我们感谢 m. 页脚, r. Lamason, m. Rengaranjan, 以及 Theriot 实验室成员的讨论和实验支持。这项工作得到了 NIH 的 R01AI036929 (J.A.T.)、HHMI (J.A.T.)、HHMI 研究中心 (F.E.O.)、斯坦福大学研究生联谊会 (F.E.O.) 和美国心脏协会 (E.E.B.) 的支持。流式细胞术是在斯坦福共享的资产管制设施进行的。

作者没有什么可透露的。

细胞可以感知各种物理环境的线索, 这不仅会影响细胞的形态, 而且会对它们的基因表达和蛋白质活性产生作用, 从而影响关键细胞功能和行为45,46。细胞的电解质的刚度越来越被视为细胞动力, 分化, 增殖, 最终细胞命运47,48,49的重要调制器。虽然在了解细胞与电解加工之间复杂的生物力学相互作用方面已经有了许多进展, 但对于环境刚度如何影响细胞对细菌感染的敏感性却知之甚少。为便于此类研究, 我们开发了这种新型的多井检测方法, 其基础是制备了可调谐刚度的聚丙烯酰胺水凝胶, 与50的感染测定方法相吻合。传统上, 在玻璃或聚苯乙烯表面对组织培养细胞的细菌感染进行了研究, 其强度比大多数黏附细胞8、51的自然 ECM 要硬 1-3 级。这里所描述的试验开辟了新的公路, 使研究细菌宿主相互作用的生理相关的环境刚度制度。
为了证明所提出的试验的概念, HMEC-1 细胞被选择为模型黏附宿主细胞和 Lm 作为模型细菌病原体。然而, 如果进行适当的修改, 则可以对该化验进行进一步的研究。这种研究可能涉及不同的哺乳动物黏附宿主细胞类型感染的其他病原体, 包括细菌和病毒。对于这种特殊的化验, 凝胶是蛋白质涂层的胶原蛋白 i, 但根据宿主细胞类型, 有可能使用不同的 ECM 蛋白涂层, 如层黏蛋白或纤连蛋白, 以促进附着的宿主细胞的水凝胶52. 另外一个取决于宿主细胞类型的考虑是水凝胶刚度范围的研究。刚度范围应取决于与特定宿主细胞类型相关的生理因素, 以及宿主在给定的刚性水凝胶中形成单层的良好程度.同样, 根据所需检查的模型病原体, 可能需要对此处描述的感染化验进行轻微的修改。
与以往制造聚丙烯酰胺凝胶24、50、53的方法相比, 该试验的创新之处在于所提出的感染试验中纳入了某些独特的特征。首先, 水凝胶是建立在多井玻璃底板上的, 既能同时进行多种条件的筛选, 也能实现某些工序的自动化。同时监测多个条件或检查多个复制是至关重要的, 因为这种方法的结果可能受到宿主细胞通道的影响, 以及添加到感染宿主的细菌的确切数量, 这往往会改变在独立实验之间。另外一个独特的特点是, 水凝胶的高度约40µm, 这是足够薄, 以图像使用常规显微镜。我们表明, 我们可以成功地执行活细胞显微术和细胞固定, 染色后, 影像, 不引入高背景荧光。最后, 水凝胶由两层组成, 上部有嵌入荧光微球, 受限于单个焦平面。此属性确保在成像过程中不会出现焦点光线干扰。珠的存在使两个检查凝胶的表面形貌, 以确保它们是一致的, 并提高性能的 TFM 和 MSM24,32,41。与 TFM 和 MSM, 有可能计算细胞 ECM 和细胞细胞的力量分别对居住在不同刚度矩阵的细胞。利用这种新的检测方法, 可以通过对环境刚度和感染的贡献同时进行比较, 从而对物理力进行测量。在这种方法下, 感染对宿主细胞力学的影响在整个过程中都可以确定。此外, 细胞内应力的演变可以用 MSM 计算, 并可作为衡量的屏障完整性的单层。最后, 鉴于该试验的多井性质, 有可能同时引入药理和遗传扰动与细菌感染, 更深入地研究宿主细胞力学和感染之间的复杂相互作用。
该技术的一个固有局限性在于基底刚度对细胞增殖速率的影响。通常, 在感染化验中, 我们需要确保在不同条件下的宿主细胞密度是相同的。这是因为细胞密度本身会影响寄主对感染的敏感性。作为宿主细胞的 HMEC-1 细胞在水凝胶上播种24小时时, 它们的数量没有显著的差异。然而, 不同的细胞类型可能表现出差异增殖, 取决于水凝胶的硬度, 可以偏倚感染研究。同样, 当细胞不形成单分子膜或不附着在水凝胶上时, 就会出现感染偏差, 如某些细胞类型在非常软的基质上播种 (例如, 人脐内皮细胞或 Madin-达比犬肾上皮细胞种子在 0.6-帕水凝胶54,55)。就病原体而言, 某些细菌 (例如,螺旋体 burdgorferi) 可以附着在宿主细胞上, 并侵入它们, 但也可以通过它们来刺字56。我们还没有测试, 如果这一化验将工作的病原体轮回研究, 但似乎有可能, 因为以前的研究, 对中性粒细胞 transmigrating 内皮细胞种子在 PA 水凝胶已被证明工作成功57。通过混合丙烯酰胺和双丙烯酰胺24、25、26 的适当浓度, 进行了大量的研究, 展示了如何生产给定杨氏模量的聚丙烯酰胺水凝胶. ,27。然而, 特别是当一个人想要对居住在不同刚度的水凝胶上的细胞进行 TFM 实验时, 通过 AFM 或其他压痕技术来确认水凝胶的预期刚度是至关重要的.58。由于使用了不同的溶剂或老化的丙烯酰胺溶液, 或者用 afm 测量的方式 (例如afm 尖端的形状), 可能会产生与预期值稍有偏差的情况。最后, 本文提出的方法是基于2D 矩阵上的种子宿主细胞, 它可以不同于更现实和生理相关的3D 场景。然而, 制造3D 凝胶具有可调谐的硬度, 将其与宿主细胞一起播种, 然后感染病原体, 仍然包含一定的技术困难。然而, 我们预计, 在不久的将来, 我们将能够扩大目前的检测, 以研究感染的3D 设置。
综上所述, 所描述的协议连同初步的结果提供了证据表明, 这一新的检测可以成为一个非常有用的工具, 研究感染的黏附宿主细胞与致病细菌在数量的方式, 并在更生理相关的环境比以前检查。在所提出的装置中制备聚丙烯酰胺水凝胶的威力在于, 该方法与流式细胞术、染色后光镜、牵引力显微镜等多种技术的性能相适应。该方法可用于研究感染不同黏附宿主细胞类型的病原体, 我们预计将有重大影响, 以解开的策略, 其中病原体感染宿主和促进发展对感染的治疗干预。

