This work was supported by the Canadian Institutes of Health Research (Grant no 7077), and by the Bellini Foundation and Roby Fondazione.

1.中等和解决方案的准备（所有无菌过滤） <ol><li> C2C12小鼠肌肉细胞（分化的研究） <ol><li>制成500毫升之生长培养基中对C2C12细胞培养维护:补充有10％FBS和2mM L-谷氨酰胺的DMEM高糖。 </li><li>对C2C12分化制备100ml分化培养基（DM）:补充有2％马血清的DMEM高糖。 </li></ol></li><li> HeLa细胞（在有丝分裂细胞研究） <ol><li>制成500毫升之αMEM对于海拉RFP-H2B细胞培养维持和实验:αMEM补充有10％FBS和2mM L-谷氨酰胺（αMEM10％）。 </li><li>制成500毫升之αMEM-减去（无脱氧核糖核苷/核糖核苷），用于海拉RFP-H2B细胞同步:αMEM-减去补充有10％FBS和2mM L-谷氨酰胺（αMEM减10％）。 </li><li>准备20毫升αMEM无酚红的海拉-RFP-H2B升IVE细胞采集:αMEM无酚红补充有10％FBS和2mM L-谷氨酰胺。 </li><li>准备100毫米胸苷:涡旋1毫升水2 O溶解24.2毫克，37℃。过滤消毒，并保持在4℃。 </li></ol></li><li> 共同的解决办法 <ol><li>制备0.05％胰蛋白酶/ EDTA:0.05％胰蛋白酶，0.625毫摩尔EDTA于1×磷酸盐缓冲盐水（PBS）中。过滤消毒和储存等分在-20℃。一旦解冻，等分保持在4℃。 </li><li>制备HBS2x:280毫摩尔NaCl，50毫米的HEPES，1.5毫摩尔的 Na 2 HPO 4。调节pH恰好之间7.01-7.05用10N的NaOH。过滤消毒，并保持在4℃。 </li></ol></li></ol> 2.海拉-RFP-H2B细胞纤维连接蛋白和电镀细胞培养板的涂层注意:在此之前的实验中，每个操作器应设置的最佳细胞电镀条件的细胞达到一个适当的密度，因为变化可以OC每个机械手和每个不同的细胞线之间CUR。 <ol><li>在实验的前一天，展开的HeLa-RFP-H2B细胞，以确保它们的增长上电镀的天指数期之内。使10厘米板块连续2 1/3稀释从80％汇合1×10厘米板开始。 注:计划在实验板的数量。计算每个条件为重复，一个用于短期活细胞成像，一个用于蛋白提取物，以确定击倒和外源蛋白的表达通过Western印迹分析的效力。计划一个附加板，以确定前，病毒转导的细胞数。 </li><li>事先用10微克/毫升纤连蛋白与细胞电镀，涂层玻璃底菜肴。 1ml的1加100稀释的1mg / ml的纤连蛋白？35毫米（稀释在无菌1×PBS中）菜以及覆盖整个表面，并在37℃，5％的CO 2下孵育1小时。 </li><li>在培育期间，预温αMEM补充的Wi第10％FBS（αMEM10％）和0.05％胰蛋白酶/ EDTA中，在37℃。 </li><li>孵育45分钟后，开始准备的细胞溶液。在无菌罩，吸从10cm平板培养基中，用1.5ml 0.05％胰蛋白酶/ EDTA轻轻漂洗两​​次，并留下0.5毫升胰蛋白酶/ EDTA的在最后愿望。 </li><li>孵育2-3分钟将细胞在37℃，5％ 的 CO 2。轻轻拍打在显微镜下板和观察，验证所有细胞已被分离。加入10毫升αMEM10％和吸管轻轻几次以分离细胞。算使用血球细胞悬液的10微升样品。 </li><li>从玻璃底菜吸纤维连接蛋白的解决方案。不要让纤连蛋白细胞接种之前干。 </li><li>板1.5×10每35mm培养皿5个细胞于2mlαMEM10％，并在37℃，5％的CO 2下孵育。后在显微镜下30-45分钟验证细胞以及国家环保总局额定的，因为它们在板的中心积聚的倾向。 </li><li>如果有必要，搅动板轻轻交叉方式重新分配细胞。板一次35毫米的钢板附加一些条件才能算之前，病毒转导的细胞数量。 </li><li>生长细胞直到第二天，以达到至少50％的细胞密度。的细胞密度在病毒转导效率显著降低低于50％的结果，增加每细胞的蛋白表达的变化，以及增加的细胞毒性。 注:使用纤连蛋白的玻璃涂层的菜肴改善细胞的生长和形态，促进有丝分裂细胞的正常发展。