我们感谢Matthew Blosser的宝贵讨论和建议。这项工作得到了海军研究办公室（N00014-16-1-2382）和国家科学基金会（PHY-1915017）的支持。

1. 蛋白质标记
<ol><li>允许NHS-罗丹明，5-HT1A 膜碎片和一个7 K MWCO自旋脱盐柱在室温下平衡。</li>
	<li>将1mg NHS-罗丹明溶解在100μL二甲基亚砜（DMSO）中。</li>
	<li>加入5μL1M碳酸氢钠溶液，将5-HT1AR溶液的pH值提高到pH 8。</li>
	<li>将3.66μLNHS-罗丹明溶液加入50μL5-HT1AR溶液中，并在微量离心管中轻轻上下移液。 注意：确保NHS-罗丹明的摩尔含量至少为10倍。</li>
	<li>保持混合物避光，并在室温下放在旋转器上1小时。</li>
	<li>每次洗涤用200μL1x磷酸盐缓冲盐水（1x PBS）洗涤7 k MWCO离心柱三次，每次洗涤1.5分钟。。</li>
	<li>将标记的蛋白质加入一个色谱柱中，并在另一个微量离心管中平衡量。</li>
	<li>在1.5 RCF下旋转标记的蛋白质一次5分钟。</li>
	<li>使用纳米滴形分光光度计在280nm和554nm处获取紫外可见光度计，并按照制造商的手册计算标记效率。</li>
	<li>将标记的蛋白质储存在5°C下，直到进一步使用。该溶液在贴标后约一周内稳定。</li>
</ol>2. 采用膜 5-HT 1A 的 GUV
<ol><li>材料和试剂的制备
	<ol>
<li>让蛋白质，脂质和BSA（牛血清白蛋白）平衡到室温。</li>
		<li>在此期间，通过将盖玻片置于甲醇中并在40°C下超声处理30分钟来清洁盖玻片。 确保甲醇完全覆盖盖玻片，并且水浴中的水位高于容器中甲醇的水平。 注意：甲醇是有毒的，应在适当的化学罩中处理。</li>
		<li>用柔和的气流擦干盖玻片上多余的甲醇。将盖玻片架盖在40°C的烤箱中15分钟，以确保多余的盖玻片干燥。</li>
		<li>开始等离子清洗过程。首先，将盖玻片放入等离子清洗机中，并关闭进气阀以排出腔室内的所有空气。</li>
		<li>一旦腔室处于真空状态，使用高RF功率设置和接近完全的真空清洁盖玻片5分钟，仅将轻微的空气吸入真空室。为确保等离子体的适当水平，请调整真空室的开口，使等离子体的最终颜色为稳定，明亮的粉红色。 注意：当使用空气时，等离子体在等离子体处理步骤期间保持明亮的粉红色至关重要，因为深紫色表示腔室中存在不适当的空气量，并将导致次优等离子体处理。</li>
		<li>5分钟后，关闭射频电源并释放真空。 注：从等离子体室取出后，请确保盖玻片保持覆盖。</li>
	</ol></li>
	<li>水凝胶制备
	<ol>
<li>将 6 mg 超低温琼脂糖与 300 μL 超纯水（即 2% （w/v） 琼脂糖）混合。 注意：2%琼脂糖将用于制造无蛋白GUV。琼脂糖溶液可以在45°C下保存两天。</li>
		<li>将9mg超低温琼脂糖与300μL超纯水混合，制成3 w / v%琼脂糖，如步骤3.1中制备。3%琼脂糖将用于制造蛋白质掺入的GUV。</li>
		<li>在将溶液置于90°C加热块上10分钟之前，先对溶液进行短暂的涡旋。然后，在将其转移到45°C加热块之前再次涡旋管，以使其保持熔融形式，直到进一步使用。</li>
	</ol></li>
	<li>琼脂糖和蛋白质混合
	<ol>
<li>将21μL3%琼脂糖与7μL5-HT1AR膜片段混合。多次缓慢地上下移液，以确保充分混合。然后，在45°C下孵育1分钟。</li>
	</ol></li>
	<li>水凝胶和脂质沉积
	<ol>
<li>对于无蛋白GUV：使用20μL2%琼脂糖在新鲜血浆清洁的盖玻片上制作薄膜。快速将另一个盖玻片滴在琼脂糖液滴的顶部，轻轻地将盖玻片分开，在两个盖玻片上形成一层薄膜。 注意：这一步很棘手，因为液滴的滑动必须发生在琼脂糖仍处于熔融形式时。</li>
