我们感谢洛朗Limozin，皮尔·迪拉德和阿斯特丽德·瓦赫勒继续有关手机应用富有成效的讨论。我们也从PLANETE洁净室设备感谢弗雷德里克·贝杜他与SEM观察的帮助。这项工作是部分由欧洲研究委员会通过批准号307104 FP / 2007-2013 / ERC资助。

1.清洗玻璃盖，幻灯片和观察室<ol><li>安排在诸如聚四氟乙烯（PTFE）由惰性材料制成的多滑动托盘玻璃盖滑动。 </li><li>浸入表面活性剂溶液与滑动托盘和观察室（推荐用于清洁石英比色杯中是合适的任何产物）。 </li><li>使用在室温下30分钟的表面活性剂溶液的超声波浴，超声处理（20和30℃之间）。 </li><li>漂洗用超纯水5倍（18.2MΩ.cm，0.059μS/ cm）的。 </li><li>超声处理中，在室温下30分钟表面活性剂溶液（20和30℃之间）。 </li><li>冲洗用超纯水10倍。 </li><li>重复步骤1.5和1.6的两倍。 </li><li>储存在室温下（20和30℃之间）的水长达一周。 </li></ol> 2.蛋白质纳米点的制备<ol><li>珠沉积<ol><li> Deposi吨悬浮液的以15度的倾斜保持二氧化硅珠（2％V / V，70μL）通过在盖滑动滴（24×24毫米，厚170微米）。 </li><li>让该悬浮液散布约1分钟，同时以90°每隔15秒翻转玻璃。 </li><li>允许液体在环境条件下蒸发。 </li><li>储存在清洁的玻璃或PTFE室中为2天环境条件。 </li></ol></li><li>的铝沉积<ol><li>放置一个射频内的载玻片（2.1节中制备）（RF）磁控溅射设备8，在旋转台在约105 mm处的铝（99％）的距离:硅（1％）的目标。 </li><li>使用涡轮分子泵将淀积室抽空到2.6×10 -4 Pa以下。这一步对于去除可能的气体杂质重要。 </li><li>与0.8帕（6.6毫托）的压力下的10sccm的助熔剂引入纯氩气气氛（5N，99.999％）。 <br/&gt;注:传入的氩气流量由质量流控制器控制，并使用压力表进行监测的过程中的压力。氩气流量是连续的，并且通过入口和出口打开阀之间的平衡控制。 </li><li>交换机上的RF功率发生器。 注意:在此，使用400-600W¯¯射频功率的在13.56MHz频率的典型范围。 RF发生器一起使用以电容耦合等离子体模式中的匹配网络。反射功率被监控，并且使用网络适配最小化。 </li><li>等离子体稳定后，溅射2分钟保持关闭，以从靶的表面除去可能的杂质快门。 </li><li>打开快门并允许溅射继续60分钟，在玻璃上沉积滑动铝至厚度为200纳米。 </li><li>切断氩气的流动，关闭闸阀来隔离从沉积腔室中的涡轮分子泵和排气用干净n中的腔室itrogen获得了房间的压力。回收的铝 - 包被的载玻片。存储长达一个月的环境条件下，清洁和密封的玻璃或PTFE框。 </li></ol></li><li>有机硅烷的气相沉积<ol><li>沉浸在步骤2.2.6在超纯水中，在室温下和超声处理在超声浴中制备的30秒铝包覆滑动件（50 W，50/60赫兹，20和30℃之间）。 </li><li>存款0.5 mL，在干燥器的底部（3-氨基丙基） - 三乙氧基硅烷（APTES）的。 注意:APTES为有机硅烷，其蒸发容易且是有毒的。 APTES只应在流罩和手套进行处理。 </li><li>把玻璃载片（在2.3.1的方法制备）在干燥器内的陶瓷网格和地点。 </li><li>干燥器连接到隔膜泵和在最大功率下运行30分钟，以产生低真空。 </li><li>合上干燥器的阀并关闭泵。 </li><li>加热干燥器至约50℃，持续1个小时。 </li><li>打开干燥器和收集的幻灯片。 </li><li>转移到另一个清洁干燥器中储存在室温下（20至30℃）达48小时。 </li></ol></li><li>蛋白的第一层的沉积 - 用生物素标记的牛血清白蛋白（BSA - 生物素） <ol><li>放置在PTFE支撑在第2.3节中制备的载玻片中的一个。 </li><li>存款1毫升溶解在磷酸盐缓冲盐水（PBS）25微克/毫升BSA - 生物素的。留在室温下（20至30℃）30分钟。 </li><li>用PBS冲洗10次。在4℃下存储最多24个小时将样品从光源远。 </li></ol></li><li>铝掩模的去除<ol><li>孵育载玻片在NaOH在PBS过夜，在室温（20至30℃）溶液（通过滴加1M NaOH中的制备约100毫升PBS中，以得到pH≈12）。 警告。氢氧化钠具有腐蚀性，应使用手套来处理。 </li><li>冲洗在超纯水中的10倍。 </li></ol></li></ol> 3.支持的脂质双层的沉积（SLB） <ol><li>清洁朗缪尔槽<ol><li>在使用泵的朗缪尔槽的PTFE外壳除去水。 </li><li>清洁用氯仿中浸过的无绒无纺一次性毛巾。 警告。氯仿是有毒的，并应在通风良好的区域进行操作，用掩模（或下一个流罩）中，用合适的手套。 </li><li>清洁4次用微温的超纯水（40〜50℃）。 </li><li>清洁至少6次用冷超纯水。 </li></ol></li><li>第一脂质层的沉积<ol><li>放置PTFE托盘在朗缪尔槽的PTFE外壳，然后用超纯水填充它。 </li><li>使用朗缪尔装置的控制软件来设置所测量的压力为0 mN / m的。 </li><li>使用气密的玻璃/金属注射器以沉积1mg / mL的脂质溶液的30微升（1，2- dioleoyl- SN中的水的表面上-glycero -3-磷酸胆碱（DOPC）的氯仿溶液）。将氯仿蒸发和脂质分子自发形成一个单层。 </li><li>使用控制软件通过关闭PTFE屏障直到所希望的压力（DOPC 27 mN / m的）达到以压缩脂质单层。 </li><li>使用控制软件浸在节2.5.2制备成使用机动夹具的PTFE外壳中的载玻片上。 </li><li>保持滑动，在夹具，垂直于空气 - 水界面。使用控制软件通过接口，以提高它慢慢（15毫米/分钟），同时保持恒定的压力在27 mN / m的。 </li><li>在干燥的环境的地方或者立即使用或储存于室温下（20和30℃之间）至24个小时。 </li></ol></li><li>第二脂质层的沉积<ol><li>维持朗缪尔槽的表面压力在27 mN / m的所需的值。压缩使用朗缪尔设备和/或控制软件添加脂质少量以实现期望的压力。 </li><li>放置携带脂质单层，在节3.2.6制备的，水平的水的表面上的玻璃玻片。确保每个滑动是浮动的PTFE托盘的上方。 </li><li>推玻璃向下滑动，一次一个滑动，到使用PTFE或金属镊子，使得它们被浸没在水中其相应的PTFE托盘。避免倾斜的幻灯片，同时推动。 </li><li>使用PTFE或金属镊子包含幻灯片的PTFE托盘转移到填充有超纯水确保幻灯片不暴露于空气中的结晶器中。 </li><li>使用PTFE或金属镊子来传输，而工作水下，双层涂覆的玻璃载片进观察室中。 注:观察腔室放置在水中结晶内部。观察室被定制，并且由PTFE环的用橡胶垫圈的ð钢外壳，该可组装的水下，使水密室，其底部表面从预先涂覆有脂质双层载玻片制成。 </li><li>合室中，同时继续在水下工作，并确保约1mL的水被截留在室的内部。 </li><li>就拿组装室出来的水。请确保它是防水的和无泄漏。 </li><li>通过添加和删除的PBS中的10倍500μL取代1mL的存在于与PBS的观察室的超纯水。确保该室是从未缺乏液体。 </li></ol></li><li> SLB封闭步骤<ol><li>在包含在3.3.6节中制备的载玻片上的观察室引入100微克/毫升的牛血清白蛋白（BSA）和孵育在室温下（20和30℃之间）30分钟。 </li><li>通过删除和添加的PBS中的10倍500μL冲洗双层。该双层可在4℃下保持24个小时。 </li></ol></li></ol> 4.功能化与配体<ol><li>在2μg/ mL的添加荧光或非荧光中性（NAV，抗生物素蛋白的去糖基化版本不在pH 7充电）到含有在室温下在部分3.4.2制备30分钟的滑动件（20和30°之间的腔室C）。 </li><li>通过添加和删除的PBS中的10倍500μL冲洗。 </li><li>在2μg/ mL的生物素化的添加抗CD3到含有在3.4.2节中制备的载玻片室中。对于SLB所述点的双官能化，以5μg/ mL的（这里，双层由DOPC的次氮基三乙酸（NTA）的脂质的+ 1％）添加的Fc-ICAM1 His标签。留在室温下（20和30℃之间）30分钟。 </li><li>通过添加和删除的PBS500μL的10倍冲洗</li><li>通过添加和移除细胞培养基的500微升10倍替换细胞培养基的PBS（PBS + 0.1％BSA）。 </li><li>离开细胞培养基的200微升的chambeR 2与滑动。 </li><li>添加细胞前，放置室，用于在37℃下10分钟。 </li></ol> 5.细胞沉积（详见参考文献7） <ol><li>仔细沉积400μL细胞悬浮液的腔室中。温育30分钟将细胞在37℃。 </li><li>固定用多聚甲醛的2％（PFA）的细胞在37℃下15分钟。通过反复删除和添加的PBS的500微升替换用PBS PFA。 </li></ol> 6.观察<ol><li> PROTEO脂质的纳米图案和SLB流动性<ol><li>使用适当的照明波长（639纳米）和滤波器立方体使用荧光显微镜图像在落射荧光模式的蛋白质图案（ 例如这里，EX TBP 483 + 564 + 642; BS TFT 506 + 582 + 659; EM TBP 526 + 601 688）。 注:该信号的强度可以被量化以估计内部和外部点的蛋白质的量。使用适当的照明波长（330纳米）和fILTER立方体图像双层（BP12分之365，FT 395，LP 397），这似乎与亮暗孔。 </li><li>使用连续光漂白（CPB）技术13,15以测量示踪剂的脂质双层中的扩散常数。这一观察刚沉积SLB后优选由。 注意:为了量化扩散常数，两个参数在CPB方法测定:将染料（拉紧）和周围的漂白区（TAUD）形成晕圈的荧光分布的衰减长度的特定漂白时间。首先是通过在照明场的期间CPB过程中心绘制的荧光强度随时间的变化而获得。第二个是通过在照射区（在达到稳态后平均超过12行和第5个图像）的边缘绘制的强度分布而获得。曲线被装配以获得绷紧和TAUD。扩散常数由TA给出UD 2 /拉紧（D = <img alt="方程" src="/files/ftp_upload/55060/55060eq1.jpg" /> ）。 </li></ol></li></ol>

