Name: how to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 13 min 18 s
Description: Watch this Scientific Journal Video about How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases at JoVE.com

We thank Simon Hayward (Vanderbilt University Medical Center) for the BPH-1 cells; and Thomas Cox and Janine Erler (Biotech Research &amp; Innovation Centre, University of Copenhagen) for their assistance with rheological measurements. MR-T was funded by Worldwide Cancer Research, formerly The Association of International Cancer Research (Grant 08-0803 to JS), The British Embassy Montevideo and Agencia Nacional de Investigacion e Innovacion (UK_RH_2015_1_2 to MR-T). MC was supported by Prostate Cancer UK (Grant S14-017 to JS and GS). KW was funded by The China Scholarship Council. MAM was funded by The Saudi Arabian Cultural Bureau.

1. BM刚度的诱导GLA可诱导（非酶交联） <ol><li>通过在4℃下温育解冻骨髓基质（10毫升）的冷冻小瓶直至小瓶的内容物已成为液体（8-16小时）（在冷室或冰箱在冰上静置）。 注意:如果不使用冷室/冰箱，覆盖整个瓶冰。这将防止BM的原液固化。 </li><li>对于以后的实验，并避免反复冻融循环，从每个新型10 mL容量瓶BM基质准备25×0毫升分装。商店小瓶在-80°C，直到由制造商指定的到期日。当需要时，解冻在4℃下的小瓶静置在冰上2小时。 </li><li>准备冰的表面平整。放置在冰之上的8孔腔室载玻片上涂布过程中保持4℃的温度。在4℃下解冻骨髓基质的小瓶。 注意:一个0.4毫升小瓶BM矩阵足以COAt的整1个以及8商会幻灯片。保持冰雪覆盖的同时处理，以防止在BM矩阵从凝固的小瓶。 </li><li>切断用剪刀200微升枪头的分配端。酷钝端200微升枪头4℃，并将其放置到一个200微升容量移液援助。占用40微升冷BM基质溶液进入枪头，并转移到一个很好的冷藏8孔室载玻片。 注:40微升的BM溶液是足以覆盖0.8 平方厘米的表面积。保持涂布过程中冷却，以避免在BM溶液的凝固枪头。不引入气泡插入BM基质溶液，并确保良好的均匀涂而不在边缘处形成一个可见的弯月面。 </li><li>根据所需井和腔数重复步骤1.4。 </li><li>涂布后，放置在37℃的8孔腔室载玻片30分钟，以促进聚合的BM。非常仔细地关闭大门孵化器，以避免液体成果管理制度不必要的干扰。不要超过30分钟孵育时间，以避免RBM的凝胶脱水。 注意:得到的凝胶是天然重组BM（RBM）。在37℃温育步骤不需要5％的CO 2。然而，为方便起见，执行在设定为37℃和5％的CO 2（和加湿）组织培养箱此步骤。 </li><li>制备在0.2M磷酸缓冲液（pH 7.8）稀释的50mM乙醇醛（GLA）。通过使用50毫升注射器将其通过0.22微米注射器过滤灭菌溶液。 <ol><li>在250微升的50mM的GLA的终体积的交联反应，加25微升的0.5M氰基硼氢化钠或2.5M的氨基胍至100mM的GLA（2×股票）的125微升和100微升0.2M磷酸缓冲液（pH值7.8的）。通过使用50毫升注射器将它们通过0.22微米注射器式滤器消毒原液。 注意:处理氰基硼氢化钠穿着白大褂，手套，面罩及呼吸器在通风橱工作时。 </li></ol></li><li>加入250微升的GLA溶液以覆盖聚合的代rBM凝胶，并在37°C孵育6小时，以产生一种半刚性代rBM凝胶或14小时，以产生一个硬代rBM凝胶。 注意:GLA的体积加入必须覆盖聚合代rBM凝胶，应作相应的调整。如果使用不同的GLA的孵育时间，RBM的凝胶进行分析，以确定成果管理制的刚性折叠式增长（见步骤2.4）。 <ol><li>通过在37℃下培养在250μl的14小时的无菌磷酸盐缓冲盐水（PBS）的天然代rBM凝胶制备阴性对照。 </li><li>制备，其中席夫碱或阿马道里加合物的重排的交联反应过程中形成被抑制两个额外的控制，通过加入50mM的氰基硼氢化钠或250mM的氨基胍。 </li></ol></li><li>准备1M甘氨酸Ë基苯乙基酯（GEE）在PBS中稀释。通过使用50毫升注射器将其通过0.22微米注射器过滤灭菌溶液。 </li><li>指定的培养时间后，小心地取出从交联代rBM凝胶GLA的解决方案，包括从控制原生代rBM凝胶控制代rBM凝胶和PBS抑制剂GLA的解决方案。加入250微升GEE解决所有RBM的凝胶，并在37℃孵育1小时。 注意:这一步淬火交联反应。 </li><li>清洗所有代rBM凝胶的10倍，500微升PBS去除GLA和GEE的所有痕迹。孵育过夜RBM的凝胶在37℃的400微升的PBS，以防止其脱水。 <ol><li>分析AGE的积累，非酶交联和粘弹性能（步骤2.1-2.4）RBM的凝胶。对于他们的粘弹性能的流变分析准备在克隆环RBM的凝胶（步骤2.4）。 </li><li>对于细胞培养，冲洗代rBM凝胶2次500微升文化我接种细胞之前直径（步骤5和6）。轻轻执行洗涤而不枪头触摸凝胶表面。 </li></ol></li></ol> 2.非酶交联和成果管理制度刚性的量化处理与GLA <ol><li>光度分析<ol><li>测量GLA治疗AGE积累并利用光度分析，以确定该美拉德反应的程度上控制代rBM凝胶。 <ol><li>步骤1.11后，从代rBM凝胶移除的PBS在8孔室玻片并添加250微升冰冷却的双蒸水。孵育在4℃下16-24小时，以确保基质被完全液化。 注:在此解决方案代rBM肽含有与自体荧光性年龄。 </li><li>转移液化BM溶液到1.5ml管中，测量使用分光光度计（激发波长= 370纳米;发射波长= 440纳米）的溶液的荧光发射。 </li></ol></li></ol></li><li>溴化氰肽的SDS-PAGE分析<ol><li>解决在聚丙烯酰胺凝胶中的GLA的处理，并控制代rBM凝胶确认GLA已诱导交联和宏观纤维的形成。 <ol><li>离心在步骤2.1.1.2以10,000 xg离心收集，在室温下5分钟的液化BM溶液。 </li><li>制备含有2微克/毫升的溴化氰的乙腈稀释的储备溶液。 注意:始终处理溴化氰在通风橱，而穿着白大褂，手套，面罩和呼吸器。 </li><li>于500μl20mg / ml的溴化氰+ 70％体积/体积的甲酸除去上清液，重新悬浮在BM凝胶沉淀，并在室温下孵育过夜。 </li><li>使用1毫升一次性注射器的再悬浮的BM凝胶粒料与切断3.5 kDa的分子量转移到透析盒。 </li><li>淹没盒到含有500毫升双蒸水和磁搅拌棒的500毫升玻璃烧杯中。放置此上磁力搅拌器和透析过夜，在4℃（16小时）（在冷室），以除去溴化氰和甲酸的所有痕迹。 </li><li>使用1毫升的一次性注射器从盒透析BM溶液转移到1.5ml试管。 </li><li>分析在12％体积/体积的聚丙烯酰胺凝胶10,11- 25微升每种BM样品。 SDS-PAGE后，进行聚丙烯酰胺凝胶12的银染可视化溴化氰基质肽13的电泳图案。 </li></ol></li></ol></li><li>免疫荧光显微镜分析<ol><li>执行的GLA免疫荧光染色处理和控制代rBM凝胶与抗AGE /戊糖，抗IV型胶原和抗层粘连蛋白抗体，随后通过共聚焦显微镜可视化累积的AGEs和IV型胶原/层粘连蛋白纤维结构重排的交联代rBM胶凝13。 注意:一定要用足量的覆盖ENTI再孵育和洗涤不枪头接触RBM的表面时代rBM凝胶。对于通过免疫荧光分析3D腺泡文化的细节见参考文献6和3D腺泡共聚焦显微镜见参考文献14。 <ol><li>洗涤GLA用PBS + 300微升（含有0.1mM 氯化钙和0.5mM MgCl 2的PBS）中5分钟，在RT治疗组和对照代rBM凝胶在8孔室玻片的2倍。 </li><li>取出PBS +再加入4％的300微升W / PBS中稀释+覆盖各代rBM凝胶v多聚甲醛（PFA）。温育30分钟，在室温下固定RBM的组件。 </li><li> w / v的PFA液中取出4％。添加添加300微升75毫摩尔氯化铵 + 0.5mM的MgCl 2溶液和在室温下孵育5分钟（重复5次）以猝灭固定。 </li><li> 130毫摩尔NaCl，7毫摩尔的 Na 2 HPO 4，3.5毫摩尔的NaH:通过在无菌水下述溶液制备免疫缓冲液（IF缓冲液）2 PO 4，7.7毫NaN 3的，0.1％w / v的牛血清白蛋白，0.5％V / V聚乙二醇叔辛基苯基醚和0.05％V / V聚乙二醇山梨糖醇单月桂酸酯。 </li><li>制备的IF通过补充封闭缓冲液的IF，用20％体积/体积山羊血清缓冲器。 </li><li>取出淬火液，加入300微升IF阻止缓冲区RBM的凝胶，以防止非特异性反应。孵育用颤抖的平台上在室温2小时。 </li><li>取出中频封闭缓冲液并在4℃下用初级抗体的300μl的稀释孵育16小时RBM的凝胶中的IF封闭缓冲液（1:500的小鼠抗戊糖单抗; 1/250兔抗IV型胶原的pAb; 1 / 250兔抗层粘连蛋白的A / C PAB）。 注意:超过20小时孵育在4℃可盘活成果管理。 </li><li>除去第一抗体和洗涤3次（每次10分钟），在室温下用300μl的中频缓冲器的摇动平台上。 </li><li>取出IF缓冲区添加二次抗体的300微升（山羊抗 - 兔或抗小鼠IgG [H + L）]用稀释1荧光染料缀合:在中频封闭缓冲液500。在室温下孵育用颤抖的平台上2小时。 </li><li>去除二级抗体并在300μl的中频缓冲器的孵育在室温下10分钟。取出中频缓冲器并在室温下在300微升PBS +的洗涤3×10分钟。 </li><li>修复和淬火的第二时间，上述（步骤2.3.1.2和2.3.1.3）中所述。 </li><li>装入染色代rBM凝胶在安装介质和分析使用啶或共聚焦显微镜主要部件的密集束的形成。 注意:对于通过免疫荧光分析3D腺泡文化的细节见参考文献6和3D腺泡啶和共聚焦显微镜见参考文献14。 </li></ol></li></ol></li><li>流变分析<ol><li>执行处理GLA的流变分析和控制代rBM凝胶来衡量其脆弱性iscoelasticity（刚性）。 <ol><li>设置代rBM凝胶是1mm厚的有直径为8mm的圆形的模具。要做到这一点，将一个克隆环（直径8mm）24孔培养板的孔内，并添加BM按照步骤1.3-1.6中所述制备基质溶液。 注意:用于实验成果管理凝胶的准确概括，对于流变仪分析准备RBM的凝胶需要具有相同的表面面积和厚度在8孔室设立RBM的凝胶。在图3中分析RBM的凝胶分别为1毫米厚和直径8mm。 </li><li>上述（步骤1.8至1.11）所描述的处理RBM的凝胶在用PBS，GLA 6小时和GLA的克隆环设置为14小时。 </li><li>测量直径8毫米代rBM凝胶上流变仪的弹性模量（E）与8毫米的平行板锯齿几何形状，在一定范围的1-3％应变，以1Hz的固定频率振荡，21℃的温度。有关其他详细信息ECM凝胶的流变分析，请参阅参考资料见参考文献13,15,16。 注:E从所得剪切储能模量（G&#39;）通过使用以下等式:E = 2 *的G&#39;测定*（1 + V）其中v是在0.5的泊松比，如在参考13,15描述16。 </li></ol></li></ol></li></ol> 3.文化和正常PEC线的处理，RWPE-1 <ol><li>生长RWPE-1细胞在角质细胞无血清培养基（KSFM）补充有5毫微克/毫升的表皮生长因子（EGF），50微克/ ml牛垂体提取物（BPE）和50U / ml青霉素，50微克/毫升链霉素（完成KSFM）。 注意:为了避免上皮细胞向间质诱导（EMT）样过渡不公开RWPE-1细胞血清。允许完整KSFM在4℃下保存，从取出后达到室温30分钟，并在37℃水浴，因为这将灭活EGF和BPE不热情。 </li><li>吸出完整KSFM从RWPE-1细胞的汇合10cm 2的板，冲洗用5ml预热的PBS并加入5毫升0.05％体积/体积的胰蛋白酶确保所有细胞都覆盖着该溶液中。 <ol><li>放置细胞的设定在5到10分钟，37℃和5％的CO 2（和加湿）标准条件组织培养箱。检查胰蛋白酶消化的程度5分钟后，轻轻敲击培养板分离细胞。 注:因此，建议不要在同时处理两个以上的板块RWPE-1细胞不耐受的胰蛋白酶长时间。同样重要的是解离的所有单元从板以避免克隆选择。 </li></ol></li><li>当所有RWPE-1细胞已经解离，加入5毫升温的PBS含有2％体积/体积的胎牛血清（FCS）以猝灭胰蛋白酶。轻轻吸取上下细胞转移到离心管中之前，打破了细胞聚集体。 </li><li>离心离解细胞在125-150点¯x克，在25℃5分钟，弃去上清液并重新悬浮细胞在5ml完全KSFM的粒料直到获得单细胞悬浮液。 </li><li>转移1毫升重悬细胞到新的管中，可加9个ml完全KSFM中的一个1来传播细胞:5通道稀释用于随后的实验中使用。算使用血球设立腺泡细胞的其余部分（参见第5.1节）。 注意:不要文化RWPE-1细胞以来培养经过长时间的时间超过10代，他们没有形成正确的体系结构腺泡。 </li><li>更换培养基每48小时，以保证EGF和BPE保持活跃。 注:包括该超出48小时的治疗这种媒介的变化。 </li></ol> 4.文化和BPH细胞株的处理，BPH-1 <ol><li>培养BPH-1细胞在RPMI 1640培养基补充有5％体积/体积的FCS，50U / ml青霉素和50μg/ ml链霉素。