大多数动物组织中的细胞通常附着在相邻细胞和细胞外基质 (ECM) 上。细胞的锚固对他们的 ECM 是关键的许多细胞过程, 从细胞的运动到细胞增殖和生存1,2。细胞锚定对 ecm 取决于 ecm 构成和刚性。细胞对后者的变化作出反应, 动态地重新排列细胞骨架, 电池 ECM 和细胞的粘连, 这反过来批判地改变细胞生物力学和功能3,4,5,6.ECM 刚度可以在空间 (即解剖位置)、时间 (即衰老) 和病理生理学过程 (如动脉硬化、癌症、感染等) 中变化。例如, 人们普遍认为, 与软矩阵相比, 居住在更硬的内皮细胞会增加它们的 ECM 和相互之间的作用力, 并表现出增加的运动和增殖7,8。同样, 居住在较硬矩阵上的成纤维细胞会向 ecm 传递高收缩力, 并显示增加的增殖、运动和 ECM 产量9、10、11。虽然细胞力学和对 ECM 刚度的反应已被广泛研究的各种细胞类型, 黏附宿主细胞之间的关系, 其 ECM 的硬度, 和细菌感染仍然是很大的未知。
为了研究 ECM 刚度在细菌-宿主细胞相互作用中的作用, 本文选择了李斯特菌 (Lm) 作为模型病原体。Lm 是一种无处不在的食物传播细菌, 它能在各种各样的哺乳动物宿主体内引起全身感染。这种兼性的胞内病原体通过遍历不同类型的血管内皮, 可以从肠道上皮转移到远端器官。如果它破坏血脑屏障, Lm 会引起脑膜炎, 当它穿过胎盘时, 它会导致自发性流产12,13。Lm 可以感染不同的宿主细胞类型, 并可以使用不同的致病策略。lm 感染主要是在上皮细胞的背景下进行的, 而对于 lm 如何感染和绕过血管腔内血管内皮细胞14、15、16的研究, 则知之甚少。此外, 在很大程度上还不知道内皮细胞的 ECM 的刚度是如何调节 Lm 入侵这些宿主细胞并传播的能力。Lm 和几个额外的细菌物种(即立克次体 parkeri) 利用它们感染的宿主细胞的肌动蛋白细胞骨架侵入他们的细胞质, 促进细胞对细胞的传播17,18,19. 它们通过表达蛋白质, 使它们能够干扰宿主肌动蛋白聚合通路, 并产生肌动蛋白彗星尾巴, 促进其向前推进16,20。由于感染, 宿主细胞的肌动蛋白细胞骨架需要动态重新排列的方式, 仍然没有充分的特点, 可能影响的生物力学的宿主细胞, 包括他们所施加的物理力量对他们的 ECM 和每个其他。为了检查这些过程, 人类微血管内皮细胞 (HMEC-1) 被选择为模型宿主细胞三的原因: 1. 内皮细胞被称为高度 mechanosensitive, 因为它们经常暴露在不同的物理线索21;2. 我所使用的用于感染内皮细胞的策略在很大程度上仍然未知22;和3。HMEC-1 是一个永生化的细胞系, 因此, 可以很容易地培养和受到遗传操作。
宿主细胞的细菌感染主要是通过在玻璃或聚苯乙烯基质上的播种细胞来研究的, 它们比大多数细胞12、14、23的生理 ECM 明显僵硬。为了检查其硬度与生理相关的基质细胞的感染情况, 并阐明 ECM 刚度对细菌感染细胞的作用, 我们遵循了一种基于制备薄microbead 聚丙烯酰胺水凝胶在多井板上可调谐刚度。所提出方法的新颖之处在于, 它允许同时监测多个条件, 因为它的多井格式和它是兼容的多种技术, 由于特定的方式, 基板的建立。HMEC-1 细胞被播种在这些蛋白涂层的水凝胶, 然后感染了不同的 Lm 菌株, 要么成为荧光在内化或组成性荧光。采用流式细胞仪评价 ECM 刚度对宿主 HMEC-1 细胞感染敏感性的影响。此外, 染色和荧光显微术用于区分附着和内化细菌。最后, 对牵引力显微镜 (TFM) 进行了成功的表征, 表明 Lm 感染对宿主内皮细胞在感染过程中对其基质施加牵引应力的影响。所提出的检测方法可以很容易地进行修改, 以便进一步研究 ECM 刚度对不同细胞系或病原体对黏附细胞感染敏感性的影响。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium hydroxide pellets</td><td>Fisher</td><td>S318-500</td><td></td></tr><tr><td>(3-Aminopropyl)triethoxysilane</td><td>Sigma</td><td>A3648</td><td></td></tr><tr><td>25% gluteraldehyde</td><td>Sigma</td><td>G6257-100ML</td><td></td></tr><tr><td>40% Acrylamide</td><td>Sigma</td><td>A4058-100ML</td><td></td></tr><tr><td>Bis-acrylamide solution (2%w/v)</td><td>Fisher Scientific</td><td>BP1404-250</td><td></td></tr><tr><td>Fluorospheres carboxylate-modified microspheres, 0.1 &amp;mu;m, yellow-green fluorescent (505/515)</td><td>Invitrogen</td><td>F8803</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Fisher</td><td>BP17925</td><td></td></tr><tr><td>TEMED</td><td>Sigma</td><td>T9281-25ML</td><td></td></tr><tr><td>Sulfo-SANPAH</td><td>Proteochem</td><td>c1111-100mg</td><td></td></tr><tr><td>Collagen, Type I Solution from rat</td><td>Sigma</td><td>C3867-1VL</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>J.T. Baker</td><td>9224-01</td><td></td></tr><tr><td>HEPES, Free acid</td><td>J.T. Baker</td><td>4018-04</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 medium, no phenol red</td><td>Thermofischer</td><td>21083027</td><td></td></tr><tr><td>MCDB 131 Medium, no glutamine</td><td>Life