Name: fabricating multi-component lipid nanotube networks using the gliding kinesin motility assay
Uploaded: 2021-07-26T13:00:00
Description: Watch this Scientific Journal Video about Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay at JoVE.com

这项工作得到了美国能源部，基础能源科学办公室，材料科学与工程部（BES-MSE）的支持。Kinesin合成和荧光显微镜是通过综合纳米技术中心的用户项目（ZIM）进行的，该中心是为美国能源部（DOE）科学办公室运营的科学用户设施办公室。

1. 储备微管溶液的制备
注意：在整个实验方案中应始终佩戴安全护目镜、手套和实验室外套。
<ol><li>准备5x BRB80缓冲液：将24.19克烟斗（哌嗪-N，N′-双[2-乙烷磺酸]）和0.38克EGTA（乙二醇-双[β-氨基乙基醚]-N，N，N′，N′-四乙酸）加入1升玻璃瓶中。加入1毫升1MMgCl2 ，并用KOH将pH调节至6.9。加入去离子水，使溶液达到500 mL终体积。</li>
	<li>准备100 mM GTP溶液储备：称取52mg ...

<ol>
	<li>Bouxsein, N. F., Carroll-Portillo, A., Bachand, M., Sasaki, D. Y., Bachand, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+network+of+lipid+nanotubes+fabricated+from+the+gliding+motility+of+kinesin+powered+microtubule+filaments.">A continuous network of lipid nanotubes fabricated from the gliding motility of kinesin powered microtubule filaments.</a> Langmuir. 29 (9), 2992-2999 (2013).</li><li>Paxton, W. F., Bouxsein, N. F., Henderson, I. M., Gomez, A., Bachand, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+assembly+of+polymer+nanotube+networks+via+kinesin+powered+microtubule+filaments.">Dynamic assembly of polymer nanotube networks via kinesin powered microtubule filaments.</a> Nanoscale. 7 (25), 10998-11004 (2015).</li><li>Leduc, 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=Cooperative+extraction+of+membrane+nanotubes+by+molecular+motors.">Cooperative extraction of membrane nanotubes by molecular motors.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (49), 17096-17101 (2004).</li><li>Lippincott-Schwartz, J., Roberts, T. H., Hirschberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+protein+trafficking+and+organelle+dynamics+in+living+cells.">Secretory protein trafficking and organelle dynamics in living cells.</a> Annual Review of Cell and Developmental Biology. 16, 557-589 (2000).</li><li>Belting, M., Wittrup, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotubes,+exosomes,+and+nucleic+acid-binding+peptides+provide+novel+mechanisms+of+intercellular+communication+in+eukaryotic+cells:+implications+in+health+and+disease.">Nanotubes, exosomes, and nucleic acid-binding peptides provide novel mechanisms of intercellular communication in eukaryotic cells: implications in health and disease.</a> Journal of Cell Biology. 183 (7), 1187-1191 (2008).</li><li>Rustom, A., Saffrich, R., Markovic, I., Walther, P., Gerdes, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotubular+highways+for+intercellular+organelle+transport.">Nanotubular highways for intercellular organelle transport.</a> Science. 303 (5660), 1007-1010 (2004).</li><li>Onfelt, B., Nedvetzki, S., Yanagi, K., Davis, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+Membrane+nanotubes+connect+immune+cells.">Cutting edge: Membrane nanotubes connect immune cells.</a> Journal of Immunology. 173 (3), 1511-1513 (2004).</li><li>Sciaky, N., 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=Golgi+tubule+traffic+and+the+effects+of+brefeldin+A+visualized+in+living+cells.">Golgi tubule traffic and the effects of brefeldin A visualized in living cells.</a> J Cell Biol. 139 (5), 1137-1155 (1997).</li><li>Sprong, H., van der Sluijs, P., van Meer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+proteins+move+lipids+and+lipids+move+proteins.">How proteins move lipids and lipids move proteins.</a> Nature Reviews Molecular Cell Biology. 2 (7), 504-513 (2001).</li><li>Keenan, T. W., Morre, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+class+and+fatty+acid+composition+of+golgi+apparatus+isolated+from+rat+liver+and+comparison+with+other+cell+fractions.">Phospholipid class and fatty acid composition of golgi apparatus isolated from rat liver and comparison with other cell fractions.</a> Biochemistry. 9 (1), 19-25 (1970).</li><li>Imam, Z. I., Bachand, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicomponent+and+Multiphase+Lipid+Nanotubes+Formed+by+Gliding+Microtubule-Kinesin+Motility+and+Phase-Separated+Giant+Unilamellar+Vesicles.">Multicomponent and Multiphase Lipid Nanotubes Formed by Gliding Microtubule-Kinesin Motility and Phase-Separated Giant Unilamellar Vesicles.</a> Langmuir. 35 (49), 16281-16289 (2019).</li><li>Wesolowska, O., Michalak, K., Maniewska, J., Hendrich, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+unilamellar+vesicles+-+a+perfect+tool+to+visualize+phase+separation+and+lipid+rafts+in+model+systems.">Giant unilamellar vesicles - a perfect tool to visualize phase separation and lipid rafts in model systems.</a> Acta Biochimica Polonica. 56 (1), 33-39 (2009).</li><li>Momin, N., 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=Designing+lipids+for+selective+partitioning+into+liquid+ordered+membrane+domains.">Designing lipids for selective partitioning into liquid ordered membrane domains.</a> Soft Matter. 11 (16), 3241-3250 (2015).</li><li>Fygenson, D. K., Braun, E., Libchaber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+diagram+of+microtubules.">Phase diagram of microtubules.</a> Physical Review E. 50, 1579 (1994).</li><li>Greene, A. C., Sasaki, D. Y., Bachand, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forming+Giant-sized+Polymersomes+Using+Gel-assisted+Rehydration.">Forming Giant-sized Polymersomes Using Gel-assisted Rehydration.</a> Journal of Visualized Experiments. (111), (2016).</li><li>Bachand, 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=Directed+self-assembly+of+1D+microtubule+nano-arrays.">Directed self-assembly of 1D microtubule nano-arrays.</a> Royal Society of Chemistry Advances. 4 (97), 54641-54649 (2014).</li></ol>