		<li>对于蛋白质掺入的GUV：再次上下移取蛋白质/琼脂糖混合物，然后将20μL2%琼脂糖沉积在血浆清洁的盖玻片上。按照上述滑移操作说明进行操作。</li>
		<li>让琼脂糖在室温下凝胶避光30分钟。</li>
		<li>将脂质滴滴沉积在琼脂糖层的顶部。在琼脂糖薄膜上使用总共10μL2mg / mL的1-棕榈酰基-2-油酰基-甘油-3-磷酸胆碱（POPC）和0.4摩尔%的1，2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺（DPPE），标记有ATTO 488（ATTO-488-DPPE）（或感兴趣的脂质混合物）。使用气相色谱针沉积液滴，并通过温和的气流一次散布一个液滴。 注意：此步骤需要小心，以便在水凝胶顶部形成相对均匀的脂质层。此外，氯仿是有毒的，应该在适当的化学罩中处理。</li>
		<li>通过将O形圈放在盖玻片的顶部，然后将腔室的顶部组件放在O形圈的顶部，来组装Sykes-Moore（S-M）腔室。使用制造商提供的钥匙，通过将腔室组件拧在一起来组装腔室，以密封腔室并防止任何泄漏。 注意：腔室的顶部应在O形圈上拧紧，但需要小心以确保盖玻片保持完好无损，因为如果O形圈不能正确放置在腔室中，盖玻片可能会破裂。此外，确保腔室密封得足够紧密，以便在添加膨胀溶液时腔室不会泄漏。如果腔室拧得不够紧，将导致泄漏和样品丢失。</li>
	</ol></li>
	<li>囊泡肿胀和收获
	<ol>
<li>通过在1x PBS中轻轻移取450μL200mM蔗糖并轻轻敲击腔室以确保水凝胶脂质层的足够缓冲液覆盖，来水合整个系统。 注意：蔗糖溶液可以用含有目标生物探针的补液缓冲液代替。</li>
		<li>将腔室置于45°C，并用盖玻片覆盖腔室的顶部以防止蒸发。让样品膨胀，防止碎屑和光线照射1小时。</li>
		<li>将100μL 1 mg / mL BSA在超纯水中加入到打算使用的96孔板的每个孔中。在室温下孵育1小时。</li>
		<li>用超纯水洗涤三次，用200 mM蔗糖在1x PBS中洗涤一次。</li>
		<li>最后，在1x PBS中加入200mM葡萄糖，直到加入GUV样品溶液。 注：BSA用于阻断GUV吸附。</li>
		<li>在让水凝胶膨胀后，轻轻摇晃并敲击腔室以移开可能仍然附着在水凝胶表面的任何GUV。然后，小心地移取GUV-蔗糖溶液。 注意：作为确保所有囊泡从表面分离的可选步骤，轻轻地将一些蔗糖悬浮液移回水凝胶表面。</li>
		<li>将悬浮液移动到先前制备的微量离心管中，该离心管在1x PBS中含有700μL200mM葡萄糖。 注意：密度梯度将导致囊泡沉降到离心管的底部。</li>
		<li>让囊泡再沉降一小时，以确保囊泡可以沉入微量离心管的底部，从而实现最佳收集。</li>
		<li>在GUV沉降在葡萄糖中后，将300μL从离心管底部（沉降的囊泡）转移到先前制备和BSA处理的96孔板中，以在共聚焦显微镜下检查囊泡。 注意：一定要避开微量离心管的最底部，以尽量减少最终样品中收集的碎屑量。</li>
	</ol></li>
	<li>在显微镜下检查样品。
	<ol>
<li>在样品上照射488nm激光（这使我们能够可视化膜，因为双层已用ATTO-488-DPPE标记）。</li>
		<li>在样品上照射561nm激光（这使我们能够可视化蛋白质，因为它已被NHS-罗丹明标记）。 注意：对样品进行成像时需要小心，因为光氧化会破坏囊泡的稳定性。在同一天观察到囊泡。</li>
	</ol></li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62830/62830fig01.jpg"> 图 1：详细协议步骤的说明。创建 BioRender.com <a href="https://www.jove.com/files/ftp_upload/62830/62830fig01large.jpg" target="_blank">请单击此处查看此图的放大版本。</a>

