Name: detection and quantification of tunneling nanotubes using 3d volume view images
Uploaded: 2022-08-31T14:00:00
Description: Watch this Scientific Journal Video about Detection and Quantification of Tunneling Nanotubes Using 3D Volume View Images at JoVE.com

D.K.V和A.R感谢马尼帕尔高等教育学院的TMA Pai奖学金。我们感谢印度科学与工程研究委员会为 SERB-SRG （#SRG/2021/001315），以及印度印度医学研究委员会 （#5/4-5/ad-hoc/Neuro/216/2020-NCD-I） 和印度马尼帕尔高等教育学院的校内基金。我们感谢JNCASR（印度贾瓦哈拉尔·尼赫鲁高级科学研究中心）的共聚焦设施和B. Suma在JNCASR中进行共聚焦显微镜。

注意：在DMEM / F-12培养基中培养的SH-SY5Y细胞用10μM视黄酸分化7天，并在37°C（5%CO 2）下用1μM oAβ低聚物处理2小时。处理后，用Karnovsky的固定溶液固定细胞，并用磷酸化PAK1（Thr423）/ PAK2（Thr402）抗体和F-肌动蛋白结合鬼笔环肽进行双重免疫染色。后来，使用共聚焦激光扫描显微镜拍摄共聚焦z-stack图像。TNTs通过手动计数进行定量，并通过构建3D体积视图图像并从它们悬停在两个细胞之间而不?...