LNT网络是体外研究膜性质和生物分子（如跨膜蛋白）转运的有用工具。此外，使用复杂的脂质制剂来制造LNT网络可以进行更多生物学相关的研究。其他制备研究已经使用了1）简单的脂质制剂和多层囊泡或2）更繁琐的运动技术来制造由复杂脂质制剂组成的GUV的网络。这里描述的方法能够使用廉价的试剂和设备，从复杂的脂质制剂和GUV中有效地制备大规模LNT网络。因此，该方法提供了研究一系列生?...

脂质纳米管（LNT）网络的制备对于非平衡脂质结构的体外检查越来越感兴趣1，2，3。细胞使用脂质小管进行蛋白质4 和核酸5 的扩散转运以及细胞间通讯6，7。内质网和高尔基体特别有趣，因为这些膜结合的细胞器是脂质和蛋白质合成以及这些整体生物分子在细胞质8，9内运输的主要位置。这些细胞器的膜由多种脂质物质组成，包括鞘脂，胆固醇和磷脂10 ，最终有助于定义其功能。因此，为了更密切地复制和研究这些细胞器，体外LNT必须由具有日益复杂的脂质制剂11的囊泡制造。
巨型单层囊泡（GUV）广泛用于研究脂质膜行为，因为它们可以与复杂的制剂可靠地合成，包括胆固醇，磷脂酰胆碱（PC），磷脂酰乙醇胺（PE），磷脂酰丝氨酸（PS）和磷脂酰肌醇（PI）12，13。这里描述的是一种使用滑动运动度测定（GMA）从具有不同脂质配方的GUV制造LNT的方法，其中LNT基于作用于GUV的驱动蛋白马达和微管细丝所进行的工作进行挤出。在该系统中，吸附在表面的驱动蛋白推动生物素化的微管，将来自ATP水解的化学能转化为有用的功（特别是从生物素化囊泡中挤出LNT）11。由此产生的LNT网络提供了一个模型平台，用于研究脂质相差异对LNT形态变化的影响。
简而言之，将驱动蛋白运动蛋白在含有酪蛋白的溶液中引入流动室，这使得电机能够吸附到腔室的玻璃表面上。接下来，含有ATP的溶液中的生物素化微管流过腔室并使其与驱动蛋白马达结合并开始运动。然后将链霉亲和素溶液引入腔室并使其与微管非共价结合。最后，将含有生物素化脂质的GUV引入腔室并与链霉亲和素包被的微管结合，然后在15-30分钟内挤出LNT以形成大规模网络。该方法使用标准实验室设备和试剂以低成本生产大型分支LNT网络11。