它通常可通过市售的明胶是便宜更换。 </li></ol> 3.腺病毒转导和内源蛋白通过siRNA转染在HeLa-RFP-H2B细胞击倒使用CAPI沉淀 注意！工作与病毒ING需要特殊的预防措施，并且一直在与病毒接触的所有材料的妥善处置。 注意！在我们的手中，CAPI沉淀往往有更多的不良影响，例如对涉及囊泡运输（ 例如 ，自噬）生物过程。因此，建议使用阳离子脂质转染试剂（见下文）和分析前等待至少48小时。 注:对照腺病毒携带不相关的基因（ 即，LacZ基因）或没有基因被用来达到使用重组腺病毒携带目的基因的最低量的最小的MOI在所有Adenofections（10-20 PFU /细胞）。 注意:此过程已被证明有助于在大细胞群体每个细胞正火表达。 <ol><li>细胞铺板后一天，计数细胞从额外镀盘的数目。吸出介质并用1ml 0.05％胰蛋白酶/ EDTA冲洗一次。加再次1毫升0.05％胰蛋白酶/ EDTA中，并在37℃，5％CO 2孵育，直到所有的细胞脱离。分离细胞使用1ml的吸管孔和计数使用血球每毫升的细胞数。 </li><li>确定转导的细胞所需的病毒的量在感染复数的兴趣点（POI）的蛋白质的每单元2噬斑形成单位（PFU），并每一个空载体的细胞18 PFU（MOI）（ 例如，LacZ基因）有一个总每个细胞20 PFU的。同时转导各杆状病毒（肌动蛋白和αTubulin，GF​​P和RFP标记分别）的4 PFU。使用以下adenofection协议转导的细胞。如在Fuchs 等人所述，使用病毒。14 </li><li>在无菌罩准备1.5毫升的塑料管各自的条件，并添加400微升温暖αMEM减10％。 </li><li>在冰上解冻的病毒的等分试样慢慢和，如果需要的话，稀释吨他病毒的股票，以吸取体积大于1微升，以尽量减少移液错误。 </li><li>每个条件计算出的病毒量加入含400微升αMEM减10％，每次1.5毫升的塑料管中，吹打轻轻拌匀。放置病毒库存立即回在-80℃，以保持它的活性。 </li><li>吸从细胞培养基中并轻轻吸取病毒混合逐滴。孵育细胞在37℃，5％的CO 2，并仔细搅动板无菌罩下每15分钟1小时，以及覆盖该病毒的细胞。使用介质的少量促进病毒与细胞的接触。 </li><li>孵育后，轻轻吸取1.6毫升αMEM减10％至各板，以获得为2ml的总体积，加入2毫胸苷开始单元同步，并孵育另外2小时的细胞在37℃，5％CO 2的。 </li><li>同时，准备转染混合followi纳克的CAPI转染方法（ 见图1）。如果没有的siRNA是必要的，通过无菌水取代的siRNA的量来执行空转染 。 </li><li>计算200微升混合物为每个条件，导致400微升每重复。下面的例子给出了为50nM的终siRNA的浓度，并以适于每个感兴趣的蛋白质。在1.5ml的塑料管，移液管加入50μl1M的氯化钙2和11微升20μM的siRNA导入139微升无菌的 H 2 O和通过涡旋混合。一个快速旋转后，小心加入逐滴加入200μlHBS2x（280毫米氯化钠，50毫米的HEPES，1.5毫米的 Na 2 HPO 4，pH值7.01-7.05）。 注:较小的液​​滴越小是析出物，从而得到更好的转染效率。轻轻空气喷射用200μl移液器混合三次。 </li><li>在室温下孵育30分钟的混合。缓慢加入逐滴加入200μl转染混合物到每个板搅拌交叉明智的。传送板在37℃，5％CO 2的16小时。 </li><li>第二天，冲洗细胞两次用2毫升温的HEPES（6.7毫米氯化钾，150mM的氯化钠，10mM的HEPES，pH值7.3）和加2mlαMEM减10％。具有四个以上的细胞板在一时间不继续进行，因为在温度变化影响的细胞周期的长度。 </li><li>可视化倒置荧光显微镜下的感染效率在20-40x（空气）的放大倍数，并在文档荧光和传输信道获取每个条件三个有代表性的图像。七小时后，在37℃，5％CO 2的添加2mM的胸苷并孵育另外16小时。 </li><li>第二天，转染和病毒感染后48小时，冲洗细胞两次用2毫升温的磷酸盐缓冲盐水（PBS），并在2毫升αMEM10％释放7小时的w / o酚红对活细胞成像或酚红蛋白的提取。 </li><li>收获细胞每个条件48小时转染后用于制备蛋白质提取物和Western印迹分析，以确定击倒的效率和内源性蛋白15的表达。 注意:使用抗体对感兴趣的蛋白质以及作为装载对照适当抗体。