这项工作由劳伦斯利弗莫尔国家实验室LDRD 18-ERD-062（C.R.）资助。感谢 John Muschler 赠送的 DgKO 和 DgKI 细胞。感谢加州大学伯克利分校电子显微镜实验室的工作人员在电子显微镜样品制备和数据收集方面提供的建议和帮助。

1. 解冻和等分试样层粘连蛋白-111
注意：纯化的层粘连蛋白-111 可在市面上买到（通常从作为基质出售的 EHS 基质中纯化），但并非所有来源都能提供完整、未降解的蛋白质。
<ol><li>通过获取 Ln1 和运行尺寸排阻色谱26 或聚丙烯酰胺凝胶电泳26 确认 Ln1 未降解。变性 Ln1 应具有 337 kDa、197 kDa 和 177 kDa 的条带，而天然 Ln1 应具有 711 kDa 的单条带。</li>
	<li>在冰箱中将 Ln1 在冰上解冻过夜，并使用低蛋白结合尖端和无菌技术等分到低蛋白结合微量离心管中（通常以 100 μg/管等分试样进行培养覆盖，或 10 μg 用于包被）27。冷冻等分试样并储存在-80°C。</li>
</ol>2. 使用低pH缓冲液进行层粘连蛋白聚合

<ol><li>制备polyLM聚合缓冲液：称取1 mM CaCl2 和20 mM乙酸钠在超纯水中混合。然后使用HCl或乙酸将pH调节至4.0。通过0.2μm过滤器进行无菌过滤，并储存在4°C。</li>
	<li>制备对照聚合缓冲液：混合 1 mM CalCl2 和 20 mM Tris，并根据需要使用 10 N HCl 或 12 N NaOH 将 pH 调节至 7.0。通过0.2μm过滤器进行无菌过滤，并储存在4°C。 注意：协议可以在此处暂停。</li>
	<li>在冰箱中解冻所需数量的 Ln-1 等分试样过夜。</li>
	<li>使用无菌细胞培养技术，将适当的缓冲液（步骤2.1中的polyLM聚合缓冲液或步骤2.2中的对照聚合缓冲液）加入到终浓度为100μg/ mL的层粘连蛋白等分试样中（即向50μg等分试样中加入0.5mL），并通过移液轻轻混合。</li>
	<li>如果使用荧光标记的层粘连蛋白进行可视化，则重新配制并以 1：50 wt/wt 的比例加入（例如 2 μg/100 μg），并通过移液轻轻混合。 注意：如果使用层粘连蛋白作为细胞附着的底物，请按照步骤2.6-2.8操作：</li>
	<li>在适当的缓冲液中稀释后，立即将层粘连蛋白转移到培养塑料或玻璃器皿上，并在37°C的细胞培养箱中孵育过夜。 请注意，涂覆盖玻片时，加入足够大的体积以产生平衡液滴（通常，13 mm圆形盖玻片使用50μL），并在37°C的加湿室中保存过夜。</li>
	<li>使用无菌技术27，用1x磷酸盐缓冲盐水（PBS）洗涤。</li>
	<li>小心地取出液体。不要让表面完全干燥。 注意：如果使用层粘连蛋白覆盖细胞以诱导牛奶蛋白，请按照步骤2.9-2.12操作：</li>
	<li>培养乳腺上皮细胞。在补充有 2% FBS、0.01 mg/mL 胰岛素、0.005 μg/mL EGF、0.05 mg/mL 庆大霉素和 0.05 g/mL 正霉素的 DMEM-F12 中培养 MepG 抗肌萎缩聚糖敲入 （DgKI） 或 MepG 抗肌萎缩聚糖敲除细胞 （DgKO）。</li>
	<li>在孔板或盖玻片底部培养皿中以 10,500 个细胞/cm2 的速度接种用于层粘连蛋白刺激的 DgKO 和 DgKI 细胞</li>
	<li>将稀释的层粘连蛋白在37°C的细胞培养箱中孵育30分钟。</li>
	<li>将polyLM等分试样以2,000× g 离心20分钟，但由于蛋白质实体的尺寸较小，并且需要更高的速度才能有效收集，因此在20,000× g 的中性pH下离心LM20分钟。</li>
	<li>使用无菌技术27，轻轻除去上清液，并重悬于补充有3μg/mL催乳素，2.8μM氢化可的松和5μg/mL胰岛素的适当体积的DMEM-F12诱导培养基中</li>
	<li>立即将层粘连蛋白转移到细胞培养基中以刺激细胞。我们已经成功地用50-800μg/mL Ln1刺激细胞</li>
</ol>3. 使用分离的糖蛋白进行层粘连蛋白聚合
<ol><li>将重组 nidogen-1 和 Ln1 在冰箱中的冰上轻轻解冻过夜。</li>
	<li>使用低蛋白结合尖端和试管并使用无菌技术，在细胞培养基中以 1：1 wt/wt 的比例将 Ln1 与 nidogen-1 混合。使用细胞培养基中的纯 Ln1 作为对照。</li>
	<li>如果使用荧光标记的层粘连蛋白进行可视化，则以 1：50 wt/wt 的比例（例如，2 μg/100 μg）复溶并添加。用移液器轻轻混合。</li>
	<li>在37°C的细胞培养箱中孵育Ln1-nidogen或对照Ln130分钟。</li>
	<li>将Ln1-nidogen共聚物以2,000× g 离心30分钟，将对照Ln1以20,000× g 离心30分钟。</li>
	<li>轻轻除去上清液并重悬于细胞培养基中，终浓度为 100-800 μg 总蛋白/mL。</li>
	<li>立即用于刺激细胞，或转移到盖玻片上，并在37°C湿润的细胞培养箱中粘附过夜。</li>
	<li>从盖玻片中取出培养基，用 1x PBS 洗涤盖玻片，然后在室温下用 10% 福尔马林（即 4% 甲醛在 PBS 中）固定 15 分钟。</li>
</ol>4. 使用抗肌萎缩聚糖表达细胞的层粘连蛋白聚合
<ol><li>培养乳腺上皮细胞。在补充有 2% FBS、0.01 mg/mL 胰岛素、0.005 μg/mL EGF、0.05 mg/mL 庆大霉素和 0.05 g/mL 正霉素的 DMEM-F12 中培养 MepG 抗肌萎缩聚糖敲入 （DgKI） 或 MepG 抗肌萎缩聚糖敲除细胞 （DgKO）。</li>
	<li>由于生长速率的差异，以 5,000 个细胞/cm2 接种 DgKI 细胞，以 8,000 个细胞/cm2 接种 DgKO 细胞。在37°C湿润培养箱中培养，每2-3天更换一次培养基，每4-5天使用严格的无菌技术传代一次。使用血细胞计数器或自动细胞计数器27 仔细计数细胞，以确保适当的生长速率。</li>