technologies</td><td>10372019</td><td></td></tr><tr><td>Foundation Fetal Bovine Serum, Lot: A37C48A</td><td>Gemini Bio-Prod</td><td>900108 500ml</td><td></td></tr><tr><td>Epidermal Growth Factor, EGF</td><td>Sigma</td><td>E9644</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H0888</td><td></td></tr><tr><td>L-Glutamine 200mM</td><td>Fisher</td><td>SH3003401</td><td></td></tr><tr><td>DPBS 1X</td><td>Fisher</td><td>SH30028FS</td><td></td></tr><tr><td>Gentamicin sulfate</td><td>MP biomedicals</td><td>194530</td><td></td></tr><tr><td>Cloramphenicol</td><td>Sigma</td><td>C0378-5G</td><td></td></tr><tr><td>DifcoTM Agar, Granulated</td><td>BD</td><td>214530</td><td></td></tr><tr><td>BBL TM Brain-heart infusion</td><td>BD</td><td>211059</td><td></td></tr><tr><td>Hoechst 33342, trihydrochloride, trihydrate - 10 mg/ul solution in water</td><td>Invitrogen</td><td>H3570</td><td></td></tr><tr><td>Formaldehyde 16% EM grade</td><td>Electron microscopy</td><td>15710-S</td><td></td></tr><tr><td>Anti-Listeria monocytogenes antibody</td><td>Abcam</td><td>ab35132</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&amp;reg; 546 conjugate</td><td>Thermofischer</td><td>A-11035</td><td></td></tr><tr><td>0.25% trypsin-EDTA , phenol red</td><td>Thermofischer</td><td>25200056</td><td></td></tr><tr><td>COLLAGENASE FROM CLOSTRIDIUM HISTOLYTIC</td><td>Sigma</td><td>C8051</td><td></td></tr><tr><td>Streptomycin sulfate</td><td>Fisher Scientific</td><td>3810-74-0</td><td></td></tr><tr><td>Sucrose</td><td>Calbiochem</td><td>8510</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Thermofischer</td><td>28364</td><td></td></tr><tr><td>MES powder</td><td>Sigma</td><td>M3885</td><td></td></tr><tr><td>KCl</td><td>J.T. Baker</td><td>3040-05</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>J.T. Baker</td><td>2444-1</td><td></td></tr><tr><td>EGTA</td><td>Acros</td><td>40991</td><td></td></tr><tr><td>&lt;strong&gt;Disposable lab equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>12 mm circular glass coverslips</td><td>Fisherbrand</td><td>12-545-81</td><td>No. 1.5 Coverslip | 10 mm Glass Diameter | Uncoated</td></tr><tr><td>Glass bottom 24 well plates</td><td>Mattek</td><td>P24G-1.5-13-F</td><td></td></tr><tr><td>5 ml polystyrene tubes with a 35 &amp;mu;m cell strainer cap&amp;nbsp;</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>T-25 flasks</td><td>Falcon</td><td>353118</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>15 ml conicals tubes</td><td>Falcon</td><td>352196</td><td></td></tr><tr><td>Disposable Serological Pipettes (1 ml, 2 ml, 5 ml, 10 ml, 25 ml)</td><td>Falcon</td><td>357551</td><td></td></tr><tr><td>Pasteur Glass Pipettes</td><td>VWR</td><td>14672-380</td><td></td></tr><tr><td>Pipette Tips (1-200 &amp;mu;l, 101-1000 &amp;mu;l)</td><td>Denville</td><td>P1122, P1126</td><td></td></tr><tr><td>Powder