击倒的效率应通过加载控制细胞裂解物减少量（与对照siRNA转染的）来确定，以提供一个滴定曲线（即 1，½，¼，⅛）。 </li></ol> 4.腺病毒转导和内源蛋白通过siRNA转染中使用阳离子脂质转染试剂HeLa细胞击倒注意:在这里，我们提出了适于不涉及细胞同步实验的协议和/或当siRNA转染不能由CAPI的方法进行，例如，以避免在某些细胞林不希望的毒性作用上课。该协议还包括为了在合适的细胞密度工作adenofection后的细胞replating步骤。我们只测试了阳离子脂质转染试剂。 注意！有病毒的工作需要特殊的预防措施，并且一直在与病毒接触的所有材料的妥善处置。 <ol><li>板每35mm培养皿1.75×10 5个细胞在2.5mlαMEM10％，并在37℃下孵育，5％ 的 CO 2。板一次35毫米的钢板附加一些条件才能算之前，病毒转导的细胞数量。 </li><li>生长细胞直到第二天，以达到至少50％的细胞密度。在病毒转导效率的显著下降小于50％的结果的细胞密度，增加每细胞的蛋白表达的变化，以及增加的细胞毒性。 </li><li>细胞铺板后一天，计数细胞从额外镀盘的数目。吸气培养基并用1ml 0.05％胰蛋白酶/ EDTA冲洗一次。加再次1毫升0.05％胰蛋白酶/ EDTA中，并在37℃，5％CO 2孵育，直到所有的细胞脱离。单独的细胞以及用1毫升移液管和计数每ml的细胞数。 </li><li>确定病毒的转导的感染复数的兴趣点（POI）的蛋白质的每个细胞20-40空斑形成单位（PFU），并每一个空载体的细胞0-20 PFU（MOI）所需的量（ 例如， LacZ的）有一个总的每个细胞40 PFU的。 </li><li>在无菌罩准备1.5毫升的塑料管各自的条件，并添加400微升温暖αMEM10％。 </li><li>在冰上解冻的病毒的等分试样慢慢和，如果需要的话，稀释的病毒原液以吸取的体积大于1微升，以尽量减少移液误差。 </li><li>每个条件计算出的病毒量加入含400微升αMEM10％，每次1.5毫升的塑料管中，吹打轻轻拌匀。第广场病毒的股票立刻回在-80°C至保持其活性。 </li><li>吸从细胞培养基中并轻轻吸取病毒混合逐滴。孵育细胞在37℃，5％的CO 2，并仔细搅动板无菌罩下每15分钟1小时，以及覆盖该病毒稀释细胞。使用介质的少量促进病毒与细胞的接触。 </li><li>同时，准备下转染法的siRNA转染组合。如果没有的siRNA是必要的，由介质替换的siRNA的量无血清来执行空转染 。 <ol><li>下面的例子给出了为50nM的终siRNA的浓度，并以适于每个感兴趣的蛋白质。对于每个adenofection，准备一份1.5毫升含416纳米的siRNA在150微升培养基无血清（ 例如 ，6.25微升20μM的siRNA + 144微升无血清培养基）和一个1.5毫升塑料管containi塑料管NG 6.25微升阳离子脂质体转染试剂+ 144无血清培养基微升。 </li><li>通过上下吹打数次用200微升吸管混合各塑料管的内容。结合两管的内容和通过上下吹打数次用200微升吸管混合。在室温下孵育5分钟。 </li></ol></li><li>与病毒孵育后，轻轻吸取1.8毫升αMEM10％至各板，以获得2.2毫升总体积，并立即添加慢慢逐滴所述siRNA转染混合物，以每盘和搅拌交叉明智的。传送板在37℃，5％CO 2的24小时。 </li><li>第二天，再培养板上的细胞，从35个毫米的钢板成四个新35毫米板在2.5mlαMEM各占10％和孵育另外24小时，在37℃，5％ 的 CO 2。 </li><li>第二天，转染和病毒感染，由一个板收获细胞用于制备的每个条件后48小时蛋白质提取物和Western印迹分析，以确定击倒的效率和内源性蛋白15的表达。 注意:对于其余板块，继续进行细胞固定，使用所选的协议，并接受了样品与关注14抗体免疫荧光分析。 </li></ol>有丝分裂细胞和数据分析的5活细胞成像<ol><li>用配有加湿/ 5％CO 2 /调温室的倒置显微镜上执行分裂细胞短期活细胞成像实验。 注意:在此研究中，使用了旋转盘共聚焦显微镜（40X，0.75 NA），配备有EMCCD冷却电荷耦合照相机在-50℃下。 </li><li>到收购前，请检查室已经达到37℃，适宜的温度。 注意:这可能需要几个小时，这取决于微观系统。 </li><li>放置在培养皿中的MICR的Oscope腔室采集前1小时，以使媒体的适当的平衡，并避免焦点因温度变化漂流。