暖培养基，PBS中，0.25％w / v的使用前胰蛋白酶0.53摩尔EDTA溶液至37℃。 注:细胞也可以在介质用2.5％体积/体积的FCS 8培养。 </li><li>从BPH-1细胞的汇合10cm 2的板吸去培养基和洗涤细胞2×3毫升的PBS以除去培养基血清所有迹线可以猝灭胰蛋白酶反应。 </li><li>吸出PBS并加入3ml胰蛋白酶-EDTA溶液，以覆盖细胞。放置板在设定为5分钟，37℃和5％的CO 2（和加湿）的孵化器。除去胰蛋白酶-EDTA溶液，当细胞呈圆形，但保持附接到所述盘。洗涤细胞用5ml PBS中。 </li><li>除去PBS之后，加入5毫升培养介质并轻轻吸取上下以产生单细胞悬液。转移细胞到15毫升管。 </li><li>取2毫升的细胞悬浮液到一个新的离心管用8ml完全培养基和板的BPH-1细胞ONT5道稀释为后续实验使用:OA 10日1 平方厘米培养板。算使用血球设立腺泡细胞的其余部分（见5.2节）。 注意:保持传代次数的记录，因为旧的BPH-1细胞不形成一个适当的架构腺泡。的通道数超过10个不希望。 </li><li>更换培养基每72小时。 </li></ol>前列腺腺泡对本地和僵硬代rBM 5. 3D文化<ol><li>如果正在使用RWPE-1细胞，以形成腺泡，稀释在步骤3.5中300微升完全KSFM制备5,000个细胞补充有BM的溶液2％体积/体积。 </li><li>如果BPH-1细胞被用于形成腺泡，稀释在步骤4.5中的RPMI 1640培养基以300μl补充有BM的溶液2％体积/体积制备2500细胞。 注意:BPH-1细胞比RWPE-1细胞较大BPH-1细胞，使数下使用后的小路6天，以获得腺泡的类似的分布TURE。 </li><li>轻轻种子细胞上天然和AGE-硬挺代rBM和小心地将培养物在设定在37℃和5％CO 2的培养箱（具有加湿），以确保在井内和每个细胞分裂偶数生长腺泡分布生产一acina。 </li><li>每2天更换用含有2％体积/体积的BM溶液新鲜培养基的培养基，以确保细胞已经所需的正常acina生长因子 动态平衡。 </li><li>监控腺泡形态使用明显微镜13日益增长的文化。 注:在培养3 3d的个体的细胞会形成&gt; 3小区的集群和后，用直径为〜1周前列腺腺泡50μm的将观察到。 </li><li>按照2.3描述了使用特异性细胞基质粘连，细胞间粘连，apico -基底极性和侵袭13的标记抗体进行免疫荧光协议。 <ol><li&gt;使用安装媒体用4&#39;，6-二脒基-2-苯基吲哚（DAPI），或包括一个额外的步骤（2.3.12之后）通过用DAPI孵育5分钟以染色细胞核和洗2×5分钟，用PBS +。 </li></ol></li></ol>在本地和僵硬代rBM 6.前列腺的三​​维培养肿瘤细胞聚集<ol><li>培养PC3细胞在RPMI 1640培养基中含10％v / v的FCS和50U / ml青霉素，50微克/毫升链霉素。使用前温热的培养基，PBS中，0.25％w / v的胰蛋白酶0.53摩尔EDTA溶液至37℃。 </li><li>从PC3细胞的汇合10cm 2的培养皿中吸出培养基并洗涤细胞用3ml PBS中2倍以除去FCS的所有痕迹，可以猝灭胰蛋白酶反应。 </li><li>吸出PBS并加入3ml胰蛋白酶-EDTA溶液，以覆盖细胞和孵育1分钟。 </li><li>当细胞变成圆形，但仍附着于皿中，小心地吸出胰蛋白酶-EDTA溶液，并用3毫升的PBS洗除去胰蛋白酶的所有痕迹。 </li><li>除去PBS之后，加入5毫升培养介质并轻轻吸取上下以产生单细胞悬液。转移细胞到15毫升管。 </li><li>取1毫升的PC3细胞悬浮液到一个新的离心管中，可加9个ml培养介质。板在10cm 2培养皿（1:10稀释）用于随后的实验使用细胞。算使用血球剩余细胞。 </li><li>稀释在步骤6.6制得的RPMI 1640培养基以300μl补充有BM的溶液2％体积/体积，以便在培养物中形成的梯度凝胶2500 PC3细胞。 </li><li>轻轻种子细胞上天然和AGE-硬挺代rBM和小心地将培养到设定在37℃和5％的CO 2（和加湿）培养箱，以确保即使在井生长球状体的分布。 </li><li>更换培养基每72小时。 </li><li>研究僵硬（富AGE-）注重结果的管理效果对前列腺肿瘤细胞的迁移，使用温度使用/ CO 2控制和湿盒17明的视频时间推移显微镜图像PC3细胞。 注意:PC3细胞生长在对本地代rBM链，并且不形成具有内腔腺泡，但如果从左上生长本地代rBM超过72小时，就会形成三维球状体。 </li><li>下列数据采集，手动跟踪PC3细胞，并计算出它们的迁移速度，形状（延伸率）和迁移17-19的持久性。 注:持久性比率= D / T，D =距离从开始到细胞的轨迹，T =细胞的轨迹总长度结束。 </li></ol>

