Name: cell membrane repair assay using a two-photon laser microscope
Uploaded: 2018-01-02T15:00:00
Description: Watch this Scientific Journal Video about Cell Membrane Repair Assay Using a Two-photon Laser Microscope at JoVE.com

这项工作得到了阿尔伯塔大学医学院和牙科学院的支持, 加勒特卡明研究主席基金的朋友, HM Toupin 神经科学研究椅子基金, 肌肉萎缩症加拿大, 加拿大创新基金会 (CFI),艾伯塔高级教育和技术 (AET), 加拿大卫生研究院 (研究院), 杰西的旅程-基因和细胞治疗的基础, 妇女和儿童健康研究所 (WCHRI) 和艾伯塔省创新健康解决方案 (AIHS).
我们要感谢 Dr. 史蒂芬拉伐尔为我们提供了全长 dysferlin 质粒。我们还要感谢 Dr. 冈田克三宅一生的技术咨询。

本研究中使用的人成纤维细胞在阿尔伯塔大学医学和牙科学院人类伦理委员会的批准下使用。
1. 全长 DYSF 质粒的细胞转染
<ol>
	<li>培养成纤维细胞的 T225 瓶含有40毫升的生长培养基-36 毫升 Dulbecco 氏改良鹰培养基 (DMEM), 4 毫升的胎儿牛血清 (FBS), 和20µL 青霉素/链霉素 (P/s)-在一个 CO2孵化器在37° c。 注意: 细胞应培养到约 70-80% 70-100, 不应允许成为完全汇合。</li>
	<li>要...