监测细胞的有丝分裂的状态。此时，该细胞的10-15％应在有丝分裂（前期-前中期）的早期阶段。 </li><li>在平衡时间，如在现有实验确定设置的采集参数。典型地，曝光时间为.2-3秒的两个通道（488和594）为100％的激光强度和121-130灵敏度是在我们手中合适的参数。 注意:首先测试应确定最小激光强度和采集时间/间隔导致与最小光漂白的适当的分辨率，导致细胞损伤和扰动丝分裂进程。 </li><li>选择每种条件几个字段来获得细胞的显著数不超过采集间隔来分析。使用旋转盘共聚焦系统，一个典型的安装将在CLUDE四种不同的条件下，每板7场和2个颜色通道（488和594）与1.5-2分钟的时间间隔超过75分钟周期。 </li><li>选择是在进入有丝分裂的细胞，重新设置，一旦所有字段已被选择的焦点并尽可能快地开始采集。 </li><li>监视系统的稳定性为至少三个时间点，并在必要重新设置的焦点。 </li><li>的75分钟的第一个短线活细胞成像后，有丝分裂细胞的新字段可被选择以获得第二组的电影的增加所分析细胞的数量。 </li><li>确定良好定义的标准来分析所使用的细胞的有丝分裂的表型，这取决于荧光标记。在有丝分裂的缺陷可能包括延长在有丝分裂所花费的时间（从核击穿，直到后期），染色体错位，主轴摇摆，和皮层起泡14。 </li></ol> 6. LifeAct-TagGFP2腺病毒转导Differentiatin摹C2C12成肌细胞鼠注:adenofection协议也适用于难以感染小鼠C2C12细胞进行分化。 <ol><li>板2×10 5的C2C12细胞35毫米培养皿中生长培养基上的塑料或选择的基片上。 注:细胞分化是在gelatine-或基质胶涂层的菜肴改善。计划一个附加板，以确定前，病毒转导的细胞数。 </li><li>次日，将细胞应已达到会合的80％。用温热的PBS洗涤细胞两次，并加入2ml的分化培养基（DM）的诱导成肌细胞分化。 </li><li>第二天，标记为分化的第1天（D1），转导腺病毒LifeAct-TagGFP2肌细胞可视化在活细胞肌动蛋白细胞骨架。根据3.2的描述从附加板计数细胞数。计算考试下面LifeAct-TagGFP2腺病毒5 PFU /细胞和45 PFU /细胞AdLacZPLE 表1中描述，总病毒量是50 PFU /细胞。描述14用于病毒</li><li>在无菌罩准备1.5毫升的塑料管各自的条件，并添加400微升温暖的DM。 </li><li>在冰上解冻的病毒的等分试样慢慢和，如果需要的话，稀释的病毒原液以吸取的体积大于1微升，以尽量减少移液误差。 </li><li>每种条件的病毒颗粒的适量添加到含有400微升的DM每次1.5毫升的塑料管中并通过移液轻轻混匀。放置病毒库存立即回在-80℃，以保持它的活性。 </li><li>吸从菜介质，轻轻吸管混合病毒滴。孵育细胞在37℃，5％的CO 2，并仔细搅动板无菌罩下每15分钟，共1小时，以良好覆盖的病毒的细胞。 </li><li>孵育后，轻轻吸取1.6毫升DM到每个板，并继续我在37℃下ncubation另外2小时，5％ 的 CO 2。 </li><li>同时，准备继CAPI转染法空染混合物。计算每个条件200微升组合。使用下面的示例为400μl的混合物的总体积。 <ol><li>在1.5ml的塑料管，移液管加入50μl的1M的CaCl 2到150微升无菌的 H 2 O和通过涡旋混合。一个快速旋转后，小心加入逐滴加入200μlHBS2x（280毫米氯化钠，50毫米的HEPES，1.5毫米的 Na 2 HPO 4，pH值7.01-7.05）。轻轻空气喷射三次用200μl移液器混匀。 </li></ol></li><li>在室温下孵育30分钟的混合。慢慢地加入逐滴加入200μl转染混合物到每个板块和搅拌交叉明智的。传送板在37℃，5％CO 2的16小时。 </li><li>第二天，冲洗细胞两次用2毫升温的HEPES（6.7毫米氯化钾，150mM的氯化钠，10mM的HEPES，pH值7.3）和加2ml DM。可视化INFE在20-40X（空气）的放大倍数倒置荧光显微镜下ction效率，并在文档荧光和传输信道获取每个条件三个有代表性的图像。 </li><li>根据所需的实验设置，请分化成肌管了好几天。细胞可以是固定的，并随后进行免疫荧光分析或活细胞成像的研究可以进行。 </li></ol>