	<li>在孔板或盖玻片底部培养皿中以 10,500 个细胞/cm2 的强度接种用于层粘连蛋白刺激的细胞并培养 2 天，使用 DgKI 细胞产生分形 Ln1 和 DgKO 细胞作为阴性对照。在冰箱中将 Ln-1 在冰上轻轻解冻过夜，然后与补充有 3 μg/mL 催乳素、2.8 μM 氢化可的松和 5 μg/mL 胰岛素的 DMEM-F12 诱导培养基混合。对于 35 mm 培养皿，使用 1 mL 含有 50-800 μg Ln1 的诱导培养基。</li>
	<li>如果使用荧光 Ln1，则以 50：1 wt/wt 的比例混合。</li>
	<li>从细胞中取出培养基，并用补充有 3 μg/mL 催乳素、2.8 μM 氢化可的松和 5 μg/mL 胰岛素的 DMEM-F12 层粘连蛋白和诱导培养基覆盖</li>
	<li>培养细胞 2 天，然后用 10% 福尔马林固定。</li>
</ol>5. 通过光学显微镜表征层粘连蛋白微观结构
<ol><li>收集固定样品，并在室温下用1x PBS洗涤3次5分钟。</li>
	<li>在室温下，用0.5%Triton X-100,3%牛血清白蛋白，10%山羊血清和40μg/ mL抗小鼠阻断抗体片段在1x PBS中封闭和透化样品1小时。 注意： Triton X-100 是危险的。戴上手套和护目镜进行处理并妥善处理。</li>
	<li>将稀释度为1：100的抗层粘连蛋白抗体与1x PBS与0.5%Triton X-100和3%BSA混合，并在室温下或在4°C下在加湿室中加入样品2小时（通常使用底部带有湿实验室湿巾的尖端盒）。</li>
	<li>取出一抗，用 1x PBS 和 0.5% Triton X-100 和 3% BSA 洗涤 3 次。</li>
	<li>在含有0.5%Triton X-100和3%BSA的1x PBS样品中以1：500的稀释度向样品中加入适当的二抗，并在室温下在加湿室中避光孵育。</li>
	<li>去除二抗，用 1x PBS 和 0.5% Triton X-100 和 3% BSA 洗涤 3 次。</li>
	<li>对于含有细胞的样品，加入 6 μM 鬼笔环肽 45 分钟，然后用含有 0.5% Triton X-100 和 3% BSA 的 PBS 洗涤 3 次，然后用 PBS 洗涤 3 次。然后用 0.1 μg/mL DAPI 在 PBS 中染色 5 分钟。</li>
	<li>如果合适，可以挂载样品。</li>
	<li>使用高分辨率共聚焦显微镜对 ln-1 结构进行成像。使用配备浸没物镜的激光扫描显微镜（水浸复消色差计40X/1.1NA或油浸复消色差计63X/1.4NA）。</li>
</ol>6. 通过盒数维度表征层粘连蛋白结构
<ol><li>使用 Matlab 工具箱或 ImageJ 中提供的箱计数维度算法分析分形属性。这些方法对层粘连蛋白的图像进行阈值测量，并在对数对数上绘制含有 Ln1 的盒数与盒的大小。这些参数之间关系的斜率是盒数维数：非分形几何的维数为 0、1 或 2，而分形几何的非整数维数为28。 注意：酸处理、糖蛋白处理和 DgKI 细胞附着的 Ln1 的盒计数分形维数均为 ~1.7，而对照 Ln1 的分形维数为 2.0。</li>
</ol>7. 通过电子显微镜表征层粘连蛋白构建体
<ol><li>将层粘连蛋白重悬于细胞培养基中，并在潮湿的37°C环境中吸附到二氧化硅晶片上90分钟。</li>
	<li>将基质固定在 pH 7.2 的 0.1 M 二甲胂酸钠缓冲液中的 2.5% 戊二醛中。 注意：戊二醛是危险的，应在通风橱中处理，并戴上手套和护目镜，并作为危险废物处理。</li>
	<li>在pH 7.2的0.1M二甲胂酸钠缓冲液中的1%四氧化锇中建立后缀基质。 注意： 四氧化锇是危险的，应在通风橱中处理，并戴上手套和护目镜，并妥善处理。二甲胂酸盐是危险的，应在通风橱中处理，并戴上手套和护目镜，并妥善处理。</li>
	<li>在pH 7.2的二甲胂酸钠缓冲液中洗涤3次。</li>
	<li>随着乙醇浓度的增加而脱水。</li>
	<li>溅射涂层基质上一层薄薄的金。</li>
	<li>使用扫描电子显微镜进行图像处理。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Palmero, C. Y., 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=The+follicular+thyroid+cell+line+PCCL3+responds+differently+to+laminin+and+to+polylaminin,+a+polymer+of+laminin+assembled+in+acidic+pH.">The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH.</a> Molecular and Cellular Endocrinology. 376 (1), 12-22 (2013).</li><li>Hochman-Mendez, C., Lacerda de Menezes, J. R., Sholl-Franco, A., Coelho-Sampaio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polylaminin+recognition+by+retinal+cells.">Polylaminin recognition by retinal cells.</a> Journal of Neuroscience Research. 92 (1), 24-34 (2014).