<ol>
	<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>Desir, 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=Chemotherapy-induced+tunneling+nanotubes+mediate+intercellular+drug+efflux+in+pancreatic+cancer.">Chemotherapy-induced tunneling nanotubes mediate intercellular drug efflux in pancreatic cancer.</a> Scientific Reports. 8, 9484 (2018).</li><li>Cordero Cervantes, D., Zurzolo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peering+into+tunneling+nanotubes-The+path+forward.">Peering into tunneling nanotubes-The path forward.</a> The EMBO Journal. 40 (8), 105789 (2021).</li><li>Dieriks, B. V., 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=&#945;-synuclein+transfer+through+tunneling+nanotubes+occurs+in+SH-SY5Y+cells+and+primary+brain+pericytes+from+Parkinson's+disease+patients.">&#945;-synuclein transfer through tunneling nanotubes occurs in SH-SY5Y cells and primary brain pericytes from Parkinson's disease patients.</a> Scientific Reports. 7, 42984 (2017).</li><li>Chen, J., Cao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-to-neuron+transportation+of+enhanced+green+fluorescent+protein+in+cerebral+cortex+requires+F-actin+dependent+tunneling+nanotubes.">Astrocyte-to-neuron transportation of enhanced green fluorescent protein in cerebral cortex requires F-actin dependent tunneling nanotubes.</a> Scientific Reports. 11, 16798 (2021).</li><li>Mittal, 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=Cell+communication+by+tunneling+nanotubes:+Implications+in+disease+and+therapeutic+applications.">Cell communication by tunneling nanotubes: Implications in disease and therapeutic applications.</a> Journal of Cellular Physiology. 234 (2), 1130-1146 (2019).</li><li>Polak, R., de Rooij, B., Pieters, R., den Boer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+precursor+acute+lymphoblastic+leukemia+cells+use+tunneling+nanotubes+to+orchestrate+their+microenvironment.">B-cell precursor acute lymphoblastic leukemia cells use tunneling nanotubes to orchestrate their microenvironment.</a> Blood. 126 (21), 2404-2414 (2015).</li><li>Panasiuk, M., Rychlowski, M., Derewonko, N., Bienkowska-Szewczyk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunneling+nanotubes+as+a+novel+route+of+cell-to-cell+spread+of+herpesviruses.">Tunneling nanotubes as a novel route of cell-to-cell spread of herpesviruses.</a> Journal of Virology. 92 (10), 00090 (2018).</li><li>Austefjord, M. W., Gerdes, H. H., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunneling+nanotubes:+Diversity+in+morphology+and+structure.">Tunneling nanotubes: Diversity in morphology and structure.</a> Communicative &amp; Integrative Biology. 7 (1), 27934 (2014).</li><li>Han, X., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+and+challenges+in+tunneling+nanotubes+research:+How+far+from+clinical+application.">Opportunities and challenges in tunneling nanotubes research: How far from clinical application.</a> International Journal of Molecular Sciences. 22 (5), 2306 (2021).</li><li>Zurzolo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunneling+nanotubes:+Reshaping+connectivity.">Tunneling nanotubes: Reshaping connectivity.</a> Current Opinion in Cell Biology. 71, 139-147 (2021).</li><li>Dilna, 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=Amyloid-beta+induced+membrane+damage+instigates+tunneling+nanotube-like+conduits+by+p21-activated+kinase+dependent+actin+remodulation.">Amyloid-beta induced membrane damage instigates tunneling nanotube-like conduits by p21-activated kinase dependent actin remodulation.</a> Biochimica et Biophysica Acta (BBA)- Molecular Basis of Disease. 1867 (12), 166246 (2021).</li><li>Chang, 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=Formation+of+cellular+close-ended+tunneling+nanotubes+through+mechanical+deformation.">Formation of cellular close-ended tunneling nanotubes through mechanical deformation.</a> Science Advances. 8 (13), (2022).</li><li>Bukoreshtliev, N. V., 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=Selective+block+of+tunneling+nanotube+(TNT)+formation+inhibits+intercellular+organelle+transfer+between+PC12+cells.">Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells.</a> FEBS Letters. 583 (9), 1481-1488 (2009).