细胞形状大大不同于在2D基板，CT26细胞里，他们通常有几个前缘与宽阔平坦的大伪足定义的丝状伪足10。 要在这项工作中获得的图像处理，Imaris里被使用。此软件会自动将收购大Z堆栈成3D投影，轻松导航和可视化在所有平面（XY，XZ和YZ）和不同的角度（ 图1，图2和3，orthogona升视图）。使用"剪裁三维"功能，不必要的z平面的感兴趣的区域的上方或下方可以被删除。如在图3中表示的，关键的是从所有平面视图，以确保均匀的细胞的三维分布，分析所收集的Z图库。在这种情况下，CT26细胞生长为球粒似乎入侵广泛的三维胶原基质时，可视化作为最大的xy投影。然而，XZ视图显示所有细胞都入侵限制Z区间相比，球体的体积，建议优先地区入侵。这实际上是在太靠近的菜，细胞快速移动朝向和刚性的2D基板的玻璃底部的旋转椭球体。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/50763/50763fig1.jpg" width="600px" /> 图1。 CT26细胞癌细胞骨架嵌在TAMRA标记的胶原蛋白一上校腺癌CT26细胞作为单细胞被嵌入在2毫克/毫升TAMRA标记的I型胶原（红）。标记微管与微管蛋白抗体（蓝色），鬼笔环肽的可视化的Alexa-488 F-肌动蛋白（绿色）和DAPI进行核染色（青色）染色的培养物的免疫荧光图像。 3D图像对应XY最大投影栈Z-38微米。正交XZ（合并底部）和YZ（右侧的合并）合并的意见。比例尺，20微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50763/50763fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50763/50763fig2.jpg" width="600px" /> 图2。骨架的CT26细胞癌细胞侵入TAMRA标记的胶原蛋白I。CT26细胞生长细胞球体被嵌入在2毫克/毫升TAMRA标记的I型胶原（红）。经过2天的文化，细胞开始vading胶原蛋白的三维矩阵​​，远离小区球体。标记微管与微管蛋白抗体（蓝色），鬼笔环肽的可视化的Alexa-488 F-肌动蛋白（绿色）和DAPI进行核染色（青色）染色的培养物的免疫荧光图像。 3D图像对应XY最大投影栈Z-66微米。正交XZ（合并底部）和YZ（右侧的合并）合并的意见。比例尺，50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50763/50763fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/50763/50763fig3.jpg" width="600px" /> 图3。 CT26细胞入侵的胶原蛋白I是在玻璃上的距离而定。CT26细胞生长细胞球状体和2毫克/毫升胶原蛋白I。嵌入在培养2天之后，细胞显着动议相差的细胞旋转椭球体，而不是由入侵吨3D胶原基质，但2D刚性玻璃上爬行。文化的荧光图像可视化的F-肌动蛋白的Alexa-488的鬼笔环肽染色）3D图像对应XY最大投影栈Z-200微米。）正交XZ视图。比例尺，150微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50763/50763fig3highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D at JoVE.com

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

本文提出了一种荧光标记的胶原蛋白，可以进一步用于修复和实时成像的三维细胞培养的方法。我们还提供了一个可视化优化的协议在3D的环境中培养的细胞内源性细胞骨架蛋白。

在3D揭示侵入性癌症细胞骨架组织

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

revealing-the-cytoskeletal-organization-of-invasive-cancer-cells-in-3d

Nanyang Technological University

Research

JoVE Journal

Medicine

15.6K 次观看.  Institut Curie. 本文提出了一种荧光标记的胶原蛋白，可以进一步用于修复和实时成像的三维细胞培养的方法。我们还提供了一个可视化优化的协议在3D的环境中培养的细胞内源性细胞骨架蛋白。 

视频: Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

科研

医学

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

小鼠模型的非侵入性成像检测和监测卵巢癌复发

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

鸡心脏侵袭试验试验癌细胞的侵袭和潜在的反侵入性化合物的活性

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...