<table border="0" cellpadding="0" cellspacing="0" style="width:1071px;" width="1071">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="64">
			<td height="64" style="height:65px;width:328px;">100x/1.4 Numerical Aperture Oil Immersion Objective</td>
			<td style="width:104px;">Olympus</td>
			<td style="width:119px;">1-U2B836</td>
			<td style="width:520px;">Olympus UPlanSApo 100x/1.40 Oil Objective Infinity Corrected, RMS Thread 
			Working Distance 0.12mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">3.0 ND Filter</td>
			<td style="width:104px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Neutral Density Filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">AMP-PNP</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2647</td>
			<td>(&#946;,&#947;-imidoadenosine 5&#8242;-triphosphate)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">ATP</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7699</td>
			<td>Adenosine 5&#39;-triphosphate disodium salt hydrate BioXtra</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:328px;">Brightline Pinkel DA/FI/TR/Cy5/Cy7-5X-A000 filter set</td>
			<td style="width:104px;">Semrock</td>
			<td style="width:119px;">LED-DA/FI/TR/Cy5/Cy7-5X-A-000</td>
			<td style="width:520px;"> 
			BrightLine Pinkel filter set, optimized for DAPI, FITC, TRITC, Cy5 &#38; Cy7 and other like fluorophores, illuminated with LED-based light sources</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Casein</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">22090</td>
			<td>Casein hydrolysate for microbiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Catalase</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">C9322</td>
			<td>Catalase from Bovine Liver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Chloroform</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">288306</td>
			<td>Chloroform anhydrous contains 0.5-1.0% ethanol as stabilizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Cholesterol</td>
			<td style="width:104px;">Avanti</td>
			<td style="width:119px;">700000P</td>
			<td>cholesterol (ovine wool, &#62;98%) (powder)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">D-Glucose</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7021</td>
			<td style="width:520px;">D-(+)-Glucose powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, &#8805;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DOPC</td>
			<td style="width:104px;">Avanti</td>
			<td style="width:119px;">850375C</td>
			<td>1,2-Dioleoyl-sn-glycero-3-phosphocholine (in chloroform)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DOPE-Biotin</td>
			<td style="width:104px;">Avanti</td>
			<td style="width:119px;">870282C</td>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DPPC</td>
			<td style="width:104px;">Avanti</td>
			<td style="width:119px;">850355P</td>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (powder)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DPPE-Biotin</td>
			<td style="width:104px;">Avanti</td>
			<td style="width:119px;">870285P</td>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DTT</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">43816</td>
			<td>DL-Dithiothreitol solution 1 M</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">EGTA</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4378</td>
			<td style="width:520px;">EGTA, Egtazic acid, Ethylene-bis(oxyethylenenitrilo)tetraacetic acid, Glycol ether diamine tetraacetic acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">Glucose Oxidase</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">G6125</td>
			<td style="width:520px;">Glucose Oxidase from Aspergillus niger 
			Type II, &#8805;10,000 units/g solid (without added oxygen)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Glycerol</td>
			<td style="width:104px;">Fisher</td>
			<td style="width:119px;">G33</td>
			<td>Glycerol (Certified ACS), Fisher Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">GTP</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8877</td>
			<td>Guanosine 5&#8242;-triphosphate sodium salt hydrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">IX-81 Olympus Microscope</td>
			<td style="width:104px;">Olympus</td>
			<td style="width:119px;">N/A</td>
			<td>IX81 Inverted Microscope from Olympus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">KOH</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1050121000</td>
			<td>Potassium Hydroxide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Magnesium Chloride</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1028</td>
			<td>1.00 M magnesium chloride solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Orca Flash 4.0 Digital Camera</td>
			<td style="width:104px;">Hamamatsu</td>
			<td style="width:119px;">C13440-20CU</td>
			<td>ORCA-Flash 4.0 V3 Digital CMOS camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Oregon Green-DHPE</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">O12650</td>
			<td>Oregon Green 488 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Paclitaxel</td>
			<td style="width:104px;">ThermoFisher</td>
			<td style="width:119px;">P3456</td>
			<td>Paclitaxel (Taxol Equivalent) - for use in research only</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">PIPES</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6757</td>
			<td style="width:520px;">1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N&#8242;-bis(2-ethanesulfonic acid)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">Texas Red-DHPE</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">T1395MP</td>
			<td style="width:520px;">Texas Red 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, 
			Triethylammonium Salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Trolox</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">238813</td>
			<td>(&#177;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tubulin, Biotin</td>
			<td style="width:104px;">Cytoskeleton</td>
			<td style="width:119px;">T333P</td>
			<td>Tubulin protein (biotin) porcine brain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tubulin, Hy-Lite 488</td>
			<td style="width:104px;">Cytoskeleton</td>
			<td style="width:119px;">TL488M</td>
			<td>Tubulin protein (fluorescent HiLyte 488) porcine brain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tubulin, Unlabeled</td>
			<td style="width:104px;">Cytoskeleton</td>
			<td style="width:119px;">T240</td>
			<td>Tubulin protein porcine brain</td>
		</tr>
	</tbody>
</table>

fabricating multi-component lipid nanotube networks using the gliding kinesin motility assay