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+tunneling+nanotubes+within+human+mesenchymal+stem+cell+spheroids.">Direct observation of tunneling nanotubes within human mesenchymal stem cell spheroids.</a> The Journal of Physical Chemistry B. 122 (43), 9920-9926 (2018).</li><li>Hanna, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Rho-GTPases+and+actin+polymerization+during+Macrophage+Tunneling+Nanotube+Biogenesis.">The role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis.</a> Scientific Reports. 7, 8547 (2017).</li><li>Raghavan, A., Rao, P., Neuzil, J., Pountney, D. L., Nath, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+Rho+GTPases+in+the+biogenesis+of+tunnelling+nanotubes:+Implications+in+disease+and+therapy.">Oxidative stress and Rho GTPases in the biogenesis of tunnelling nanotubes: Implications in disease and therapy.</a> Cellular and Molecular Life Sciences. 79, 36 (2021).</li><li>Abounit, S., Delage, E., Zurzolo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+tunneling+nanotubes+for+intercellular+trafficking.">Identification and characterization of tunneling nanotubes for intercellular trafficking.</a> Current Protocols in Cell Biology. 67, 11-21 (2015).</li><li>Hodneland, E., 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=Automated+detection+of+tunneling+nanotubes+in+3D+images.">Automated detection of tunneling nanotubes in 3D images.</a> Cytometry A. 69 (9), 961-972 (2006).</li><li>Wang, X., Bukoreshtliev, N. V., 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=Developing+neurons+form+transient+nanotubes+facilitating+electrical+coupling+and+calcium+signaling+with+distant+astrocytes.">Developing neurons form transient nanotubes facilitating electrical coupling and calcium signaling with distant astrocytes.</a> PLoS One. 7 (10), 47429 (2012).</li><li>Nath, 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=Spreading+of+neurodegenerative+pathology+via+neuron-to-neuron+transmission+of+beta-amyloid.">Spreading of neurodegenerative pathology via neuron-to-neuron transmission of beta-amyloid.</a> Journal of Neuroscience. 32 (26), 8767-8777 (2012).</li><li>Domert, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spreading+of+amyloid-beta+peptides+via+neuritic+cell-to-cell+transfer+is+dependent+on+insufficient+cellular+clearance.">Spreading of amyloid-beta peptides via neuritic cell-to-cell transfer is dependent on insufficient cellular clearance.</a> Neurobiology of Disease. 65, 82-92 (2014).</li><li>Pawley, J. B., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Points,+Pixels,+and+Gray+Levels:+Digitizing+Image+Data.">Points, Pixels, and Gray Levels: Digitizing Image Data.</a> Handbook Of Biological Confocal Microscopy. , 59-79 (2006).</li><li>Pontes, B., 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=Structure+and+elastic+properties+of+tunneling+nanotubes.">Structure and elastic properties of tunneling nanotubes.</a> European Biophysics Journal. 37 (2), 121-129 (2008).</li><li>Haimovich, G., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+mRNA+transfer+between+mammalian+cells+in+coculture+by+single-molecule+fluorescent+in+situ+hybridization+(smFISH).">Detection of mRNA transfer between mammalian cells in coculture by single-molecule fluorescent in situ hybridization (smFISH).</a> Methods in Molecular Biology. 2038, 109-129 (2019).</li><li>Janardhan, K. S., Jensen, H., Clayton, N. P., Herbert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+in+investigative+and+toxicologic+pathology.">Immunohistochemistry in investigative and toxicologic pathology.</a> Toxicologic Pathology. 46 (5), 488-510 (2018).</li><li>Qin, Y., 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=The+combination+of+paraformaldehyde+and+glutaraldehyde+is+a+potential+fixative+for+mitochondria.">The combination of paraformaldehyde and glutaraldehyde is a potential fixative for mitochondria.</a> Biomolecules. 11 (5), 711 (2021).</li><li>Graham, R. C., Karnovsky, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histochemical+demonstration+of+monoamine+oxidase+activity+by+coupled+peroxidatic+oxidation.">The histochemical demonstration of monoamine oxidase activity by coupled peroxidatic oxidation.</a> Journal of Histochemistry &amp; Cytochemistry. 13 (7), 604-605 (1965).</li></ol>