Free Examination Gloves</td><td>Microflex</td><td>XC-310</td><td></td></tr><tr><td>Cuvettes bacteria</td><td>Sarstedt</td><td>67.746</td><td></td></tr><tr><td>Razors</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Syringe needle</td><td>BD</td><td>305167</td><td></td></tr><tr><td>0.2um sterilizng bottles</td><td>Thermo Scientific</td><td>566-0020</td><td></td></tr><tr><td>20 ml syringes</td><td>BD</td><td>302830</td><td></td></tr><tr><td>0.2um filters</td><td>Thermo Scientific</td><td>723-2520</td><td></td></tr><tr><td>wooden sticks</td><td>Grainger</td><td>42181501</td><td></td></tr><tr><td>Saran wrap</td><td>Santa Cruz Biotechnologies</td><td>sc-3687</td><td></td></tr><tr><td>Plates bacteria</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>&lt;strong&gt;Large/non-disposable lab equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tissue Culture Hood</td><td>Baker</td><td>SG504</td><td></td></tr><tr><td>Hemacytometer</td><td>Sigma</td><td>Z359629</td><td></td></tr><tr><td>Bacteria incubator</td><td>Thermo Scientific</td><td>IGS180</td><td></td></tr><tr><td>Tissue culture Incubator</td><td>NuAire</td><td>NU-8700</td><td></td></tr><tr><td>Inverted Nikon Diaphot 200 epifluorescence microscope</td><td>Nikon</td><td>NIKON-DIAPHOT-200</td><td></td></tr><tr><td>Cage Incubator</td><td>Haison</td><td>Custom</td><td></td></tr><tr><td>Scanford FACScan analyzer&amp;nbsp;</td><td>Stanford and Cytek upgraded FACScan</td><td>Custom</td><td></td></tr><tr><td>Pipette Aid</td><td>Drummond</td><td>4-000-110</td><td></td></tr><tr><td>Pipettors (10 &amp;mu;l, 200 &amp;mu;l, 1000ul)</td><td>Gilson</td><td>F144802, F123601, F123602</td><td></td></tr><tr><td>pH meter</td><td>Mettler Toledo</td><td>30019028</td><td></td></tr><tr><td>forceps</td><td>FST</td><td>11000-12</td><td></td></tr><tr><td>1 L flask</td><td>Fisherbrand</td><td>FB5011000&amp;nbsp;</td><td></td></tr><tr><td>Autoclave machine</td><td>Amsco</td><td>3021</td><td></td></tr><tr><td>Stir magnet plate</td><td>Bellco</td><td>7760-06000</td><td></td></tr><tr><td>Magnet stirring bars</td><td>Bellco</td><td>1975-00100</td><td></td></tr><tr><td>Spectrophotometer</td><td>Beckman</td><td>DU 640</td><td></td></tr><tr><td>Scanford FACScan analyzer</td><td>Cytek Biosciences</td><td>Custom Stanford and Cytek upgraded FACScan</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microscope Software (&amp;mu;Manager)</td><td>Open Imaging</td><td></td><td></td></tr><tr><td>Matlab</td><td>Matlab Inc</td><td></td><td></td></tr><tr><td>Flowjo</td><td>FlowJo, LLC</td><td></td><td></td></tr><tr><td>Automated image analysis software, CellC</td><td>https://sites.google.com/site/cellcsoftware/</td><td></td><td>The software is freely available. Eexecutable files and MATLAB source codes can be obtained at https://sites.google.com/site/cellcsoftware/</td></tr></tbody></table>