<ol>
	<li>Cong, X., Hubmayr, R. D., Li, C., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+wounding+and+repair+in+pulmonary+diseases.">Plasma membrane wounding and repair in pulmonary diseases.</a> Am J Physiol Lung Cell Mol Physiol. 312 (3), L371-L391 (2017).</li><li>Demonbreun, A. R., McNally, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+Membrane+Repair+in+Health+and+Disease.">Plasma Membrane Repair in Health and Disease.</a> Curr Top Membr. 77, 67-96 (2016).</li><li>McNeil, P. L., Ito, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+cell+plasma+membrane+wounding+and+resealing+in+vivo.">Gastrointestinal cell plasma membrane wounding and resealing in vivo.</a> Gastroenterology. 96 (5 Pt 1), 1238-1248 (1989).</li><li>McNeil, P. L., Kirchhausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+emergency+response+team+for+membrane+repair.">An emergency response team for membrane repair.</a> Nat Rev Mol Cell Biol. 6 (6), 499-505 (2005).</li><li>Cooper, S. T., McNeil, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Repair:+Mechanisms+and+Pathophysiology.">Membrane Repair: Mechanisms and Pathophysiology.</a> Physiol Rev. 95 (4), 1205-1240 (2015).</li><li>Geeraerts, M. D., Ronveaux-Dupal, M. F., Lemasters, J. J., Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytosolic+free+Ca2++and+proteolysis+in+lethal+oxidative+injury+in+endothelial+cells.">Cytosolic free Ca2+ and proteolysis in lethal oxidative injury in endothelial cells.</a> Am J Physiol. 261 (5 Pt 1), C889-C896 (1991).</li><li>McDade, J. R., Archambeau, A., 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=Rapid+actin-cytoskeleton-dependent+recruitment+of+plasma+membrane-derived+dysferlin+at+wounds+is+critical+for+muscle+membrane+repair.">Rapid actin-cytoskeleton-dependent recruitment of plasma membrane-derived dysferlin at wounds is critical for muscle membrane repair.</a> FASEB J. 28 (8), 3660-3670 (2014).</li><li>Benninger, R. K., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+excitation+microscopy+for+the+study+of+living+cells+and+tissues.">Two-photon excitation microscopy for the study of living cells and tissues.</a> Curr Protoc Cell Biol. Chapter 4 (Unit 4), 11-24 (2013).</li><li>Demonbreun, A. R., 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=An+actin-dependent+annexin+complex+mediates+plasma+membrane+repair+in+muscle.">An actin-dependent annexin complex mediates plasma membrane repair in muscle.</a> J Cell Biol. 213 (6), 705-718 (2016).</li><li>McNeil, P. L., Khakee, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruptions+of+muscle+fiber+plasma+membranes.+Role+in+exercise-induced+damage.">Disruptions of muscle fiber plasma membranes. Role in exercise-induced damage.</a> Am J Pathol. 140 (5), 1097-1109 (1992).</li><li>Rodriguez, A., Webster, P., Ortego, J., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomes+behave+as+Ca2+-regulated+exocytic+vesicles+in+fibroblasts+and+epithelial+cells.">Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells.</a> J Cell Biol. 137 (1), 93-104 (1997).</li><li>Reddy, A., Caler, E. V., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+repair+is+mediated+by+Ca(2+)-regulated+exocytosis+of+lysosomes.">Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes.</a> Cell. 106 (2), 157-169 (2001).</li><li>Bansal, D., 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=Defective+membrane+repair+in+dysferlin-deficient+muscular+dystrophy.">Defective membrane repair in dysferlin-deficient muscular dystrophy.</a> Nature. 423 (6936), 168-172 (2003).</li><li>Han, R., 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=Dysferlin-mediated+membrane+repair+protects+the+heart+from+stress-induced+left+ventricular+injury.">Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury.</a> J Clin Invest. 117 (7), 1805-1813 (2007).</li><li>Huynh, C., Roth, D., Ward, D. M., Kaplan, J., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+lysosomal+exocytosis+and+plasma+membrane+repair+in+Chediak-Higashi/beige+cells.">Defective lysosomal exocytosis and plasma membrane repair in Chediak-Higashi/beige cells.</a> Proc Natl Acad Sci U S A. 101 (48), 16795-16800 (2004).</li><li>Corrotte, M., Castro-Gomes, T., Koushik, A. B., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+for+plasma+membrane+wounding+and+assessment+of+lysosome-mediated+repair+responses.">Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses.</a> Methods Cell Biol. 126, 139-158 (2015).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>Jontes, J. D., Buchanan, J., Smith, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+cone+and+dendrite+dynamics+in+zebrafish+embryos:+early+events+in+synaptogenesis+imaged+in+vivo.">Growth cone and dendrite dynamics in zebrafish embryos: early events in synaptogenesis imaged in vivo.</a> Nat Neurosci. 3 (3), 231-237 (2000).</li><li>Galbraith, J. A., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+damage+in+thick+specimens+by+multiphoton+excitation.">Controlled damage in thick specimens by multiphoton excitation.</a> Mol Biol Cell. 14 (5), 1808-1817 (2003).</li><li>McNeil, P. L., Miyake, K., Vogel, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endomembrane+requirement+for+cell+surface+repair.">The endomembrane requirement for cell surface repair.</a> Proc Natl Acad Sci U S A. 100 (8), 4592-4597 (2003).</li><li>Weisleder, 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=Visualization+of+MG53-mediated+cell+membrane+repair+using+in+vivo+and+in+vitro+systems.">Visualization of MG53-mediated cell membrane repair using in vivo and in vitro systems.</a> J Vis Exp. (52), (2011).</li><li>Matsuda, C., Kiyosue, K., Nishino, I., Goto, Y., Hayashi, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysferlinopathy+Fibroblasts+Are+Defective+in+Plasma+Membrane+Repair.">Dysferlinopathy Fibroblasts Are Defective in Plasma Membrane Repair.</a> PLoS Curr. 7, (2015).</li><li>Philippi, S., 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=Dysferlin-deficient+immortalized+human+myoblasts+and+myotubes+as+a+useful+tool+to+study+dysferlinopathy.">Dysferlin-deficient immortalized human myoblasts and myotubes as a useful tool to study dysferlinopathy.</a> PLoS Curr. 4, RRN1298 (2012).</li><li>Cai, 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=MG53+nucleates+assembly+of+cell+membrane+repair+machinery.">MG53 nucleates assembly of cell membrane repair machinery.</a> Nat Cell Biol. 11 (1), 56-64 (2009).</li><li>Swaggart, K. 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=Annexin+A6+modifies+muscular+dystrophy+by+mediating+sarcolemmal+repair.">Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.</a> Proc Natl Acad Sci U S A. 111 (16), 6004-6009 (2014).</li><li>Roostalu, U., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+molecular+interactions+at+damaged+sarcolemma.">In vivo imaging of molecular interactions at damaged sarcolemma.</a> Dev Cell. 22 (3), 515-529 (2012).</li><li>Azakir, B. 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=Modular+dispensability+of+dysferlin+C2+domains+reveals+rational+design+for+mini-dysferlin+molecules.">Modular dispensability of dysferlin C2 domains reveals rational design for mini-dysferlin molecules.</a> J Biol Chem. 287 (33), 27629-27636 (2012).</li><li>Lee, J., Yokota, T., Takeda, S., Miyagoe-Suzuki, Y., Mori-Yoshimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+6.">Ch 6.</a> Translational Research in Muscular Dystrophy. , 87-102 (2016).</li><li>Barthelemy, F., Wein, N., Krahn, M., Levy, N., Bartoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+research+and+therapeutic+perspectives+in+dysferlinopathies.">Translational research and therapeutic perspectives in dysferlinopathies.</a> Mol Med. 17 (9-10), 875-882 (2011).</li></ol>