在过去的20年中，一些研究人员一直在试图理解和表征TNT的结构18。缺乏特异性标志物阻碍了进展，并且对可用于识别，表征和定量TNT的方便，标准化方法的需求不断增加。 TNT被定义为徘徊在两个细胞之间的基于F-肌动蛋白的膜导管。研究表明，β-微管蛋白阳性，封闭式，发育中的神经突在两个远处细胞之间徘徊，类似于TNT样结构12，13...

隧道纳米管（TNT）是基于F-肌动蛋白的，主要是开放式膜导管，在货物和细胞器的细胞间转移中起着至关重要的作用1。TNT的独特之处在于它们在不与基质接触的情况下连接相邻的细胞;它们的长度超过 10-300 μm，直径在 50 nm 到 1 μm之间变化 2，3。TNT是瞬态结构，其寿命持续几分钟到几小时。TNTs首先在PC12神经元细胞中得到证实1;后来，大量研究表明它们存在于几种体外和体内细胞类型中 4，5.一些研究揭示了TNT在各种疾病模型中的病理意义，例如神经退行性疾病，癌症和病毒感染6，7，8。
TNT的结构异质性已被各种细胞系统中的几项研究证明9。差异基于细胞骨架组成、形成机制和转移的货物类型10。首先，徘徊在两个相邻细胞之间并转移细胞器的开放式F-肌动蛋白阳性膜连续性被认为由TNTs组成11。然而，在TNTs的形成中观察到的缺乏明确性或多样性增加了开发TNT特异性标志物的难度。因此，很难通过常规检测方法识别TNT结构，也难以区分膜纳米管的开端和闭端突起12。然而，TNTs作为两个细胞之间的F-肌动蛋白膜突起徘徊的特征相对容易，使用常规成像技术进行识别也更可行。其他基于肌动蛋白的细胞突起，如丝状伪足和背丝状伪足，不能在两个远处细胞之间徘徊，特别是当细胞固定时。值得注意的是，封闭式、电耦合、发育中的神经突通常被称为TNT样结构13。
众所周知，F-肌动蛋白在TNT形成中起重要作用，多项研究表明，F-肌动蛋白抑制剂细胞松弛素D抑制TNTs的形成14，15。相比之下，微管抑制剂对TNT形成没有任何影响16。在过去的20年中，已经有几份关于TNT在病理学和肿瘤耐药性和治疗的传播中发挥的重要作用的报告17。因此，对更好的TNT表征技术的需求永无止境。
缺乏TNT的特异性标志物以及形态和细胞骨架组成的多样性使得难以开发独特的表征方法。一些研究使用了自动图像检测和TNT量化技术18，19。然而，与用于检测和定量TNT的自动图像分析相比，目前的3D体积手动分析方法有几个优点。 通常，训练有素的人眼比自动图像检测方法更容易发现这些悬浮的纳米结构。此外，在缺乏算法专业知识的实验室中，自动检测方法可能难以实施。本方法因其精密度和重现性而被研究人员广泛采用。
在最近的一项研究中，我们发现oAβ通过PAK1介导的肌动蛋白依赖性内吞机制 促进 神经元细胞中TNT的生物发生12。oAβ诱导的TNTs也表达活化的PAK1（或磷酸化PAK1）。我们开发了一种3D体积视图图像重建方法来区分oAβ诱导的，F-肌动蛋白和磷酸化PAK1免疫染色的TNT。 β-III微管蛋白阳性，发育中的神经突通常类似于TNT样悬浮结构20。因此，我们进一步区分了基于F-肌动蛋白的TNTs与β-III微管蛋白阳性神经突起和其他TNT样突起。3D体积视图图像已被用于根据TNT悬停在基质上并在两个相邻细胞之间保持连接的特征来识别TNT。本文描述了使用共聚焦z-stack图像识别和检测含肌动蛋白的膜导管或TNT，最后从3D体积视图重建图像中手动定量识别的结构。所提出的方法无法区分开放式适当的TNT和封闭式TNT样结构;该方法有助于在平坦基质上的 体外 2D细胞培养物中鉴定TNT。然而，该方法易于实施和复制，可广泛用于仅基于肌动蛋白的TNT的精确定量，并将其与神经突和β-微管蛋白阳性TNT样结构区分开来。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm dish with 14 mm well size made of #1.5 cover slip</td>
			<td>Cellvis</td>
			<td>D35-14-1.5-N</td>
			<td>Imaging dish used to seed cells for staining experiments</td>
		</tr>
		<tr>
			<td>A&#946; (1-42) 1 mg</td>
			<td>AnaSpec</td>
			<td>#64129</td>
			<td>Oligomers of amyloid beta to treat the cells</td>
		</tr>
		<tr>
			<td>Alexa flour 488 Goat Anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11070</td>
			<td>Secondary antibody for phospho-PAK1</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet</td>
			<td>Thermo Scientific (MSC Advantage)</td>
			<td>51025411</td>
			<td>Provide aspetic conditions duirng cell culture</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Electron Corporation (Heraeus Hera Cell 240)</td>
			<td>51026556</td>
			<td>For growing cells at or near body temperature</td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>ZEISS (Carl Zeiss)</td>
			<td>LSM 880</td>
			<td>Able to generate three-dimensional images of large specimen at super-resolution</td>
		</tr>
		<tr>
			<td>DABCO [1,4-Diazobicyclo-(2,2,2) octane]</td>
			<td>Merck</td>
			<td>8034560100</td>
			<td>Anti-bleaching reagent</td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-diamidino-2-phenylindole)</td>
			<td>Sigma</td>
			<td>D9542-1MG</td>
			<td>Neuclear stainer</td>
		</tr>
		<tr>