a multi-well format polyacrylamide-based assay for studying the effect of extracellular matrix stiffness on the bacterial infection of adherent cells

细胞外基质刚度包括多种环境机械刺激, 众所周知, 影响细胞的行为, 功能, 和命运的一般。虽然越来越多的黏附细胞类型的反应矩阵的刚度已经被描述, 如何黏附的细胞对细菌感染的敏感性取决于基质刚度是很大的未知, 如细菌感染的影响宿主细胞的生物力学。我们推测宿主内皮细胞对细菌感染的敏感性取决于这些细胞所驻留的基质的刚度, 而细菌宿主细胞的感染会改变其生物力学。为了检验这两种假说, 以内皮细胞为模型宿主, 以李斯特菌为模型致病菌。通过建立一种新的多井格式检测方法, 证明了基质刚度对细胞内内皮细胞感染的影响, 可以通过流式细胞术和染色显微镜进行定量评价。此外, 利用牵引力显微技术, 可以研究l-李斯特菌感染对宿主内皮细胞生物力学的影响。该方法可以分析组织相关力学对黏附细胞细菌感染的影响, 这是了解细胞间生物力学相互作用的关键步骤, 它们的细胞外基质和病原菌。这种方法也适用于多种其他类型的细胞生物力学和反应基板刚度的研究, 其中重要的是能够在每个实验并行执行许多复制。