作者没有利益冲突需要披露。

我们已经确定了对整体方案的成功至关重要的两个步骤：血浆处理和脂质沉积。盖玻片的血浆清洗对于确保琼脂糖水凝胶有足够的覆盖和粘附到玻璃盖玻片上至关重要。等离子清洗可以完成两件事：首先，它可以去除玻璃表面的有机物痕迹;其次，它激活盖玻片表面，随着玻璃表面亲水性的增加，润湿性增加62，63。等离子体清洁后触摸盖玻片表面会失活...

合成模型膜是研究生物膜基本性质和功能的强大工具。巨型单层囊泡（GUV）是研究各种质膜特性的最突出平台之一，可以设计成模仿不同的生理条件1，2，3，4，5，6，7，8。众所周知，质膜及其组织在众多细胞过程中起着关键作用，例如信号转导，粘附，内吞作用和转运9，10，11，12，13，14，15。
GUV已使用各种方法制造，包括温和的水合作用16，水凝胶溶胀17，电铸18，微流体技术19，20，21，22，喷射23和溶剂交换24，25，26。由于处理整体膜蛋白（IMP）的挑战，研究它们的 体外 平台受到限制。GUV提供了一个简化的平台，用于在模仿其原生环境的环境中研究IMP。尽管在GUV中有几种蛋白质重构的方法，但结合具有正确取向的蛋白质并保持蛋白质功能会带来挑战27。
GUV中最成功的蛋白质重构需要洗涤剂交换方法;这涉及通过洗涤剂将蛋白质从其天然环境中溶解，然后进行蛋白质纯化，然后通过各种方法用脂质代替洗涤剂分子28。虽然洗涤剂在纯化过程中用于稳定IMP的三级结构，但洗涤剂胶束对于这些蛋白质来说是一个相对不自然的环境，它们在脂质双分子层中稳定性更好，特别是对于功能研究28，29，30。此外，由于这些蛋白质的大小，细腻以及需要额外的洗涤剂交换步骤，使用传统的GUV制造技术将功能性跨膜蛋白掺入脂质双层中一直很困难27，31，32，33。使用有机溶剂去除去垢剂会导致蛋白质聚集和变性34。改进的洗涤剂介导方法一直很有希望，但是，洗涤剂去除步骤需要谨慎，并且可能需要对特定蛋白质进行优化31，35。此外，利用电铸的方法可能会限制蛋白质的选择，并且可能不适用于所有脂质组合物，特别是带电脂质31，36，37。已经使用的另一种技术是肽诱导的含有所需蛋白质的大单层囊泡（LUV）与GUV的融合，尽管它被发现是费力的，并且可能导致插入外来分子 - 梭原肽33，38，39。来自活细胞的巨型质膜囊泡（GPMV）可用于克服其中一些问题，但是它们可以对所得的脂质和蛋白质组成进行最小控制14，40，41。因此，使用我们改良的琼脂糖溶胀方法将IMPs整合到GUV的双脂层中，为在膜环境中进一步检查这些蛋白质提供了一种可靠的方法42，43，44，45。
细胞信号传导和通信涉及称为G蛋白偶联受体（GPCRs）的蛋白质家族;GPCR是最大的蛋白质家族之一，与调节情绪、食欲、血压、心血管功能、呼吸和睡眠以及许多其他生理功能有关46。在这项研究中，我们使用人类血清素1A受体（5-HT1AR），它是GPCR家族的典型成员。5-HT1AR可以在中枢神经系统（CNS）和血管中找到;它影响许多功能，如心血管，胃肠道，内分泌功能，以及参与情绪调节47。GPCR研究的一大障碍来自其复杂的两亲性结构，而GUV为研究各种感兴趣的性质提供了一个有前途的平台，包括蛋白质功能，脂质 - 蛋白质相互作用和蛋白质 - 蛋白质相互作用。已经使用各种方法来研究脂质 - 蛋白质相互作用，例如表面等离子体共振（SPR）48，49，核磁共振波谱（NMR）50，51，蛋白质脂质覆盖（PLO）测定51，52，53，54，天然质谱法55，等温滴定量热法（ITC）56，57和脂质体沉降测定法58，59。我们的实验室使用简化的GUV方法，通过包封BODIPY-GTPγS来研究脂质 - 蛋白质相互作用对蛋白质功能的影响，BODIPY-GTPγS在受体的活性状态下与Giα 亚基结合。它们的结合使荧光团解压，产生荧光信号，该荧光信号可以随着时间的推移被检测到45。此外，各种研究调查了脂质 - 蛋白质相互作用以及蛋白质在传感或稳定膜曲率中的作用60，61，利用可行的GUV方法可能是一个关键优势。
该协议展示了一种使用改性琼脂糖水凝胶系统将GPCR掺入GUV膜中的简单方法17，42。此外，基于我们之前的工作，我们的方法可能适用于可以承受短期暴露于30-40°C的IMP。 简而言之，我们铺展了一层琼脂糖薄膜与含有目标GPCR的膜片段相结合。在该层凝胶化之后，我们在琼脂糖上沉积脂质溶液，并允许溶剂蒸发。然后用水缓冲液进行系统的再水化，导致形成GUV，其中蛋白质掺入脂质双分子层。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td>Avanti Polar Lipids, Alabaster, AL</td>
			<td>850375C-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;TI-Eclipse inverted microscope</td>
			<td>Nikon, Melville, NY</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)</td>
			<td>Avanti Polar Lipids, Alabaster, AL</td>
			<td>850355C-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>13/16&#8243; ID, 1&#8243; OD silicon O-rings</td>
			<td>Sterling Seal &#38; Supply, Neptune, IN</td>
			<td>5-003-8770</td>
			<td></td>
		</tr>
		<tr>
			<td>16-bit Cascade II 512 electron-multiplied charge coupled device camera</td>
			<td>Photometrics, Huntington Beach, CA</td>
			<td>&#160;Cascade II 512</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)</td>