			<td>DMEM media</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td>Used for the preparation of 100uM of A&#946; (1-42)</td>
		</tr>
		<tr>
			<td>&#160;DMEM/F12 (1:1 mixture of DMEM and Ham&#8217;s F12)</td>
			<td>Gibco</td>
			<td>12500062</td>
			<td>Culture media for SH-SY5Y</td>
		</tr>
		<tr>
			<td>DMSO (Dimethyl sulphide) Cell culture grade</td>
			<td>Cryopur</td>
			<td>CP-100</td>
			<td>Cell culture grade used as dissolving agent for Retinoic acid</td>
		</tr>
		<tr>
			<td>DMSO (Dimethyl sulphide) Molecular grade</td>
			<td>Himedia</td>
			<td>MB058</td>
			<td>Used as one of the dissolving agent for the lyophilized A&#946; (1-42)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>&#160;Major supplement for Culture media (US origin)</td>
		</tr>
		<tr>
			<td>Formalin Fixative (Neutral buffered 10%)</td>
			<td>Sigma Aldrich</td>
			<td>HT5014-120ML</td>
			<td>Component in the Karnovsky&#39;s fixative solution</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (Grade I, 25% in H2O)</td>
			<td>Sigma</td>
			<td>G5882</td>
			<td>Component in the Karnovsky&#39;s fixative solution</td>
		</tr>
		<tr>
			<td>HFIP (1,1,1,3,3,3-hexafluoro-2-propanol ) solution</td>
			<td>TCI</td>
			<td>H024</td>
			<td>Used to dissolve A&#946; (1-42) 1 mg</td>
		</tr>
		<tr>
			<td>Image Processing/ Analysis Software: FIJI (ImageJ)</td>
			<td>National Institute of Health (NIH)</td>
			<td></td>
			<td>Used to process/analyze the images and to differentiate the TNTs from neurites using its plugin named &#34;volume viewer&#34;.</td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Christ, Alpha</td>
			<td>2.4 LDplus</td>
			<td>0.05 mg aliquots of A&#946; (1-42) can be stored in -20 &#176;C after lyophilization only</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Neomycin Mixture</td>
			<td>Thermo fisher Scientific</td>
			<td>15640055</td>
			<td>Antibiotic mixture</td>
		</tr>
		<tr>
			<td>Phalloidin-iFlor 555</td>
			<td>Abcam</td>
			<td>ab176756</td>
			<td>F-actin binding stain</td>
		</tr>
		<tr>
			<td>Phospho-PAK1 (Thr423) /PAK2 (Thr402) [Rabbit]</td>
			<td>CST</td>
			<td>#2601</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Polyclonal Antibody to Tubulin Beta 3 (TUBb3)</td>
			<td>Cloud clone</td>
			<td>PAE711Hu01</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-50MG</td>
			<td>Differentiating reagent</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Merck</td>
			<td>8047-15-2</td>
			<td>Detergent used in the Incubation buffer in immunostaining</td>
		</tr>
		<tr>
			<td>Water bath sonicator (Quart, Drain valve Heater)</td>
			<td>Ultrasonic Cleaner</td>
			<td>3.0 L/3.2</td>
			<td>Sonicator used to dissolve A&#946; (1-42) stock, after DMSO adding to it during the preparation of 100 &#181;M A&#946; (1-42)</td>
		</tr>
		<tr>
			<td>ZEN Microscopy software</td>
			<td>ZEISS (Carl Zeiss)</td>
			<td></td>
			<td>Imaging software to acquire confocal microscopy images with smart automation</td>
		</tr>
	</tbody>
</table>