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A., 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=Cell+spreading+and+focal+adhesion+dynamics+are+regulated+by+spacing+of+integrin+ligands.">Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands.</a> Biophys J. 92 (8), 2964-2974 (2007).</li><li>Schvartzman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanolithographic+Control+of+the+Spatial+Organization+of+Cellular+Adhesion+Receptors+at+the+Single-Molecule+Level.">Nanolithographic Control of the Spatial Organization of Cellular Adhesion Receptors at the Single-Molecule Level.</a> Nano Lett. 11 (3), 1306-1312 (2011).</li><li>Mossman, K., Groves, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterned+supported+membranes+as+tools+for+quantitative+studies+of+the+immunological+synapse.">Micropatterned supported membranes as tools for quantitative studies of the immunological synapse.</a> Chem.Soc.Rev. 36 (1), 46-54 (2007).</li><li>Furlan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatase+CD45+both+positively+and+negatively+regulates+T+cell+receptor+phosphorylation+in+reconstituted+membrane+protein+clusters.">Phosphatase CD45 both positively and negatively regulates T cell receptor phosphorylation in reconstituted membrane protein clusters.</a> J Biol Chem. 289 (41), 28514-28525 (2014).</li><li>Hsu, C. J., 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=Ligand+mobility+modulates+immunological+synapse+formation+and+T+cell+activation.">Ligand mobility modulates immunological synapse formation and T cell activation.</a> PloS One. 7 (2), e32398 (2012).</li><li>Yu, C., 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=Early+integrin+binding+to+Arg-Gly-Asp+peptide+activates+actin+polymerization+and+contractile+movement+that+stimulates+outward+translocation.">Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation.</a> Proc Natl Acad Sci U S A. 108 (51), 20585-20590 (2011).</li></ol>

