Name: cell membrane repair assay using a two-photon laser microscope
Uploaded: 2018-01-02T15:00:00.000+00:00
Duration: 6 min 35 s
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>

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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><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher</td><td>11320033</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F1051</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher</td><td>15140122</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Thermo Fisher</td><td>25300062</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>35mm collagen-coated glass-bottom dishes</td><td>MatTek</td><td>P53GCOL-1.5-14-C</td><td></td></tr><tr><td>Serum-deprived media</td><td>Thermo Fisher</td><td>31985070</td><td></td></tr><tr><td>Transfection reagent</td><td>Thermo Fisher</td><td>15338100</td><td></td></tr><tr><td>FM 4-64 Dye</td><td>Invitrogen</td><td>T13320</td><td></td></tr><tr><td>Tyrode&amp;rsquo;s Salts Solution</td><td>Sigma-Aldrich</td><td>T2397</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Carl Zeiss</td><td>NA</td><td></td></tr><tr><td>Chameleon Two-photon laser</td><td>Coherent</td><td>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

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

科研

癌症研究

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

遗传学

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

生物学

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

免疫与感染

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

实时成像检测方法的评估钙的作用<sup&gt; 2 +</sup&gt;和鞘磷脂酶在成孔毒素伤口的修复

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

激光诱导的热塑性等离子体穿刺用于质膜修复研究

A Thermoplasmonic Approach for Investigating Plasma Membrane Repair in Living Cells and Model Membranes

The cell membrane is crucial for cell survival, and ensuring its integrity is essential as the cell experiences injuries throughout its entire life cycle. To prevent damage to the membrane, cells have developed efficient plasma membrane repair mechanisms. These&#160;repair mechanisms can be studied by combining confocal microscopy and nanoscale thermoplasmonics to identify and investigate the role of key proteins, such as annexins, involved in surface repair in living cells and membrane model systems.
The puncturing method employs a laser to induce highly localized heating upon nanoparticle irradiation. The use of near-infrared light minimizes phototoxicity in the biological sample, while the majority of the absorption takes place in the near-infrared resonant plasmonic nanoparticle. This thermoplasmonic method has been exploited for potential photothermal and biophysical research to enhance the understanding of intracellular mechanisms and cellular responses through vesicle and cell fusion studies. The approach has shown to be complementary to existing methods for membrane disruption, such as mechanically, chemically, or optically induced injuries, and provides a high level of control by inflicting extremely localized injuries. The extent of the injury is limited to the vicinity of the spherical nanoparticle, and no detrimental damage occurs along the beam path as opposed to pulsed lasers using different wavelengths. Despite certain limitations, such as the formation of nanobubbles, the thermoplasmonic method offers a unique tool for investigating cellular responses in plasma membrane repair in an almost native environment without compromising cell viability.
When integrated with confocal microscopy, the puncturing method can provide a mechanistic understanding of membrane dynamics in model membrane systems as well as quantitative information on protein responses to membrane damage, including protein recruitment and their biophysical function. Overall, the application of this method to reduced model systems can enhance our understanding of the intricate plasma membrane repair machinery in living cells.

作者聚焦：用创新的热等离子体穿刺探索质膜修复机制 

The thermoplasmonic puncture method integrates confocal microscopy, optical tweezers, and gold nanoparticles to study protein responses during plasma membrane repair in cells and giant unilamellar vesicles. The technique enables rapid and localized membrane puncture, allowing the identification of key proteins and their functional roles in the intricate plasma membrane repair machinery.

一种用于研究活细胞和模型膜中质膜修复的热等离子体方法

The cell membrane is crucial for cell survival, and ensuring its integrity is essential as the cell experiences injuries throughout its entire life cycle. ...

双光子激光显微镜在细胞膜修复中的应用

摘要

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此视频中的章节

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

MG53介导的细胞膜使用维修的可视化在体内在体外系统

哺乳动物细胞血浆膜检测效率的高通量测量

二维和三维活细胞成像的DNA损伤反应蛋白

实时成像检测方法的评估钙的作用

成像生物膜损伤和参与亚细胞修复过程

一种用于研究活细胞和模型膜中质膜修复的热等离子体方法

一种用于研究活细胞和模型膜中质膜修复的热等离子体方法

一种用于研究活细胞和模型膜中质膜修复的热等离子体方法