</li><li>Leal-Lopes, C., Grazioli, G., Mares-Guia, T. R., Coelho-Sampaio, T., Sogayar, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerized+laminin+incorporation+into+alginate-based+microcapsules+reduces+pericapsular+overgrowth+and+inflammation.">Polymerized laminin incorporation into alginate-based microcapsules reduces pericapsular overgrowth and inflammation.</a> Journal of Tissue Engineering and Regenerative Medicine. 13 (10), 1912-1922 (2019).</li><li>Paccola Mesquita, F. C., Hochman-Mendez, C., Morrissey, J., Sampaio, L. C., Taylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+as+a+Potent+Substrate+for+Large-Scale+Expansion+of+Human+Induced+Pluripotent+Stem+Cells+in+a+Closed+Cell+Expansion+System.">Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System.</a> Stem Cells International. 2019, 9 (2019).</li><li>Walker, J. M., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Protein Protocols Handbook. , 61-67 (2002).</li><li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Techniques. 7th edition. , (2016).</li><li>Mandelbrot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Fractal Geometry of Nature. , (1982).</li><li>Schittny, J. C., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+short+arm+domains+of+basement+membrane+laminin+are+critical+for+its+self-assembly.">Terminal short arm domains of basement membrane laminin are critical for its self-assembly.</a> Journal of Cell Biology. 110 (3), 825-832 (1990).</li><li>Schuger, L., Yurchenco, P., Relan, N. K., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+fragment+E4+inhibition+studies:+basement+membrane+assembly+and+embryonic+lung+epithelial+cell+polarization+requires+laminin+polymerization.">Laminin fragment E4 inhibition studies: basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization.</a> International Journal of Developmental Biology. 42 (2), 217-220 (1998).</li><li>Colognato, H., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+and+function:+the+laminin+family+of+heterotrimers.">Form and function: the laminin family of heterotrimers.</a> Developmental Dynamics. 218 (2), 213-234 (2000).</li><li>Kabaeva, Z., Meekhof, K. E., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemma+instability+during+mechanical+activity+in+Large+myd+cardiac+myocytes+with+loss+of+dystroglycan+extracellular+matrix+receptor+function.">Sarcolemma instability during mechanical activity in Large myd cardiac myocytes with loss of dystroglycan extracellular matrix receptor function.</a> Human Molecular Genetics. 20 (17), 3346-3355 (2011).</li></ol>