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S., Hall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+breast+and+prostate+tissues+under+compression.">Elastic moduli of breast and prostate tissues under compression.</a> Ultrason Imaging. 20 (4), 260-274 (1998).</li><li>Zhang, M., 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=Quantitative+characterization+of+viscoelastic+properties+of+human+prostate+correlated+with+histology.">Quantitative characterization of viscoelastic properties of human prostate correlated with histology.</a> Ultrasound Med Biol. 34 (7), 1033-1042 (2008).</li><li>Corona, G., 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=Benign+prostatic+hyperplasia:+a+new+metabolic+disease+of+the+aging+male+and+its+correlation+with+sexual+dysfunctions.">Benign prostatic hyperplasia: a new metabolic disease of the aging male and its correlation with sexual dysfunctions.</a> Int J Endocrinol. , (2014).</li></ol>

The authors have nothing to disclose.

一种从纯代rBM凝胶6的MECs的代3D腺体腺泡协议是由加4毫克/毫升I型胶原注重成果的管理矩阵在先前的研究修改。在加入胶原导致RBM的凝胶从175±37增加至1589±380帕斯卡的弹性模量。在刚性这个9.1倍的增加调制的MECs 21的生长，存活，迁移和分化。 （ - - ） - 核糖，以促进已被加入到RBM的凝胶中的I型胶原蛋白的非酶交联的协议是由包括带D的处理步骤再次修改。刚度得到的15倍的增长被发现的MECs致癌转化合作，以促进他们的入侵行为22。 I型胶原成果管理凝胶加式的实验方法有利于的MECs与胶原纤维，其中仅基质之间的物理屏障后，会出现人体组织的直接互动和在BM提供上皮经历蛋白水解降解。通过产生从纯代rBM凝胶中用GLA预处理胸肌3D腺腺泡，当前协议打开研究骨髓刚度本身如何可以触发其侵入行为（ 图3）的方式。 BM刚度在这个协议引起的水平有生理意义。相比，在用PBS（ 表1）处理的代rBM凝胶122±55帕斯卡孵化用50mM GLA 6小时和14小时分别增加纯代rBM凝胶的弹性模量，以175±90和322±160。这了RBM刚性为1.7至3.2倍的增加概括在恶性观察相比正常前列腺或良性前列腺增生的组织23-26刚度2.5至3.4倍。正如最近出版概述13 AGE和成果管理刚度的PEC腺泡的积累引起的形态变化可以从AR统计上显著转变量化ounded到多边形，管腔/总腺泡面积，以及突出的细胞从acina迁移到丰富的AGE-代rBM（ 图3）下降。免疫印迹也可以被用来评估的EMT标志物和收缩行为（ 例如磷酸化肌球蛋白轻链-2，pMLC2 13）在正常与僵硬代rBM生长胸肌（ 图3）（ 例如 ，E-钙粘蛋白13的损失）。使用免疫荧光染色和共聚焦显微镜进一步评价可以应用于显现在BM（ 如层粘连蛋白，胶原蛋白IV和AGE积累13），蜂窝心尖到基底极性（ 例如 EEA1根尖定位:早期内体抗原1;和GM130:130 kDa的顺式高尔基标记13）和粘附分子的细胞型态（ 例如 E-钙粘蛋白定位于细胞-细胞连接13）（ 图3）。 <img aLT ="图3"SRC ="/文件/ ftp_upload / 54230 / 54230fig3.jpg"/&gt; 图3:这里介绍不同的协议概述该图描绘了如何准备和加固重组基底膜（RBM）用乙醇醛（美拉德反应），如何种子细胞上的僵硬代rBM，如何分析僵硬代rBM（美拉德反应），可用于分析由富AGE-代rBM诱导的细胞和分子变化程序的程度。年龄，糖化终产物; BM，基底膜; DAPI，4&#39;，6-二脒基-2-苯基; EEA1，早期内涵体抗原1; GAPDH，甘油醛-3-磷酸脱氢酶; GLA，乙醇醛; GEE，甘氨酸乙酯; GM130，130 kDa的顺式高尔基体标记;对MLC2（Thr18 / Ser19），肌球蛋白轻链-2磷酸化位点18苏氨酸和丝氨酸19;代rBM，重组基底膜; SDS-PAGE十二烷基硫酸钠聚丙烯酰胺凝胶电泳。对于RWPE1腺泡比例尺= 10微米;为PC3肿瘤细胞球体比例尺= 100微米。这个数字已经从13参考修改。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54230/54230fig3large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> （ - ） - - 核糖被选择作为交联剂成果管理制若D的故障排除步骤是必要的。在协议开发后发现，用1M D处理- （ - ） -核糖72小时，如前面对代rBM /胶原凝胶22描述的，导致了RBM凝胶的脱水和收缩。 （ - ） - - 较低浓度的D的评价核糖和较短的处理时间可能有助于克服这个限制。 其中，代rBM刚度更高水平的期望在协议中的未来应用的一个潜在的限制可能会遇到。如果较长的温育时间和GLA的更高浓度来诱导更高水平的代rBM凝胶刚度将是必要<html>评估这些处理条件是否对细胞存活和增殖的影响，如先前所述13。还应当指出的是，与血清RWPE-1细胞的培养诱导应避免表型EMT状过渡和暴露于血清或含血清的材料。例如，如果实验涉及的短干扰RNA（siRNA）的寡核苷酸转染，该过程应使用KSFM生长RWPE-1细胞进行了优化，而不将细胞转换到低血清转染培养基。当在模型中使用的瞬态的siRNA接近沉默实现该缺点可能损害基因的水平。对于一些蛋白质指标，将被建议采用可调谐基因沉默和蛋白水平所需的减少诱导的shRNA载体。通过基质细胞或肿瘤细胞相关的赖氨酰氧化酶（LOX）17结合的酶交联的修改也可以被掺入到未来的模型。 jove_content"&gt;此协议将有利于通过年龄依赖性BM刚度佩奇引发亲创机制（RWPE-1，BPH-1）和侵袭性的PTC（PC3）抗肿瘤转移的目标评估未来的研究。由于前列腺增生被认为是一种代谢紊乱27，该协议也铺平了道路实现我们代谢紊乱和增加的前列腺癌的风险之间的关系的更好的了解。鉴于其暴露于AGE引起的BM刚度可以是用于其它癌症侵袭的触发类型，这将是感兴趣的使用的协议来建立，纳入正常上皮细胞和肿瘤细胞从其他器官（ 如乳腺癌，结肠癌，卵巢，胰腺）类似的模型。 在协议中的关键步骤，与其定时一起，总结于图4。在最初的步骤是，以保持成果管理原液于4℃，同时它解冻，以防止其聚合酶必需ymerization。枪头不应放入RM原液直至它们已被冷却至4℃。用于下一步骤它也是重要的，以确保它们都涂有RBM的溶液前室载玻片已平衡至4℃。只要RBM的溶液的温度高于4℃增加会经历不可逆的聚合以形成凝胶。它必要的rBM未在聚合阶段干扰，以确保它形成均匀的表面适于细胞培养和显微镜分析。与GLA具有或不具有该美拉德反应（氰基硼氢化钠和amingoguanidine）的抑制剂孵育的持续时间将决定RBM的凝胶如何僵硬变。建议如果需要半刚性的条件使用6小时孵育GLA，和14小时孵育如果硬条件是必需的（ 表1）。 GLA的备用孵育时间或浓度可以如不同含量使用刚度是期望的。在这种情况下RBM的凝胶流变分析需要结合的重要步骤。以下淬火用GEE孵育并用PBS随后的洗涤步骤的美拉德反应的步骤中，RBM的凝胶可立即使用或它们的细胞培养使用前储存在4℃下进行最多48小时。一旦细胞培养建立其改变培养基（包括任何处理）每两天是非常重要的。建议根据接受调查的参数保持3D细胞培养3-12天。对于3D PEC腺泡建议进行分析后6天的文化，以及3D PTC球状体分析后，在一审中培养3天后建议。 <img alt="图4" src="/files/ftp_upload/54230/54230fig4.jpg" /> 图4:用关键步骤和时序议定书简单的综述指出的FLOW线图描述了如何准备和加固重组基底膜（RBM）用乙醇醛（美拉德反应）与显示关键步骤和时间。其中，该协议可以被停止，并且存储代rBM凝胶点，也被指示。代rBM，重组基底膜; GLA，乙醇醛; GEE，甘氨酸乙酯; ON，隔夜; PBS，磷酸盐缓冲盐水; RT，室温。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54230/54230fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

