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>要准备细胞播种, 用40毫升磷酸缓冲盐水 (PBS) 冲洗两次细胞, 然后加入5毫升的0.05% 胰蛋白酶分离细胞。将电池返回到37° c CO2孵化器, 并孵育5分钟的细胞。<ol>
		<li>当细胞完全分离时, 加入40毫升的生长培养基来停止胰蛋白酶反应。</li>
		<li>使用例或其他单元计数设备对单元格进行计数。</li>
	</ol>
	</li>
	<li>种子2毫升的细胞在 1x105细胞/毫升成35毫米胶原涂层的玻璃底部的菜肴。在37° c 的 CO2培养箱中过夜孵育细胞。 注意: 细胞应该是大约50-60% 汇合的第二天。</li>
	<li>去除培养基, 用2毫升无血清的培养基 (无 FBS 的培养基) 取代。</li>
	<li>用 lipotransfection 染了全长 dysferlin 质粒的细胞。<ol>
		<li>在1.5 毫升的试管中制备150µL 的无血清培养基 (每道要转染的一根管子)。</li>
		<li>将3µL 的转染试剂添加到每1.5 毫升含有血清剥夺介质的管中。在室温下孵育至少5分钟。</li>
		<li>在一个单独的1.5 毫升管, 增加175µL 的血清剥夺培养基和3.5 µg 质粒 DNA。 注: 将上述金额乘以每增加的菜品进行转染。</li>
		<li>将含有质粒 DNA 的培养基中的150µL 与150µL 的血清剥夺培养基和转染试剂相结合。在室温下孵育20分钟。</li>
		<li>均匀分布300µL 介质与质粒和转染试剂在盘中。在37° c 的 CO2孵化器中孵育24小时的盘子。</li>
	</ol>
	</li>
	<li>用含有血清的新鲜培养基取代培养基, 在37° c 的 CO2培养箱中孵育另外24小时。</li>
</ol>
2. 双光子伤试验细胞的制备
<ol>
	<li>通过移去除介质, 用1毫升 Tyrode 溶液冲洗细胞一次, 然后加入1毫升的新鲜 Tyrode 溶液, 其中含有1µL 2.5 mM FM 4-64 染料。 注: FM 4-64 染料的最终浓度为2.5 µM。</li>
	<li>如果需要评估在钙耗尽的环境中的膜修复, 冲洗细胞与1毫升 pbs, 然后加入1毫升 pbs 含有1毫米 EGTA。 注意: 由于 PBS 将导致细胞分离随着时间的推移, 伤人化验应尽快完成。</li>
</ol>
3. 双光子激光致伤法
<ol>
	<li>使用倒置共焦显微镜和相关程序软件, 创建一个0.2 µm x 2 µm 目标, 并将其放置在细胞膜的边缘, 使目标线重叠, 并与细胞膜的方向垂直 (图 2)。</li>
	<li>使用543纳米氖激光器激发 FM 染料信号, 并将检测范围设置为 600-760 nm。使用 488 nm 氩激光激发 GFP 信号 (图 4), 并将检测范围设置为 500-550 nm。<ol>
		<li>创建一个5分钟序列图像扫描, 成像细胞每5秒。</li>
		<li>为了产生一个膜病变, 漂白细胞使用双光子激光器设置为 820 nm, 使用15% 激光功率与10迭代和漂白细胞二十五年代后的时间序列的开始。</li>
	</ol>
	</li>
	<li>为了量化 FM 4-64 染料荧光, 使用该软件绘制一个6µm x 6 µm 感兴趣的区域 (ROI), 并把它毗邻受伤的位置和细胞内 (图 3)。 注意: 在软件此应用程序中使用 "范围指示器" 功能, 避免像素会的取样区域, 红色像素表示饱和像素。将原始荧光值导出到电子表格中进行统计分析。</li>
	<li>通过减去背景值 (膜伤前 timepoints 的平均荧光值) 和在 t = 0 (图 5, 6) 上的荧光值除以净增量, 计算每个时间点的相对荧光值.</li>
</ol>

<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>

作者没有什么可透露的。

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 ...

cell-membrane-repair-assay-using-a-two-photon-laser-microscope

Research

JoVE Journal

Immunology and Infection

12.3K 次观看.  University of Alberta Faculty of Medicine and Dentistry. 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。

视频: Cell Membrane Repair Assay Using a Two-photon Laser Microscope

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

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)1-3. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP4,5, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP.
Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF6-8. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition.
OBJECTIVE: To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay.
METHODS: Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMR&alpha;) with high purity9-13. The recombinant sGMR&alpha; was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions14,15. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMR&alpha; than those produced by other insects or yeasts.
RESULTS: The cell-free assay system using sGMR&alpha; yielded the data with high plasticity and reliability. GM-CSF binding to sGMR&alpha; was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMR&alpha; is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells.
CONCLUSIONS: We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.

We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.

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

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

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

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

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

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

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 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

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

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

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

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

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

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

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

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

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

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

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

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine 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

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 extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

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 ...