这项工作由劳伦斯利弗莫尔国家实验室在美国能源部的主持下根据合同DE-AC52-07NA27344进行。IM 版本 LLNL-JRNL-799443。

层粘连蛋白是上皮、肌肉和神经器官系统中ECM的关键元素，在体外已被证明在调节细胞功能分化中起着重要作用。越来越多的证据表明，向细胞展示的层粘连蛋白结构调节其信号传导和由此产生的细胞表型15,16，因此控制 Ln1 微观结构的方法应该对在这些领域工作的基础生物学家以及寻求概括体外正常行为的组织工程师感兴趣。更一般地说，已知各种细?...

与生长因子或细胞因子不同，细胞外基质 （ECM） 蛋白可以组装成结构网络。由于ECM功能障碍是许多疾病的关键因素，因此细胞从ECM组成、微观结构和生物力学中感知和转导信号的机制是治疗开发的有吸引力的靶标。越来越多的证据表明，这些网络的微观结构在这些蛋白质如何向细胞发出信号方面起着重要作用。迄今为止，ECM 微观结构与细胞功能之间的这种联系已在胶原 I1、纤连蛋白2 和纤维蛋白3 中得到证明。
层粘连蛋白-111 （Ln1） 是一种三聚体十字形蛋白质，是细胞外基质的关键结构成分，可接触上皮细胞4、肌肉细胞5 和神经细胞6。在乳腺中，Ln1 是乳腺上皮细胞功能分化为产奶细胞所必需的 7,8,9，Ln1 对于诱导肿瘤逆转至关重要 10,11。Ln1 和其他层粘连蛋白是肌肉中皮质肌动蛋白网络和肌节组织发育所必需的12,13，以及神经细胞迁移和神经突生长13 所必需的。因此，了解层粘连蛋白向细胞发出信号的机制是一个活跃的研究领域。
Ln1 包含多种受体的多个粘附结构域，包括整合素、突聚糖、肌营养不良聚糖、LBP-110 和 LamR（图 1A）4,14。含有层粘连蛋白 α1 的 c 末端球状结构域的 E8 片段是结合抗肌萎缩聚糖和整合素 α6β1 和 α3β115 所必需的，并且是上皮细胞功能分化所必需的15,16。相比之下，短臂显示出不同的生物活性17，这意味着在网络形成后识别这些不同Ln1结构域的可用性可以改变细胞行为。
我们最近证明，排列成聚合物网络的 Ln1 表现出分形特性（即在多个长度尺度上自相似的结构）18。与缺乏这种排列的 Ln1 相比，分形网络对上皮细胞发出的信号不同19。该分形网络表示特定的组装结构，其中长臂优先暴露于单元18。由于这种不同的长臂显示，上皮细胞经历功能分化为产奶细胞，具有组织良好的紧密连接和肌动蛋白应力纤维的抑制 19。
我们描述了从短臂-短臂结合相互作用中生成 Ln1 网络的 3 种方法，这些方法显示了 Ln1 长臂的高显示：1） 通过与酸性缓冲液的无细胞组装，2） 通过与糖蛋白共孵育，或 3） 通过与细胞表面抗肌萎缩聚糖的相互作用。这三种方法通过三种截然不同的机制产生相似的层粘连蛋白微观结构，从而在实验设计中具有灵活性。先前的研究表明，这三种方法可以代表生物学的稳健性：在乳腺上皮细胞中，细胞表面抗肌萎缩聚糖、糖蛋白或人工结构的层粘连蛋白都可以诱导功能分化19。因此，研究人员可以根据研究对象在这些方法之间进行选择：表达抗肌萎缩聚糖的细胞对 Ln-1 微观结构不敏感，因为细胞膜中的抗肌萎缩聚糖诱导正确的聚合成分形网络19,20。基质凝胶是层粘连蛋白和糖蛋白的混合物21，代表了Ln-1最经济的来源，并提供了诱导抗肌萎缩聚糖敲除乳腺细胞分化所需的微观结构20，但含有具有批次间变异性的复杂蛋白质混合物。PolyLM 代表了提供此功能的最干净、最还原的系统，但与Matrigel 19 相比，诱导乳腺细胞分化的成本更高，功效更低。
这三种方法具有扩散限制聚集的分形网络结构特征18,19，并且与多个实验系统中的聚集层粘连蛋白相比，显示出生物活性的改变22,23,24,25。使用这些方法的未来研究可以识别上皮分化所需的受体和信号传导，并确定在异常微环境20（例如肿瘤）中恢复细胞中的正常细胞-基质相互作用。