The basement membrane (BM) is a sheet of specialized extracellular matrix (ECM) that maintains stable tissue borders by separating layers of epithelial cells from the stroma1. Covalent crosslinking between adjacent triple helices of collagen IV in the BM stabilizes their lateral association by establishing an irregular network of super-twisted helices2. These collagen IV lattices act as a scaffold for its interaction with laminin and other BM components1. The structural arrangement of the BM provides it with the mechanical strength and rigidity necessary for the normal development of glandular epithelia3. 
During aging and disease the BM progressively thickens and stiffens3,4. For example, a 3-fold increase in the elastic modulus (E) of the ocular BM occurs between the ages of 50 and 80 in the normal population, and this stiffening is further exacerbated in metabolic disorders like diabetes5. The structural and biomechanical changes in the BM that result in its increased stiffness occur when its ECM components, collagen IV and laminin, become non-enzymatically crosslinked following their exposure to advanced glycation endproducts (AGEs).
The purpose of the method described here was to establish a model for the investigation of how BM stiffness, due to AGE exposure, promotes prostate epithelial cell (PEC) and prostate tumour cell (PTC) invasiveness in the context of the switch to metastatic prostate cancer (PCa). To do this a previous method used for generating 3D glandular acini from mammary epithelial cells (MECs) in reconstituted rBM gels6 was adapted to include an additional step where the rBM gels are pre-treated with glycolaldehyde (GLA). Several techniques for assessing GLA induced crosslinking and stiffening of pre-treated rBM gels are described, including photometric analysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), confocal microscopy and rheometric analysis. The prostate cell types selected for culture on the pre-stiffened rBM include: RWPE-1, PECs derived from a normal prostate gland7; BPH-1, PECs derived from a prostate gland affected by BPH8; and PC3, metastatic PTCs derived from a secondary tumor located in the vertebral bone of a prostate cancer (PCa) patient9. 
In addition to advancing the study of prostate gland pathology, the protocol for stiffening of rBM gels by their treatment with GLA can be adapted to investigate how BM stiffness contributes to other age-related pathologies and metabolic disorders. For example, the model can be directly applied to investigate how metastatic cancer is induced by BM stiffness in organs such as the breast, colon, ovary and pancreas by the incorporation of appropriate cell types. Furthermore, the protocol can be adapted to investigate how stiff BM promotes biomechanical mechanisms of disease progression in diabetes-related microvascular disease, retinopathy, nephropathy and neuropathy.

<table><tbody><tr><td>&lt;strong&gt;I - Material for monolayer culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BPH-1</td><td>CaP Cell Line Database</td><td>PCaCL-132</td><td>Contact: simon.hayward@mcmail.vanderbilt.edu</td></tr><tr><td>Complete keratinocyte serum-free media&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>17005-075</td><td>Do not warm at 37 &amp;ordm;C before use</td></tr><tr><td>Fetal calf serum&amp;nbsp;</td><td>First Link UK Ltd</td><td>02-00-850</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>PC3&amp;nbsp;</td><td>American Type Culture Collection</td><td>CRL-1435</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>ThermoFisher Scientific</td><td>15070-063</td><td></td></tr><tr><td>Phosphate Buffer Saline (Dulbecco A) tablets</td><td>Oxoid</td><td>BR0014G</td><td></td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Sigma-Aldrich</td><td>R5886</td><td>warm in 37 &amp;ordm;C water bath before use</td></tr><tr><td>RWPE-1&amp;nbsp;</td><td>American Type Culture Collection</td><td>CRL-11609</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich</td><td>T4049</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;II - Material for 3D culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>271004</td><td></td></tr><tr><td>Aminoguanidine hydrochloride</td><td>Sigma-Aldrich</td><td>396494</td><td>Irritating to eyes, respiratory system and skin</td></tr><tr><td>Chamber slides, 8-well&amp;nbsp;</td><td>Thermo Scientific Nunc Lab-Tek</td><td>TKT-210-816M</td><td></td></tr><tr><td>Culture Matrix reconstituted basement membrane (rBM) reduced growth factor extract&amp;nbsp;</td><td>AMS Biotechnology</td><td>3445-005-01</td><td>Store Basement membrane (BM) at -80 &amp;ordm;C in aliquots</td></tr><tr><td>Cyanogen bromide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C91492</td><td>Toxic by contact skin and inhalation</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>695076</td><td></td></tr><tr><td>Glycine ethyl ester hydrochloride (GEE)</td><td>Sigma-Aldrich</td><td>50060</td><td>Irritating to eyes</td></tr><tr><td>Glycolaldehyde dimer (GLA)</td><td>Sigma-Aldrich</td><td>G6805</td><td></td></tr><tr><td>Sodium cyanoborohydride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>71435</td><td>Highly flammable; Toxic by contact skin and inhalation</td></tr><tr><td>Syringe filter 0.22 microns</td><td>Appleton Woods</td><td>BC680</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;III - Material to quantify Maillard reaction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>ThemoFisher Scientific</td><td>D3571</td><td>light sensitive and store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Cloning cylinders</td><td>Sigma-Aldrich</td><td>C1059</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td><td>ThemoFisher Scientific</td><td>A-11001</td><td>light sensitive</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Alexa Fluor 488 conjugate</td><td>ThemoFisher Scientific</td><td>A-11034</td><td>light sensitive</td></tr><tr><td>Goat serum</td><td>Abcam</td><td>ab7481</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Vectashield mounting media&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>Mouse anti-pentosidine clone PEN-12 mAb</td><td>TransGenic Inc</td><td>KH012</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>F8775</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Rabbit anti-human collagen IV polyclonal antibody&amp;nbsp;&amp;nbsp;</td><td>Acris Antibodies</td><td>R1041</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Rabbit anti-laminin A/C pAb</td><td>Santa Cruz Biotechnology Inc</td><td>sc-7292</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Polyethylene glycol tert-octylphenyl ether (Triton-X100)</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Polyethylene glycol sorbitan monolaurate (Tween-20)</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Dialysis cassette Slide-A-Lyzer</td><td>ThemoFisher Scientific</td><td>66333</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;IV - Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ARG2 controlled strain rotational rheometer</td><td>T.A. Instruments</td><td></td><td></td></tr><tr><td>Axiovert S100 (20X magnification) microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>CO2 controlled humidified incubation chamber for Zeiss Axio S100 microscope</td><td>Solent Scientific</td><td></td><td></td></tr><tr><td>Confocal Axiovert 200M (40X, 63X magnification) microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Olympus LH50A microscope fitted with a digital camera using phase-contrast&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td></td><td></td></tr><tr><td>PHERAstar Plus plate reader spectrophotometer</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;V - Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image J 1.47v</td><td>National Institute of Health, USA</td><td></td><td></td></tr><tr><td>MetaXpress</td><td>Molecular Divices</td><td></td><td></td></tr></tbody></table>