			<td>Avanti Polar Lipids, Alabaster, AL</td>
			<td>850457C-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mW solid-state lasers at 488 nm and emission filter centered at 525 nm, and 561 nm with emission filter centered at 595 nm</td>
			<td>Coherent, Santa Clara, CA</td>
			<td>488/561-50-LS</td>
			<td></td>
		</tr>
		<tr>
			<td>5-HT1AR membrane fragments</td>
			<td>Perkin Elmer, Waltham, MA</td>
			<td>RBHS1AM400UA</td>
			<td></td>
		</tr>
		<tr>
			<td>ATTO-488-1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)</td>
			<td>ATTO-TEC, Siegen, Germany</td>
			<td>AD 488-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench top plasma cleaner</td>
			<td>Harrick Plasma, Ithaca, NY</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin (BSA)</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform (CHCl3)</td>
			<td>Millipore Sigma, Burlington, MA</td>
			<td>CX1055</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol (Chol)</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>C8667-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96-well Flat Clear Bottom</td>
			<td>Corning, Corning, NY</td>
			<td>3904</td>
			<td></td>
		</tr>
		<tr>
			<td>Elmasonic E-Series E15H Ultrasonic</td>
			<td>Elma, Singen, Germany</td>
			<td>[no longer sold on main website]</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol (MeOH)</td>
			<td>Millipore Sigma, Burlington, MA</td>
			<td>MX0485</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>NHS-Rhodamine</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>46406</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline (PBS) (10x PBS)</td>
			<td>Corning, Corning, NY</td>
			<td>21-040</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning-disc CSUX confocal head</td>
			<td>Yokogawa,Tokyo, Japan</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>standard 25 mm no. 1 glass coverslips</td>
			<td>ChemGlass, Vineland, NJ</td>
			<td>CLS-1760</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Sykes-Moore chambers</td>
			<td>Bellco, Vineland, NJ</td>
			<td>1943-11111</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low melting temperature agarose</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>A5030</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Analog Heatblock</td>
			<td>VWR International, Radnor, PA</td>
			<td>[no longer sold on main website]</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Tube Rotator</td>
			<td>VWR International, Radnor, PA</td>
			<td>10136-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>89882</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of model lipid membranes incorporating g-protein coupled receptors (gpcrs)