<table border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="665">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% formalin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">HT501128</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin primary antibody</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L9393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Rabbit Alexafluor 488 secondary antibody</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A32731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C4901 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">Heracell 150 or similar</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge for microcentrifuge tubes</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:161px;">5418 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal Microscope</td>
			<td style="width:121px;">Zeiss</td>
			<td style="width:161px;">LSM 710 or equivalent</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">25CIR-1 or similar</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">11330107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGF (Epidermal Growth Factor)</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">11376454001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescently Labeled Laminin</td>
			<td style="width:121px;">Cytoskeleton Inc</td>
			<td style="width:161px;">LMN02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gentamicin</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">VWRV0304-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">G5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">320331 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrocortisone</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">H0888-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">16634-50mg</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MepG Dystroglycan Knockout Cells</td>
			<td style="width:121px;">LBNL</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S8045 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normocin</td>
			<td style="width:121px;">Invivogen</td>
			<td style="width:161px;">ant-nr-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium Tetroxide</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">75633</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ovine Prolactin</td>
			<td style="width:121px;">Los Angeles Biomedical Research Institute</td>
			<td style="width:161px;">Ovine Pituitary Prolactin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phalloidin</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A22287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">10010023 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Purified Laminin-111</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L2020-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Recombinant human Nidogen-1, carrier free</td>
			<td style="width:121px;">R&#38;D systems</td>
			<td style="width:161px;">2570-ND-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Acetate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S2889 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Cacodylate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile filters</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">SLGP033RS or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">252859 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">X100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-low protein binding tubes</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">76322-516, 76322-522 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrapure water</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">15230253 or similar</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating a fractal microstructure of laminin-111 to signal to cells

层粘连蛋白-111 （Ln1） 是上皮、肌肉和神经系统中细胞外基质的重要组成部分。我们之前已经证明，Ln1的微观结构改变了它向细胞发出信号的方式，这可能是因为Ln1组装到网络中暴露了不同的粘附结构域。在该协议中，我们描述了三种生成聚合Ln1的方法。

层粘连蛋白-111是一种三聚体蛋白，在其短臂末端具有三个自组装结构域（图1A）。如果处理得当，短臂可以聚合成六边形晶格（图1B，1C），在更大的空间尺度上显示出扩散限制的聚集体分形特性（图2）。在含钙的中性pH缓冲液中孵育的层粘连蛋白形成用抗Ln1抗体可视化的小聚集体（图2A），其?...

Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

我们描述了三种生成具有分形特性的 Ln1 聚合物的方法，与未聚合的 Ln1 相比，这些聚合物向细胞发出的信号不同。

生成层粘连蛋白-111的分形微观结构以向细胞发出信号

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...

generating-a-fractal-microstructure-of-laminin-111-to-signal-to-cells

Research

JoVE Journal

Bioengineering

912 次观看.  Texas Heart Institute. 我们描述了三种生成具有分形特性的 Ln1 聚合物的方法，与未聚合的 Ln1 相比，这些聚合物向细胞发出的信号不同。

视频: Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

生成受控动态化学景观，研究微生物行为

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

利用软光刻改变根面微结构的仿生复制

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

增强分子化合物输送到细胞的声透流体装置的组装和操作

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

荧光蛋白中脯氨酸类似物对脯氨酸的残基特异换：骨架的“分子手术”如何影响折叠和稳定性

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...