双光子激光损伤细胞膜是一种精确而多用途的技术, 用于评估膜加盖的动力学体外。本文介绍了用双光子激光损伤法测定 dysferlinopathy 患者细胞细胞膜加盖能力的协议。我们的结果表明, dysferlinopathy 患者细胞是有缺陷的细胞膜加盖, 这是与其他研究人员的发现相一致, 进一步突出了惊人的相似之处细胞膜加盖动力学之间的肌肉和肌肉单元格类型3,11<s...

真核细胞的细胞膜由一双层蛋白质镶嵌的磷脂组成, 它定义了细胞的内/外环境, 对于维持细胞的稳态和细胞的存活至关重要。由机械或化学侮辱引起的细胞膜损伤是常见的各种哺乳动物的细胞类型, 包括骨骼和心肌, 胃, 和肺细胞1,2,3,4。除了由于日常生理机能的损伤外, 细胞膜还可能受到环境侮辱、细菌毒素和缺血再灌注的损害5。在细胞膜上重新破裂导致不受管制的细胞外钙离子的流入, 以及其他可能有毒的胞外成分--进入细胞, 触发下游信号叶栅, 从而迅速导致细胞死亡1,4,5,6。
到目前为止, 已有几种模型的膜修复细胞。根据膜破裂的大小和性质, 可以激活不同的修复机制。例如, 建议细胞膜可以利用侧流或蛋白质堵塞来修复小的干扰 (和 #60; 1 nm)。侧向融合模型建议通过 dysferlin 膜的横向征募7快速修补膜破裂, 而蛋白质堵塞模型则表明通过蛋白质聚合修补小穿孔(主要 annexins)8. 反之, 较大的膜病变触发 Ca2 +依赖性, dysferlin 介导的囊泡融合和修复补丁的形成。在修复修补程序模型中, 快速的 Ca2流入该单元会触发多种蛋白质 (dysferlin、annexins、mitsugumin-53 和 EDH 蛋白) 的招募, 这些蛋白形成一个复杂的或 "补丁", 同时融合胞内泡或溶酶体在膜损伤的部位4,9,10,11,12。重要的是要注意, 这些模型不一定是互斥的, 并可能协同工作, 以促进膜修复。未正确重新细胞膜损伤与多种疾病状态有关, 包括肌营养不良 (dysferlinopathy-包括三好肌病、肢带肌营养不良型 IIB, 以及胫骨前开始的远端肌病变)13, 心肌病14, 和 Chediak-东综合征 (社区卫生)15。
鉴于适当的细胞膜完整性和加盖在健康和疾病中起着如此重要的作用, 对细胞膜修复的基本分子机制的更深的理解将有利于寻找新的治疗策略。因此, 有必要拥有适当的实验技术来监测动力学和评价细胞膜修复能力。设计了几种用于膜修复的体外方法。其中一个策略涉及机械损伤, 通过在细胞上使用吸管/外科刀片/剃刀或滚动玻璃珠在单元格上的刮擦, 可以方便地16。然而, 这种类型的机械损伤产生更大的损害, 并创造了高度的变化, 细胞损伤, 在内部和之间的文化。
另一种产生膜伤的方法是用双光子显微镜进行激光消融。与传统的激光共聚焦显微镜相比, 采用单光子激发, 双光子激光器同时使用两个长, 低能光子, 以促进高能电子17的激发。这个非线性的过程只在焦平面内产生激发, 而不是沿着整个光路17 (图 1)。当成像活细胞18,19时, 这种减少的励磁量有助于最小化光。因此, 研究人员能够在细胞膜上产生精确的病变, 并利用荧光染料实时监测细胞膜加盖, 并观察细胞膜破裂后封装的荧光强度变化。
这种方法已被反复用于研究膜伤的体外和在体内和多细胞类型20,21。例如, 利用此技术对 dysferlinopathy 患者的成纤维细胞和管的膜修复缺陷进行了评估22,23。此外, 从小鼠分离的单肌纤维被用来监测修复补丁形成13,24,25。在单肌纤维膜修复过程中也可以观察到荧光标记蛋白的运动9。此外, 在 real-time体内26中可以观察到斑马鱼胚胎双光子激光伤后的心肌修复过程。
在这篇文章中, 我们概述了一种方法, 评估细胞膜修复动力学的双光子激光伤, 虽然这种方法可以用于各种细胞类型的目的, 以量化等离子膜加盖能力体外。在这种方法中, 细胞的孵育与 FM4-64, 一个亲油性, 细胞不透水染料, 迅速荧光, 因为它结合在细胞质内的负电荷磷脂通过膜病变进入细胞 (图 2和 #38;3). 对膜病变附近的染料荧光进行量化, 可以监测细胞膜重新本身所需的时间。为了说明这一方法的效用, 我们使用 dysferlinopathy 患者成纤维细胞转染 GFP-共轭全长 dysferlin (DYSF) 质粒, 以评估挽救细胞膜修复。