作者什么都没有透露。

上述方案中的关键步骤是通过一个支撑脂双层相关的蛋白质纳米点或这些点周围的空间的回填的形成。相对于蛋白质纳米点的第一个关键步骤是胎圈掩模的制备方法。盖滑动的清洁是至关重要的。将载玻片需要与被推荐用于清洗石英比色杯中，或用氧等离子体的洗涤剂溶液中或者清洗。其它清洁技术，如浸渍在乙醇或异丙醇处理不会使玻璃充分亲水，并因此不支持大覆盖珠单层的形成。与此同时?...

<html>细胞粘附通过专门的细胞粘附分子（凸轮），蛋白质存在于细胞膜，其能够在细胞外基质或在另一细胞结合到其对应的发生。上粘附的细胞，最粘附分子包括整联无处不和钙粘蛋白，在簇1中的形式存在。 T淋巴细胞（T细胞）与抗原呈递细胞（的APCs）的相互作用提供的在两个小区之间的界面形成受体簇的重要性特别引人注目插图 - 通常被称为免疫突触。在形成与充当信令的平台2，3，4，并最终集中的T细胞形式微米尺度簇的表面上的APC，T细胞受体（TCR）的第一接触，以形成一个较大的中心超分子簇（CSMAC ）小姑娘= "外部参照"&gt; 5，6，7。最近，已表明在APC上侧，所述TCR的配体也聚集8。 在T细胞的APC相互作用，混合动力系统的部署，其中APC通过与相关蛋白官能化的人造表面模仿的上下文中，已大大提高了我们突触接口2，3，4，5，6，7的理解。在这种情况下，它是设计APC模拟表面可捕捉到目标小区的一个或多个方面密切相关。例如，如果配体是在支持的脂质双层接枝，它们可以以双层的平面扩散，模仿APC表面上的情况，并在同一时间允许的形成CSMAC 6,7。类似地，APC上的簇已经通过在聚合物9，10，11，12，13，14的海创建配体的岛屿模仿。然而，这两个功能迄今尚未合并。 在这里，我们描述了一种新技术，以创建由脂质双层与扩散脂质包围抗CD3（靶向TCR复合物的抗体）的纳米点。双层使用朗缪尔-布罗杰特/朗缪尔-谢弗技术7，15，16沉积并如果需要的话，可以用一个特定的蛋白质被官能化-例如，T细胞整联配体（称为ICAM1）。此外，抗CD3蛋白点腠LD与另一种抗体或CAM来代替。虽然我们选择的蛋白质如T细胞黏附的研究平台将来使用，这里详细的策略可以适用于任何的蛋白质和DNA，甚至。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glass coverslips</td>
			<td>Assistent, Karl Hecht KG&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Observation chamber</td>
			<td>Home made</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline surfactant concentrate (Hellmanex)</td>
			<td>Hella Analytics</td>
			<td>9-307-011-4-507</td>
		</tr>
		<tr>
			<td>Ultra-sonicator</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Labbox</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystallizer&#160;</td>
			<td>Shott</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidine</td>
			<td>Thermo Fischer Scientifique</td>
			<td>84607</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Sigma-aldrich</td>
			<td>P3813</td>
		</tr>
		<tr>
			<td>Water MQ&#160;</td>
			<td>ELGA, Veolia France</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica beads</td>
			<td>Corpuscular Inc</td>
			<td>147114-10</td>
		</tr>
		<tr>
			<td>APTES</td>
			<td>Sigma-aldrich</td>
			<td>A3648</td>
		</tr>
		<tr>
			<td>BSA-Biotin</td>
			<td>Sigma-aldrich</td>
			<td>A8549</td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti Polar Lipids</td>
			<td>850375C</td>
		</tr>
		<tr>
			<td>Dansyl-PE</td>
			<td>Avanti Polar Lipids</td>
			<td>810330C</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-aldrich</td>
			<td>650471</td>
		</tr>
		<tr>
			<td>Gastight syringe&#160;</td>
			<td>Dominique Dustcher , France</td>
			<td>74453</td>
		</tr>
		<tr>
			<td>Film balance</td>
			<td>NIMA</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss, Germany</td>
			<td>TIRF-III system</td>
		</tr>
		<tr>
			<td>Aluminium Target&#160;</td>
			<td>Kurt J. Lesker Compagny, USA</td>
			<td></td>
		</tr>
		<tr>
			<td>Radio Frequency Magnetron sputtering Syst&#232;me&#160;</td>
			<td>modified SMC 600 tool by ALCATEL , France</td>
			<td></td>
		</tr>
	</tbody>
</table>