how to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases

在这里，我们描述了可以用于研究期间年龄相关疾病和代谢紊乱（ 如癌症，糖尿病，微血管病变，视网膜病变，肾病和神经病）相关的增加的厚度和基底膜（BM）的硬度的生物物理微环境的协议。该模型的前提是通过用乙醇醛（GLA）的重构的BM（RBM）矩阵的非酶交联通过美拉德反应以促进高级糖化终产物（AGE）生成。可用于确认AGE生成，非酶交联和在GLA增加的刚度的实验室技术的实例处理的代rBM进行了概述。这些包括制备天然代rBM和僵硬代rBM（用GLA治疗）（用磷酸盐缓冲盐水，PBS处理的）的用于测定的:由光度分析和免疫荧光显微镜，其非酶通过十二烷基硫酸钠聚丙烯酰胺的交联的AGE含量凝胶电泳（SDS PAGE），以及共焦显微镜，其增加的刚度使用流变测定。这里描述的方法可以用于增加代rBM的刚性（弹性模量，E）的高达3.2倍，并在健康与患病的人前列腺组织进行的测量一致。重建具有衰老和患病的前列腺癌相关的生物物理微腺3前列腺细胞类型的原生代rBM和僵硬代rBM介绍:从一个正常的前列腺衍生RWPE-1，前列腺上皮细胞（佩奇）; BPH-1，从受良性前列腺增生（BPH）一个前列腺衍生的胸肌;和PC3，转移性细胞从二级骨肿瘤前列腺癌起源的。多个参数可被测量，包括大小，形状和由RWPE-1和BPH-1对本地与僵硬代rBM构成的三维腺腺​​泡浸润特性，并且平均泡孔长度，迁移速度和3D spher的细胞运动的持久性由PC3细胞在相同的条件下形成的OID。细胞信号传导途径和蛋白质的亚细胞定位还可评估。

3D前列腺腺泡养在硬代rBM 后在培养6天，胸肌从正常前列腺组织的（RWPE-1）（ 图1A）和良性前列腺增生组织（BPH-1）（ 图1B）上天然（PBS处理）代rBM形式腺泡被组织为上皮的均匀球状体细胞。这些腺泡也有高度的组织佩奇心尖到基底极性和可见管腔空间13,20的特性。 通过从正常前列腺组织的胸肌形成的腺泡（RWPE-1）（ 图1A）和硬化（富AGE-）代rBM（用GLA治疗）BPH组织（BPH-1）（ 图1B）具有破坏架构（移从球形在形态和细胞多角形突起/从腺泡迁移到RBM的丰富AGE-）（ 图1A）。这些<html>腺泡的特征还在于由已失去心尖到基底极性与小的或不存在的管腔空间13高度紊乱胸肌。 <img alt="图1" src="/files/ftp_upload/54230/54230fig1.jpg" /> 图1:成长为3D腺腺泡对本地和僵硬的重组基底膜（RBM）前列腺上皮细胞 （A）RWPE-1细胞的明视场图像生长12小时6天与PBS（本机）或处理过的代rBM凝胶50毫米乙醇醛14小时（AGE-丰富;硬）;比例尺= 50微米。 （B）的BPH-1细胞，生长成在面板A中描述;标尺= 50微米;数据代表3个独立的实验。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54230/54230fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html>表1"SRC ="/文件/ ftp_upload / 54230 / 54230table1.jpg"/&gt; 表1:生长于本地，半刚性和硬重组基底膜（RBM）前列腺上皮RWPE-1腺泡特点 RWPE-1腺泡生长成果管理与PBS处理前14小时（本机），乙醇（GLA ）6小时（半硬）或GLA 14小时（硬）。为腺泡状，百分比（％）±轮的标准偏差（SD），半多边形和多边形腺泡从5个独立实验计算（50腺泡每种条件量化）。相对腺泡大小从3次独立实验计算（天然代rBM = 100％）。为侵袭，％±与一个或多个突起的细胞的SD腺泡从3次独立实验计算。倍数变化除以为天然条件对应的值的半刚性或刚性的条件下所获得的平均值计算。的P值使用学生t检验（α= 0.05）来计算。 内容】"FO:保together.within页="1"&gt;年龄依赖性增加代rBM刚度促进PC3前列腺肿瘤细胞迁移生长在原生代rBM PC3细胞保持连续细胞间接触相互独立（ 图2A）迁移，而PC3细胞丰富的AGE-（硬）长大代rBM举动。在培养PC3细胞72小时形成对本地焦点（球体）（PBS处理）代rBM，而在硬（富AGE-）代rBM PC3细胞球体不和独立迁移（ 图2B）。上僵硬（富AGE-）代rBM PC3细胞比生长在天然代rBM（ 图2C）PC3细胞更细长。僵硬代rBM PC3细胞迁移速度比生长在原生代rBM（ 图2D）PC3细胞快。相比于生长在原生代rBM（ 图2E）PC3细胞僵硬代rBM PC3细胞显示持续性的减少。 <img aLT ="图2"SRC ="/文件/ ftp_upload / 54230 / 54230fig2.jpg"/&gt; 图2:在本地和僵硬的重组基底膜（RBM）前列腺肿瘤细胞迁移 （A）生长在用PBS（本机）或50毫米乙醇醛处理14小时代rBM凝胶PC3细胞的明视图像（富含AGE-，僵硬） 。细胞用明场显微镜（10X物镜）和每小时1图像的12小时后的细胞追踪来生成轨迹采集速率成像。显示的图像对应的时间点后0,3，6,9和12小时。单个细胞的轨迹示出的12小时的时间点。比例尺= 100微米。 （B）PC3细胞对本地或僵硬代rBM培养72小时，成像为面板（A）中描述。比例尺= 100微米。详细显示了2倍放大选中的区域。 （C）的平均值±SD元长度（微米）;原生代rBM僵硬代rBM之间显著差异（p = 1.2×10 -23）。 （ <str翁&gt; D）平均值±SD速度（微米/小时）从细胞轨迹计算;原生代rBM僵硬代rBM（P = 0.004）之间的差异显著。 （E）的平均值±SD细胞运动的持久性（比D / T，其中D =从开始到细胞轨迹，T =细胞轨迹的总长度的端部的距离）;原生代rBM僵硬代rBM（P = 0.0007）之间的显著差异。对于板CE&gt; 10个细胞进行分析，数据代表的3个独立的实验。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54230/54230fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases at JoVE.com