脂质纳米管（LNT）网络代表了一种体外模型系统，用于研究分子转运和脂质生物物理学，与真核细胞中发现的无处不在的脂质小管相关。然而，体内LNT是高度非平衡的结构，需要组装，维护和重组化学能和分子马达。此外，体内LNT的组成是复杂的，包括多种不同的脂质物质。挤出LNT的典型方法是时间和劳动密集型的，它们需要光学镊子，微珠和微量移液器从巨大的脂质囊泡中强行拉出纳米管。这里介绍的是滑动运动性测定（GMA）的方案，其中使用运动蛋白驱动的微管运动从巨大的单层囊泡（GUV）中快速产生大规模的LNT网络。使用这种方法，LNT网络由各种模仿生物LNT复杂性的脂质制剂形成，使其在脂质生物物理学和膜相关转运的体外研究中越来越有用。此外，该方法能够使用常用的实验室设备在短时间内（<30分钟）可靠地产生LNT网络。LNT网络特征（如长度、宽度和脂质分区）也可通过改变用于制造网络的GUV的脂质组成来调整。

LNT网络（图4）是使用所述协议制造的，该协议使用微管的驱动蛋白转运所执行的工作来挤出GUV中的LNT。简而言之，使用蔗糖溶液使用琼脂糖凝胶再水合制备了GUV，并在GPEM溶液中聚合微管并在BRB80T中稳定。接下来，将驱动蛋白电机引入流通池中，在盖玻片表面形成一层有源电机。然后引入微管并加入链霉亲和素溶液，这促进了生物素化脂质和GUV（随后引入）之间的结合。
...

Watch this Scientific Journal Video about Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay at JoVE.com

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

该协议描述了使用滑动运动蛋白运动与巨型单层脂质囊泡一起制造脂质纳米管网络的过程。

使用滑动运动蛋白运动度测定法制造多组分脂质纳米管网络

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

该协议允许简单生成大规模脂质纳米管网络，其中单个纳米管显示类似于母囊泡的相分离行为。这种方法的主要优点是，我们利用分子马达所做的工作，以高度平行的方式自组装脂质纳米管，而无需人为干预。要建立滑动运动度测定，首先，将三组双面胶带贴在相距五毫米的载玻片上。轻轻地将盖玻片压在胶带上，并将30微升的一微摩尔驱动蛋白溶液加入制备的流通池中。五分钟后，将30微升10微克/毫升微管溶液加入细胞中，将实验室擦拭轻轻按压在流道的另一端，以促进溶液交换。再孵育五分钟后，每次洗涤用50微升室温动力溶液洗涤细胞一至三次，然后向流通池中加入30微升10微克/毫升链霉亲和素溶液。10分钟后，加入30微升10X GUV溶液，在室温下孵育30分钟。然后，加入两微升100毫摩尔AMP-PNP溶液，并用密封剂关闭腔室。将流动室转移到倒置显微镜进行成像，并选择合适的滤光片组。使用100X油物镜首先聚焦盖玻片的表面，然后再获取感兴趣网络的图像。然后在 ImageJ 中打开图像，然后单击“图像”、“颜色”和“合成”以叠加红色和绿色通道以创建复合图像。成像后，打开感兴趣的图像，然后单击“分析并设置比例”以校准显微镜的刻度。将像素填充到千分尺转换因子，然后单击确定。使用多点线工具从母体GUV开始跟踪纳米管，并按住Control和M来测量长度。图像处理工具会将每个新测量结果保存在结果窗口中。测量完所有试管后，选择“图像”、“调整”和“阈值”，然后单击“应用”以应用阈值。然后，在所需管子上绘制一个已知长度的矩形。要测量该区域的集成密度，请单击“分析并测量”以确定液体纳米管节点中的脂质分配，使用线工具在目标节点上绘制一条线，并测量绿色和红色通道中的节点强度。在这项代表性分析中，制备了液体纳米管网络，以产生液体无序和有序相，以及相分离的囊泡。例如，用45%饱和脂质和55%不饱和脂质合成的囊泡导致囊泡分离成共存的液相和固相。然而，如果包括胆固醇，则可以观察到液 -液共存，从而产生分离成共存的液序和液态无序相的GUV。此外，可以在相分离的混合物中观察到节点。要记住的最重要的事情是选择适当的曝光时间和ND滤光片集，以便能够可视化脂质纳米管而不会对荧光团进行光漂白。定制脂质纳米管的相行为组成使我们能够使用极简主义模型系统开始研究连接细胞的隧道纳米管的生物物理学。

Research

JoVE Journal

Bioengineering

Plasma Lithography Surface Patterning for Creation of Cell Networks

等离子体表面图案光刻技术的蜂窝网络创作

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

科研

生物工程

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

使用滑翔分析生物大分子的抗弯刚度测量

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

的剪切粘合地图使用SynVivo合成微血管网络代

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

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 ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-Ãƒâ€šÃ‚ÂµCP and Sequential Nucleophilic Substitution

制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

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

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

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 ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabricating Cotton Analytical Devices

制造棉分析仪器

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...