电解 HMEC-1 细胞对 Lm 感染的依赖敏感性: 不同刚度的 PA 水凝胶, 所有表面涂层的胶原 i, 是建立在多井玻璃底部板, 如协议步骤 1所述 (见图 1)。AFM 的测量是为了确认水凝胶的确切刚度, 如前所述26,27 (见图 2)。过去的研究表明, 内皮细胞基底膜的局部顺应性可以从1帕 (例如, 脑组织) 到70帕 (例如, 主动脉)36,37,38,39,40. 因此, 我们选择测试驻留在矩阵上的 HMEC-1 细胞感染, 其刚度如下: 0.6 帕, 3 帕, 20 帕, 70 帕。对于每一个水凝胶刚度, 六水凝胶的制备, 以评估结果的重现性。
流式细胞术用于评估 HMEC-1 细胞对 Lm 感染的 ECM 刚度依赖性 (见图 3)。HMEC-1 细胞感染了 Lm 应变 (JAT985), 在内化后表达荧光标记 (actAp: mTagRFP), 只允许检测细胞内细菌 (见图 1L - 1N)。JAT985 也缺乏学报, 禁止细菌从细胞扩散到细胞, 因为学报对于形成肌动蛋白彗星尾巴和随后的细菌传播是必要的。7-8 h 感染后, 使用流式细胞仪对 HMEC-1 细胞感染进行评估。为了保证对单个细胞的分析, 细胞计数分布的大部分是使用正向散射图进行封闭的, 然后再执行第二个浇口步骤, 以排除展示自体荧光的细胞 (见图 3A - 3C).图 3所示的初步结果表明, 与驻留在软0.6 人民军水凝胶上的 HMEC-1 细胞相比, Lm 的 HMEC-1 感染大约是两倍大的, 与住在柔软的70人民的帕里的细胞相比较 (见图 3B -3D). 与软矩阵相比, HMEC-1 细胞在僵硬时的 lm 感染敏感性增加可能是由于: 1. 增加 lm 对 HMEC-1 的黏附力;2. 增加 Lm 入侵 HMEC-1;或3。以上两种都是共同发生的。为了测试哪个假说持有, HMEC-1 细胞被播种在软 (3 帕) 和僵硬 (70 帕) 水凝胶和感染组成性 GFP 表示 Lm (见图 4A - 4C)。样品在感染后不久就被固定, 而外部 (附着) 的细菌则被抗体染色。使用定量显微镜, 我们发现, 当宿主细胞与软凝胶相比时, HMEC-1 的细菌附着在僵硬的情况下 (见图 4D)。与流式细胞仪数据一致, 当宿主细胞与软凝胶相比时, HMEC-1 的细菌内化程度明显增加 (见图 4E)。然而, 无论基底硬度 (见图 4F), Lm 进入 HMEC-1 细胞的入侵效率 (细菌内化/细菌总数) 是相似的。
Lm 感染 HMEC-1 细胞的牵引力显微术: HMEC-1 细胞的 Lm 感染可改变受感染宿主细胞的生物力学, 包括附着于其 ECM 或彼此的强度, 影响其屏障完整性。我们试图用牵引力显微镜32计算细胞 ECM 牵引力和单层应力显微镜41计算胞内张力, 以此来评估这种情况是否可能。图 5描述了在不同时间点感染后的 20-帕水凝胶 HMEC-1 细胞的形变场、牵引应力场和细胞内张力场的分布图。图 5A - 5D指未感染的控制 HMEC-1 细胞, 而图 5E - 5H指的是 HMEC-1 细胞感染 Lm 在多重感染等于300细菌/细胞。这项初步工作表明, 受感染的 HMEC-1 细胞降低其细胞 ECM 和细胞内应力的程度, 在感染的过程中, Lm, 而这是没有观察到未感染的控制细胞。
ECM 刚度依赖性 Lm 在 HMEC-1 单分子膜中的传播: 采用时移显微术研究了基质刚度对 Lm 通过 HMEC-1 膜传播的影响。当 Lm 通过单层传播时, 细菌会产生一种感染的焦点, 作为时间的函数而增长 (参见图 6a和视频图 1和2)。感染焦点的面积是通过绘制一个凸壳来测量的, 这个最小的凸多边形包含一组点, 围绕细菌42。在这一领域中, 没有标准的指标来衡量L. 李斯特细胞对细胞的效率, 通过一层宿主细胞传播。为了评估传播效率, 一些人计算了感染焦点41中的宿主细胞数, 另一些人在感染宿主细胞组43周围手动绘制了边界。我们选择在细菌周围画一个凸多边形, 因为它是一个自动化的、一致的、计算性很低的过程, 用来测量李斯特菌传播的效率.通过这样做, 我们发现在70帕水凝胶上播种的 HMEC-1 细胞的感染焦点区域略有减少 (见图 6B和视频图 1和2)。为了确定感染焦点的生长速率, 通过取感染焦点区域的平方根并将其与 pi 进行划分, 将径向距离绘制为时间函数。这种数学变换假定感染焦点的形状大致为圆形44。为了测量聚焦生长的速度, 测量了3和70帕矩阵的径向距离的变化率 (即斜率)。这一方法阐明, 感染焦点增长更快, 单调的 HMEC-1 细胞播种到3人民军矩阵。然而, 聚焦在 70-帕矩阵上的单元格 (见图 6C), 其对焦速度明显减慢 (前200分钟) 和稍慢 (200 至600分钟)。