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

在这里，我们解释模型生物物理微环境，其中交联和提高晚期糖基化产物（AGEs）引起的基底膜（BM）的刚度病理意义的协议。

如何学习基底膜刚度生物物理触发在前列腺癌和其他与年龄相关的病理或代谢性疾病

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

how-to-study-basement-membrane-stiffness-as-biophysical-trigger

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

9.6K 次观看.  Universidad de la República (UDELAR). 在这里，我们解释模型生物物理微环境，其中交联和提高晚期糖基化产物（AGEs）引起的基底膜（BM）的刚度病理意义的协议。 

视频: How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

基质贴壁细胞的微管吸吮的估计单元刚度

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

科研

生物工程

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

原子力显微镜悬臂式纳米压痕:空气和流体中纳米级机械性能测量

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

工程学

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

生物学

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

基于聚丙烯酰胺的多井格式实验研究细胞外基质硬度对黏附细胞细菌感染的影响

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

免疫与感染

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

近同步激光扫描在活细胞上的共和和原子力显微镜（康波卡尔）

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Extracting the Young's Modulus of Native Murine Pulmonary Basement Membranes from Atomic Force Microscopy Derived Force Maps

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of nanometers. Because mammalian cells sense and react to the mechanics of their immediate microenvironment, the characterization of biomechanical properties of tissues with high spatial resolution is crucial for understanding various developmental, homeostatic, and pathological processes. The basement membrane (BM), a roughly 100 - 400 nm thin extracellular matrix (ECM) substructure, plays a significant role in tumor progression and metastasis formation. Although determining Young&#39;s modulus of such a thin ECM substructure is challenging, biomechanical data of the BM provides fundamental new insights into how the BM affects cell behavior and, in addition, offers valuable diagnostic potential. Here, we present a visualized protocol for assessing BM mechanics in murine lung tissue, which is one of the major organs prone to metastasis. We describe an efficient workflow for determining the Young&#39;s modulus of the BM, which is located between the endothelial and epithelial cell layers in lung tissue. The step-by-step instructions comprise murine lung tissue freezing, cryosectioning, and AFM force-map recording on tissue sections. Additionally, we provide a semi-automatic data analysis procedure using the CANTER Processing Toolbox, an in-house developed user-friendly AFM data analysis software. This tool enables automatic loading of recorded force maps, conversion of force versus piezo-extension curves to force versus indentation curves, computation of Young&#39;s moduli, and generation of Young&#39;s modulus maps. Finally, it shows how to determine and isolate Young&#39;s modulus values derived from the pulmonary BM through the use of a spatial filtering tool.

This protocol visualizes how to prepare cryosections of murine lung tissue, perform atomic force microscopy force map experiments, and analyze the data to determine Young's modulus of the native murine pulmonary basement membrane.

从原子力显微镜衍生的力图中提取天然小鼠肺基底膜的杨氏模量

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The cellular mechano-sensation of extracellular mechanical stimuli is believed to be through the membrane receptors, the associated protein complex, and the cytoskeleton. Recent advances in mechanobiology demonstrate that the cell nucleus in cytoplasm itself can independently sense mechanical stimuli simultaneously. However, a mechanistic understanding of how the cell nucleus senses, transduces, and responds to mechanical stimuli is lacking, mainly because of the technical challenges in accessing and quantifying the nucleus mechanics by conventional tools. This paper describes the design, fabrication, and implementation of a new magnetic force actuator that applies precise and non-invasive 3D mechanical stimuli to directly deform the cell nucleus. Using CRISPR/Cas9-engineered cells, this study demonstrates that this tool, combined with high-resolution confocal fluorescent imaging, enables the revelation of the real-time dynamics of a mechano-sensitive yes-associated protein (YAP) in single cells as a function of nucleus deformation. This simple method has the potential to bridge the current technology gap in the mechanobiology community and provide answers to the knowledge gap that exists in the relation between nucleus mechanotransduction and cell function.

This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging.

结合3D磁力致动器和多功能荧光成像来研究细胞核力学生物学

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The ...