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The authors have nothing to disclose.

在这里，我们描述的方法使得能够进行耗尽救援实验，这是适用于那些对影响的化学计量和蛋白质复合物和大分子结构的动力学的蛋白质的过表达特别敏感细胞生物学过程的功能分析。有丝分裂的细胞分裂是涉及在细胞中的整体结构的最引人注目的和引人注目的变化微调细胞地貌的一个极端的例子。使用adenofection用市售BacMam试剂相结合，引进低，但检测的量的肌动蛋白和微管蛋白标志物细胞成像?...

在哺乳动物细胞中基因表达的功能失活的金标准解剖蛋白质的功能。新开发的基础上，利用位点特异性核酸酶如锌指核酸酶的基因组编辑的技术和聚集定期相互间隔短回文重复序列（CRISPR）/ CAS9现在允许的有针对性的基因缺失和突变1,2-细胞系的产生。这些新的方法应该彻底改变我们正在研究蛋白质的功能和我们人类疾病的遗传学的理解方式。在一些情况下，然而，长期或完整基因敲除是不希望的，并可能会引起二次电池的补偿机制。遗传修饰的细胞系的产生，也可与原代细胞培养用有限增殖的能力，或当一大组在各种细胞类型的突变的筛查寻求处理时限制。这经常需要用于确定小区b的依赖性对蛋白质的结构要求iological过程。为此，通过RNA干扰，能够在各种细胞背景短暂枯竭救援实验可逆击倒仍然是一个简单而强大的方式来执行的利率3蛋白的结构与功能分析。但是，这种方法的主要缺点是难以实现高效的沉默和重新引入的在接近生理水平的兴趣或它的变体的蛋白质中的大多数细胞群。这是至关重要的，以使全面研究试图关联在单个细胞（亚效等位基因表型）与那些在小区基于人口的分析可见，例如在蛋白质 - 蛋白质相互作用的水平可见的作用效果。 使用传统的转染方法，人们很难达到一个人口众多的细胞外源性蛋白质的同质和低表达。重组病毒的细胞转导像腺病毒往往使外源蛋白质的更标准化的表达。然而，腺病毒的摄取是由CAR受体，其是在非人类细胞中不存在或仅在某些人类细胞类型弱表达限定。此外，腺病毒的细胞进入激活信令调节细胞形状和粘附4-6通路。研究细胞地貌的调节机制时，这显然是不理想的。当我们进行了伴侣复杂，BAG3-HSPB8的功能分析，在细胞分裂和肌动蛋白，我们正面临着这个问题。开拓性的工作压力7,8期间描述的蛋白质的质量控制和自噬这个伴侣复合作用。大部分这些研究，然而，依赖于蛋白表达，假定分子伴侣被应力过程中通常上调。这已经离开开放是否BAG3的问题，在与HSPB8复杂，可以向表达个分裂的细胞的正常运行ESE伴侣像许多癌细胞类型9。特别是，无论是伴侣复合有助于控制有丝分裂的进展非常感兴趣，因为HSPB伴侣和细胞骨架的动态10之间的连接出现基于肌动蛋白结构的重塑。为了解决这个问题，我们寻求发展为耗尽救援实验一种有效的方法，将不与有丝分裂进展或细胞形态干扰，并且这将保留蛋白质稳态避免大分子复合物的动力学的二次扰动调节细胞形状的改变。因此理想情况下，应同时进行的感兴趣的基因的耗尽加回。 腺病毒的复合物与阳离子聚合物或脂质的使用已被描述为促进在体外和体内 11,12基因转移。例如，磷酸钙（CAPI）出现以形成沉淀腺病毒增强通过CAR-独立通路13病毒绑定条目。事实上，我们发现，基于腺病毒的细胞转导和转染结合的阳离子化合物可增强耗尽救援实验的效率。这允许我们通过3-降低病毒的量至20倍，这取决于从更广泛的窗口的细胞系和感兴趣的基因，并从中受益，以便在大多数附近内源水平来调整外源蛋白的表达的对细胞形态的影响降到最低的兴趣的细胞群。在这种情况下，我们也可以实现内源蛋白质表达（&gt; 75％）的高效率击倒。我们特此通过步骤描述了方法步骤，并提供证据表明，由热休克蛋白家族的应激诱导的分子伴侣的不变的水平所评估蛋白质稳态不显著扰动，使得该方法适于生理RO的功能分析按时间推移视频显微镜分子伴侣乐。该协议是适合于细胞的同步程序，并有丝分裂进展过程中使用可商购的杆状病毒为低水平荧光标记的，与正常的肌动蛋白为基础的和主轴动力学干扰最小的共表达。我们进一步显示的方法中，这是适用于"硬转导"小鼠C2C12细胞，对成肌细胞分化无显著影响到体外肌管的通用性。