事实上, 对进一步数据的分析证实, 在70帕的矩阵中, 感染焦点平均增加了两倍, 特别是在前200分钟 (见图 6D)。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57361/57361fig3.jpg"> 图 3: 应用流式细胞仪测量 HMEC-1 细胞对 Lm 入侵的依赖敏感性. HMEC-1 细胞感染 Lm (JAT985), 通过流式细胞仪对感染进行分析。这个面板显示了一个来自一个井的代表性 HMEC-1 细胞的侧向散射图。通过门控选择细胞的体积分布, 排除碎片 (左) 和细胞双峰或三胞胎 (右)。这张图显示了每个细胞的 Lm 荧光强度对数的直方图, 用于在软0.6 帕上镀 HMEC-1。这张图显示了 HMEC-1 在70帕水凝胶上镀的每细胞的 Lm 荧光强度的对数直方图。N = 4-6 复制的直方图以不同的颜色显示。控制未感染细胞的直方图是紫色的。用来定义被感染的门是用红色显示的。该语言为 100, 感染评估8小时后感染。D. 数据显示受感染的 HMEC-1 细胞与水凝胶硬度的百分比 (N = 5)。水平条形图描述数据的平均值。用非参数魏氏秩和检验计算P值。<a href="/files/ftp_upload/57361/57361fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Video Figure 1" src="/files/ftp_upload/57361/57361mov1.jpg"> 视频图 1:通过在3帕基板上播种的 HMEC-1 细胞单层 (相位) 传播胞内 Lm (红色通道). 细胞在莱博维茨的 L-15 媒介 (10% 个血清, 20 µg/毫升庆大霉素) 被成像在一个环境房间平衡到37˚C。这些图像每5分钟收集一次。电影速度是15帧/秒.<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57361/Video_Figure_1.mov" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Video Figure 2" src="/files/ftp_upload/57361/57361mov2.jpg"> 视频图 2: 通过在70帕基板上播种的 HMEC-1 细胞单层 (相位) 传播胞内 Lm (红色通道). 细胞在莱博维茨的 L-15 媒介 (10% 个血清, 20 µg/毫升庆大霉素) 被成像在一个环境房间里平衡到37°c。这些图像每5分钟收集一次。电影速度是15帧/秒.<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57361/Video_Figure_2.mov" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>杨氏模量 (E, 人民军)</td>
			<td>丙烯酰胺% (从40% 库存)</td>
			<td>Bisacrylamide% (从2% 股票)</td>
		</tr>
<tr>
<td>0。6</td>
			<td>3</td>
			<td>0.045</td>
		</tr>
<tr>
<td>3</td>
			<td>5</td>
			<td>0.075</td>
		</tr>
<tr>
<td>10</td>
			<td>10</td>
			<td>0.075</td>
		</tr>
<tr>
<td>20</td>
			<td>8</td>
			<td>0.195</td>
		</tr>
<tr>
<td>70</td>
			<td>10</td>
			<td>0.45</td>
		</tr>
</tbody></table>表1。变刚度聚丙烯酰胺 (PA) 水凝胶的组成.在这个表中, 40% 的丙烯酰胺溶液的百分比和2% 双丙烯酰胺溶液的百分比达到给定的刚度 (杨氏模量, E) 在不同的列表示。
<img alt="Supplemental Material" src="/files/ftp_upload/57361/57361supfig1.jpg"> 补充材料1。计算牵引应力计算胞内张力.<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57361/Supplemental_Material_1.docx" target="_blank">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells at JoVE.