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">11320033</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F1051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">25300062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">35mm collagen-coated glass-bottom dishes</td>
			<td style="width:71px;">MatTek</td>
			<td style="width:119px;">P53GCOL-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Serum-deprived media</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Transfection reagent</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">15338100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">FM 4-64 Dye</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">T13320</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Tyrode&#8217;s Salts Solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T2397</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Confocal laser scanning microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Chameleon Two-photon laser</td>
			<td style="width:71px;">Coherent</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell membrane repair assay using a two-photon laser microscope

许多病理生理学的侮辱会对细胞膜造成损害, 当与细胞膜修复或完整性的先天缺陷相结合时, 会导致疾病。因此, 了解细胞膜修复的基本分子机制是一个重要的目标, 以发展新的治疗策略的疾病与功能失调细胞膜动力学。许多体外和体内研究旨在了解各种疾病背景下的细胞膜加盖, 利用双光子激光消融作为测定实验治疗后的功能性结局的标准。在这种方法中, 细胞膜受到双光子激光的伤害, 导致细胞膜破裂和荧光染料渗入细胞。细胞内的荧光强度可以被监测, 以量化细胞重新本身的能力。有几种可供选择的方法来评估细胞膜对损伤的反应, 以及双光子激光伤的方法本身的巨大变化, 因此, 一个单一的, 统一的细胞伤害模型将有利于减少这些方法之间的差异。在这篇文章中, 我们概述了一个简单的双光子激光伤人协议, 评估细胞膜修复在体外的健康和 dysferlinopathy 患者成纤维细胞转染或没有一个全长 dysferlin 质粒。

健康的人成纤维细胞, 处理 dysferlinopathy 患者成纤维细胞, 和转染的质粒含有全长 dysferlin 序列的患者成纤维细胞受到双光子激光损伤, 以评估膜加盖能力实时。健康的人成纤维细胞显示低水平的 FM 4-64 荧光激活后, 激光伤和处理患者成纤维细胞表现出高度的相对荧光强度后受伤 (图 5和 #38; 6).转染全长 dysferlin 质粒的患者细胞显示, 相对于处...

Watch this Scientific Journal Video about Cell Membrane Repair Assay Using a Two-photon Laser Microscope at JoVE.com

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

双光子激光显微镜在细胞膜修复中的应用

双光子激光的细胞膜损伤是一种广泛应用的评价膜加盖能力的方法, 可应用于多种细胞类型。在这里, 我们描述了一个协议的体外活成像膜加盖在 dysferlinopathy 患者细胞后双光子激光消融。

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

Research

JoVE Journal

Immunology and Infection

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

一个细胞检测系统估算的GM - CSF抗体的中和能力，采用重组可溶性GM - CSF受体

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

科研

免疫与感染

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

的视觉检测，以监测T6SS介导的细菌竞争

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

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

调查肥大细胞分泌颗粒;从合成到胞吐作用

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...