由于质膜的复杂性以及影响活细胞中蛋白质行为的众多因素，对整体膜蛋白的结构和功能进行强有力的 体外 研究一直是一项挑战。巨型单层囊泡（GUV）是一种仿生和高度可调 的体外 模型系统，用于研究蛋白质 - 膜相互作用并以精确的刺激依赖性方式探测蛋白质行为。在该协议中，我们提出了一种廉价而有效的方法来制造GUV，其中人5-羟色胺1A受体（5-HT1AR）稳定地整合到膜中。我们使用改良的水凝胶溶胀方法制造GUV;通过将脂质膜沉积在琼脂糖和5-HT1AR的混合物上，然后对整个系统进行水润，可以将正确定向和功能性的5-HT1AR结合到膜中形成囊泡。然后，这些GUV可用于通过显微镜检查蛋白质 - 膜相互作用和定位行为。最终，该协议可以促进我们对整体膜蛋白功能的理解，提供深刻的生理洞察力。

测量蛋白质的浓度，并将标记程度计算为染料与蛋白质之间的摩尔比为1：1。通过使用共聚焦显微镜检查GUV，我们能够确认囊泡的成功形成和蛋白质整合。用0.4摩尔%ATTO 488-DPPE标记脂质，并通过伯胺的罗丹明NHS酯修饰共价标记蛋白质。 图2a 和 图2b 分别显示了ATTO 488和罗丹明通道中掺入蛋白质的囊泡。所有显微照片都经过暗电流和平坦场校正。 <strong class...

Watch this Scientific Journal Video about Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs at JoVE.com

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

该协议利用琼脂糖肿胀作为一种强大且可推广的技术，用于将整体膜蛋白（IMP）掺入巨型单层脂质囊泡（GUV）中，如本文所述，用于重建人1A 5-羟色胺受体蛋白（5-HT1AR），这是药理学上重要的G蛋白偶联受体之一。

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

由于质膜的复杂性，研究天然环境中的整体膜蛋白一直是一个挑战。巨型单层脂质囊泡或GUV是研究生物膜特性的最突出平台之一，它们可以被设计成模仿各种生理条件。该协议描述了一种基于水凝胶溶胀后自发掺入的GUV来制造蛋白质掺入GUV的方法。制造GUV的传统方法不能推广为整体膜蛋白掺入。这些方法通常产量低，方法复杂且临时，不适用于广泛的蛋白质类别。另一方面，该协议代表了一种灵活，稳健和简单的方法，以结合GUV中的功能性整体膜蛋白.我们通过制备含有蛋白质的水凝胶支架并在其上沉积一层薄薄的脂质来实现这一点。在这里给出的例子中，我们在GUV中掺入了G蛋白偶联受体或GPCR.在整个系统重新水合后，GUV从水凝胶表面萌芽，膜含有正确取向的GPCR.允许所有试剂达到室温。在此期间，通过将盖子放入甲醇中并在40下超声处理30分钟来清洁盖子。

construction-model-lipid-membranes-incorporating-g-protein-coupled

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Cell Biology

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Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Unit 1

Membrane Structure and Components

SCIENTISTS IN ACTION

3.4K 次观看.  University of Southern California. 由于质膜的复杂性，研究天然环境中的整体膜蛋白一直是一个挑战。巨型单层脂质囊泡或GUV是研究生物膜特性的最突出平台之一，它们可以被设计成模仿各种生理条件。该协议描述了一种基于水凝胶溶胀后自发掺入的GUV来制造蛋白质掺入GUV的方法。制造GUV的传统方法不能推广为整体膜蛋白掺入。这些方法通常产量低，方法复杂且临时，不适用于广泛的蛋白质类别。另一方面?...

视频: Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

使用无标记光学生物传感器由G-蛋白偶联受体，以检测钾通道的调制

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

科研

生物工程

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

一种新型声带生物反应器的构建与鉴定

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

通过结合表面接枝和修改后的过程光敏感膜的制备

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

构建模块化水凝胶片的微模式化宏观缩放3D细胞结构

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

用于动态生长测试的温度，光照和pH监测的台式藻类光合生物反应器的构建和设置

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

人工脂质纳米管网作为内质神经的底部模型的自发形成与重排

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

离子通道掺杂脂质膜组成的生物分子记忆器的组装与表征

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.