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

我们开发了一种多井格式的聚丙烯酰胺检测方法, 以探讨细胞外基质硬度对黏附细胞细菌感染的影响。该检测与流式细胞术、染色和牵引力显微镜相兼容, 可以定量测量细胞、胞外基质和致病细菌之间的生物力学相互作用。

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, ...

a-multi-well-format-polyacrylamide-based-assay-for-studying-effect

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

11.6K 次观看.  Stanford University School of Medicine. 我们开发了一种多井格式的聚丙烯酰胺检测方法, 以探讨细胞外基质硬度对黏附细胞细菌感染的影响。该检测与流式细胞术、染色和牵引力显微镜相兼容, 可以定量测量细胞、胞外基质和致病细菌之间的生物力学相互作用。

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

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

"发光法"的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

可见光和庆大霉素的协同作用<em&gt;假单胞菌假单胞菌</em&gt;微生物

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

生物工程

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user&#8217;s specific biological and technical needs.

Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

聚丙烯酰胺凝胶侵袭伪足和牵引力化验的肿瘤细胞

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

医学

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

Quantitative Time-Lapse Microscopy for Assessing Extracellular Matrix Stiffness-Dependent Bacterial Dissemination

Source: Bastounis, E. E. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57361">A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells.</a> J. Vis. Exp.&#160;(2018)
This video showcases quantitative time-lapse microscopy for evaluating extracellular matrix stiffness-dependent Listeria monocytogenes dissemination through endothelial cells. A softer hydrogel facilitates faster bacterial dissemination through endothelial cells compared to stiffer counterparts, highlighting the influence of matrix stiffness on bacterial dissemination.

用于评估细胞外基质刚度依赖性细菌传播的定量延时显微镜

1. Manufacturing Thin Two-layered Polyacrylamide (PA) Hydrogels on Multi-Well Plates

	Dissolve ammonium persulfate (APS) in distilled ultrapure water to ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

An Assay to Study the Impact of Extracellular Matrix Stiffness on Bacterial Infection

Source: Bastounis, E. E., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/57361">A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells.</a> J. Vis. Exp. (2018).
This video demonstrates an assay to study the effect of extracellular matrix (ECM) stiffness on bacterial infection of adherent cells. Polyacrylamide hydrogels of varying stiffness, overlaid with collagen, are layered in a multi-well plate to mimic the stiffness of physiological ECM. Upon overlaying human endothelial cells on the hydrogels and infecting the cells with Listeria monocytogenes bacteria engineered to express a fluorescent protein once intracellular, flow cytometry is used to compare internalized bacteria in cells residing on stiffer hydrogels versus softer hydrogels.

研究细胞外基质刚度对细菌感染影响的测定法

1. Manufacturing Thin Two-layered Polyacrylamide (PA) Hydrogels on Multi-Well Plates

	Polyacrylamide hydrogel fabrication
	NOTE: See&#160;Figure 1.
	
		...