detection and quantification of tunneling nanotubes using 3d volume view images

最近的发现表明，细胞通过纳米级肌动蛋白膜导管（即“隧道纳米管”（TNT） ）进行 直接，远程，细胞间转移。TNT被定义为开放式脂质双层包围的膜延伸，其介导直径范围在50nm至1μm之间的相邻细胞之间的连续性。 TNT最初在神经元细胞中得到证实，但连续的研究表明TNT存在于几种细胞类型和疾病中，例如神经退行性疾病，病毒感染和癌症。一些研究将相邻细胞之间的封闭式电偶联膜纳米结构称为TNT或TNT样结构。
根据终点膜连续性阐明超微结构在技术上具有挑战性。此外，由于缺乏特异性标记物，在使用常规方法表征TNT方面，细胞-细胞通讯的研究具有挑战性。TNT主要定义为基于F-肌动蛋白的开放式膜突起。然而，一个主要的局限性是F-肌动蛋白存在于所有类型的突起中，这使得区分TNT与其他突起变得具有挑战性。基于F-肌动蛋白的TNT的显着特征之一是这些结构在两个细胞之间徘徊而不接触基质。因此，根据它们在细胞之间的徘徊，可以方便地将不同的F-肌动蛋白染色的TNT与其他突起（例如丝状伪足和神经突）区分开来。
我们最近表明，通过肌动蛋白依赖性内吞作用内聚蛋白的内化低聚淀粉样蛋白-β 1-42（oAβ）刺激活化的p21激活激酶-1（PAK1），其介导SH-SY5Y神经元细胞之间含F-肌动蛋白的TNTs与磷酸化PAK1共表达的形成。该协议概述了一种 3D 体积分析方法，以从捕获的 oAβ 处理的神经元细胞中 F-肌动蛋白和磷酸化-PAK1 免疫染色膜突起的 z 堆栈图像中鉴定和表征 TNT。此外，TNTs与基于F-肌动蛋白和β-III微管蛋白免疫染色膜导管的发育神经突和神经元生长物区分开来。

在这里，我们通过从共聚焦z-stack图像构建3D体积视图来鉴定和表征SH-SY5Y神经元细胞中oAβ诱导的TNT（图1）。用F-肌动蛋白和磷酸化PAK1对细胞进行双重免疫染色。分析免疫染色细胞的共聚焦z-stack图像以鉴定TNT（图2）。此外，分析DIC图像以验证F-肌动蛋白和磷酸化PAK1染色的TNT结构是细胞之间的膜导管（图3）。此外，用F-肌动蛋白和β...

Watch this Scientific Journal Video about Detection and Quantification of Tunneling Nanotubes Using 3D Volume View Images at JoVE.com

Detection and Quantification of Tunneling Nanotubes Using 3D Volume View Images

隧道纳米管（TNTs）主要是连接相邻细胞的开放式F-actin膜纳米管，促进细胞间通讯。TNT与其他细胞突起的显着特征是细胞之间纳米管的悬停性质。在这里，我们通过构建共聚焦z堆栈图像的3D体积视图来表征TNT。

使用3D体积视图图像检测和定量隧道纳米管

Recent discoveries have revealed that cells perform direct, long-range, intercellular transfer via nano-scale, actin-membrane conduits, namely ...