<table border="0" cellpadding="0" cellspacing="0" width="1349">
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		<tr height="21">
			<td height="21" style="height:21px;width:523px;">C2C12 Mouse Myoblasts</td>
			<td style="width:331px;">ATCC</td>
			<td style="width:192px;">CRL-1772</td>
			<td style="width:304px;"></td>
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			<td height="21" style="height:21px;width:523px;">Adenovirus custom design</td>
			<td style="width:331px;">Welgen</td>
			<td style="width:192px;">Custom design</td>
			<td></td>
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			<td height="21" style="height:21px;width:523px;">Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:523px;">CellLight&#174; Actin-GFP, BacMam 2.0</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">C10582</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">CellLight&#174; Tubulin-RFP, BacMam 2.0</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">C10614</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM), High Glucose</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">EDTA</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">12483-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Fibronectin</td>
			<td style="width:331px;">Sigma</td>
			<td style="width:192px;">F1141</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Glass bottom dishes, 35mm</td>
			<td style="width:331px;">MatTek Corperation</td>
			<td style="width:192px;">P35G-1.5-20-C Case</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">HeLa-RFP-H2B</td>
			<td style="width:331px;">Kind gift of Dr Sabine Elowe, Qu&#233;bec, Canada</td>
			<td style="width:192px;"></td>
			<td>Klebig C et al. 2009</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">HEPES</td>
			<td style="width:331px;">Fisher Scientific</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Horse Serum, New Zealand</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">16050-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">KCl</td>
			<td style="width:331px;">Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">L-Glutamine</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine&#174; RNAiMAX Transfection Reagent</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">13778-150</td>
			<td></td>
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			<td height="21" style="height:21px;width:523px;">Minimal Essential Medium&#160; (MEM) Alpha&#160;</td>
			<td style="width:331px;">Wisent</td>
			<td style="width:192px;">310-101-CL</td>
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			<td height="42" style="height:42px;width:523px;">Minimal Essential Medium&#160; (MEM) Alpha without Desoxyribonuleosides/Ribonucleosides</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">12000-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Minimal Essential Medium&#160; (MEM) Alpha without Phenol Red</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">41061-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td style="width:331px;">Biobasic</td>
			<td style="width:192px;">S0404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">OptiMEM</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">11058-021</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:523px;">rAVCMV-LifeAct-TagGFP2</td>
			<td style="width:331px;">IBIDI</td>
			<td style="width:192px;">60121</td>
			<td></td>
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			<td height="21" style="height:21px;width:523px;">siRNA duplexes</td>
			<td style="width:331px;">Dharmacon</td>
			<td style="width:192px;">Custom design</td>
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			<td height="21" style="height:21px;width:523px;">Thymidine</td>
			<td style="width:331px;">Sigma</td>
			<td style="width:192px;">T9250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Trypsine 2.5%</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td>15090-046</td>
			<td></td>
		</tr>
	</tbody>
</table>