ligand nano-cluster arrays in a supported lipid bilayer

目前，处于钝化表面的细胞生物学研究的海洋产生黏附蛋白岛的有序阵列相当大的兴趣。在过去几年里，它已成为越来越清楚地看到活细胞反应，不仅能呈现给他们的分子的生化性质，而且这些分子的显示方式。创建蛋白微图案因此现在在许多生物学实验室的标准;纳米图案也更方便。然而，在细胞间相互作用的背景下，有必要图案不仅蛋白质，而且脂质双层。这种双PROTEO脂质的图案至今没有得到方便。我们提供了一个浅显的技术来创建支持玻璃蛋白质纳米点，提出了一种方法，用支撑脂双层（SLB）回填点间的空间。从示踪剂的光漂白包括在SLB荧光脂质，我们证明了双层的-PL表现出相当大的ANE流动性。用荧光基团功能化的蛋白点，使我们能够像他们，并表明他们在规则六边形网格排列。典型的点尺寸是大约800nm和这里展示的间距为2微米。这些基板有望成为用于细胞粘附，迁移和机械 - 感测研究，作为有用的平台。

的荧光图像进行分析，以测量点的间距和尺寸。典型间隔被发现是1900±80 nm和典型点大小为600±100纳米（ 图1G）。的间距用的用于掩模珠的大小设置。的点尺寸是由珠尺寸以及沉积条件设定。 SLB被唯一地围绕蛋白质点和不淀积在它们（ 图2），在SLB成像通道看到的孔，并在NAV成像通道看到的点之间的完美的互补性。连续光漂白数据的分析表明，在?...

Watch this Scientific Journal Video about Ligand Nano-cluster Arrays in a Supported Lipid Bilayer at JoVE.com

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

我们提出了一个协议官能玻璃与由流体脂质双层包围的纳米蛋白贴剂。这些基材与先进的光学显微镜兼容，并有望作为细胞黏附和迁移的研究平台。

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

ligand-nano-cluster-arrays-in-a-supported-lipid-bilayer

Research

JoVE Journal

Bioengineering

6.9K 次观看.  CNRS, UMR 7325, CINaM. 我们提出了一个协议官能玻璃与由流体脂质双层包围的纳米蛋白贴剂。这些基材与先进的光学显微镜兼容，并有望作为细胞黏附和迁移的研究平台。 

视频: Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

恒压控制的挤压方法制备纳米脂质体

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

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 ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.