adenofection: a method for studying the role of molecular chaperones in cellular morphodynamics by depletion-rescue experiments

细胞过程，例如有丝分裂和细胞分化是通过细胞形状的变化在很大程度上依赖于细胞骨架结构的适当改造管辖。这涉及到更高阶的大分子结构的在给定时间和地点的组装拆卸，这个过程是引起的蛋白质的过表达干扰特别敏感。方法，可以保留蛋白质稳态和维持近到正常细胞形态是高度理想的是确定感兴趣的蛋白在广泛的细胞过程的功能的贡献。基于RNA干扰瞬态枯竭救援实验是分析蛋白质的功能和结构要求强大的方法。然而，从它的生理水平最小偏差靶蛋白的重新引入是一个真正的挑战。在这里，我们描述了开发研究的分子伴侣的作用的方法称为adenofection的在分裂细胞的正常d动作伙伴和肌动蛋白重塑的关系。 HeLa细胞耗尽BAG3与siRNA双链体靶向3&#39;UTR区。 GFP标记BAG3蛋白同时再引入使用耦合到转染试剂重组腺病毒的细胞的&gt; 75％。启用Adenofection表示在耗尽BAG3在HeLa细胞中接近生理水平BAG3-GFP蛋白，在不存在应激反应。观察内源性热休克蛋白分子伴侣蛋白动态平衡的主要胁迫诱导监管的水平没有影响。此外，通过添加杆状病毒驱动的荧光标记物的表达在细胞转导转染时，我们可以通过时间推移显微解剖的有丝分裂细胞动力学与正常的有丝分裂进展的最小扰动的分析。 Adenofection也适用于难以感染小鼠细胞，并适合于成肌细胞的diff功能分析erentiation成肌管。因此adenofection提供了一个通用的方法来进行参与依赖于高阶骨架动力学敏感的生物过程的蛋白的结构与功能分析。

使用阳离子脂质BAG3-GFP的质粒DNA的转染HeLa细胞与异质表达相关，一些细胞显示的蛋白质的勉强可检测的水平和其他轴承很高BAG3水平（ 图2A）。在这些细胞中，蛋白稳态损耗由BAG3-GFP的积累成核周聚集体（ 图2A，箭头）证明。与此相反，用携带BAG3-GFP腺细胞转导显示更均匀的低表达和BAG3-GFP的精确定位（ 图2B，单独感染）。值得注意的是...

Watch this Scientific Journal Video about Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments at JoVE.com

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level.

Adenofection:用于研究分子伴侣在细胞地貌被耗尽，救援实验中的角色方法

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell ...

adenofection-method-for-studying-role-molecular-chaperones-cellular

Research

JoVE Journal

Biology

7.6K 次观看.  Centre de recherche sur le cancer de l'Université Laval. We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level. 

视频: Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

科研

生物学

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

龙虎斗动粒蛋白的动态行为与染色体运动，在生活学习有丝分裂关卡<em&gt;果蝇</em&gt;合胞胚胎

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS的/出租车细胞趋化测量

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

一个循序渐进的步骤方法的到Nanodisc脂质颗粒的ABC转运蛋白的重组

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和"剪切环"中的应用:一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...