缺乏隧道纳米管特异性标记限制了该领域的进展，并且对识别，表征和量化隧道纳米管的方法的需求不断增加。如果没有开发算法和/或更改现有算法的专业知识，自动检测方法很难实现，但是我们的手动方法很容易以更高的精度实现。通过隧道纳米管的长距离细胞间转移在各种疾病模型中以多种方式实现，包括神经退行性病理学、病毒传播和癌症。因此，对隧道纳米管的研究以了解其在发病机制中的作用非常重要。隧穿纳米管可能会在染色过程中断裂。避免使用盖玻片和安装在载玻片上，而是使用玻璃底部成像皿。改良的固定方案有助于保持隧道纳米管的完整。演示该程序的将是Sangeeta NAF实验室的博士生Deepak KV。首先，在固定前用PBS洗涤对照和寡聚A-β处理的细胞两次，持续两分钟。使用溶解在pH 7.2的0.1摩尔磷酸盐缓冲液中的2%福尔马林固定剂和2.5%戊二醛制备Karnovsky的固定剂溶液。然后通过在室温下加入Karnovsky的固定溶液45分钟来固定成像皿中的细胞。通过将0.1克皂苷溶解在5毫升FBS中并通过加入95毫升PBS稀释来制备孵育缓冲液。然后使用孵育缓冲液洗涤固定细胞两次，持续两分钟。固定后，加入第一个抗磷酸化-PAK1的抗体，在孵育缓冲液中加入1至250的稀释液，并在黑暗潮湿的室中以4摄氏度孵育过夜。孵育24小时后的第二天，用孵育缓冲液洗涤细胞两次，持续两分钟。然后加入与Alexa Fluor 488和鬼笔环肽555偶联的二抗。将细胞在室温下在黑暗潮湿的室中孵育1.5小时。孵育后，用孵育缓冲液洗涤细胞两次，持续两分钟。通过在 1 到 2000 稀释液中加入 DAPI 来染色细胞核，并在室温下在黑暗中孵育 5 至 10 分钟。使用25毫克DABCO在90%甘油和10%PBS中制备DABCO封片剂。为了正确溶解，使用分光光度级稀盐酸将pH调节至8.6，并将溶液保持在摇杆上进行混合。作为抗漂白剂，将DABCO封片剂直接添加到底部包含盖玻片的成像皿上。等待至少一到两个小时，然后直接进行共聚焦成像。为了识别隧道纳米管，使用共聚焦激光扫描显微镜捕获免疫染色细胞的Z-stack图像，方法是首先选择通道并在共聚焦软件中的窗口轨道一，轨道二和轨道三中依次单击所需的激光器。单击轨道一个窗口下方的选项 TPMT，以拍摄带有荧光通道的微分干涉对比图像。选择软件的“采集”选项卡，单击“Z-stack”选项卡，然后等待窗口打开。然后单击实时扫描以聚焦培养皿底部的细胞。选择该聚焦图像作为第一个堆栈，然后向上聚焦以查看单元格的最顶层，并选择该图像作为最后一个堆栈。停止实时扫描并单击“最佳”选项卡旁边的数字以固定堆栈的步长，该步长根据细胞的厚度确定切片的数量和间隔。使用 405 纳米、488 纳米和 561 纳米激光器拍摄 DAPI、FITC 和 TRITC 三个通道的连续图像，并以 1.02 微秒的像素停留时间进行捕获。使用荧光通道捕获DIC通道中的图像以观察细胞边界。打开斐济软件中以czi数据格式保存的共聚焦图像进行分析。选择“超级堆栈”选项以查看图像的每个 Z 堆栈和通道。滚动通道和 Z 堆栈滚动条以选择感兴趣的特定通道的确切堆栈。然后首先通过滚动通道栏选择 F-肌动蛋白染色通道，并手动滚动 Z 堆栈以逐个查看每个堆栈。识别 F 肌动蛋白染色结构，这些结构似乎是连接在 Z 堆栈下部可见的细胞，并且靠近成像皿表面，Z 等于 2 作为神经突。识别隧道纳米管，通过将Z堆栈从Z等于4向顶部滚动Z堆栈来识别隧道纳米管，F-肌动蛋白正悬停细胞到细胞导管。寻找靠近 Z 堆栈下部朝向表面的神经突，并观察到它们开始消失，Z 堆栈在 Z 等于 6 时向顶部滚动。通过分析来自Z堆栈的导管的悬停性质，鉴定磷酸化-PAK1正隧道纳米管与F-肌动蛋白正隧道纳米管类似。由于磷酸化-PAK1染色比F-肌动蛋白染色弱，因此在Z等于4和Z等于6处寻找磷酸化PAK1染色的隧道纳米管。此外，观察DIC图像以验证F-肌动蛋白和磷酸化PAK1染色隧道纳米管结构是细胞之间的膜导管。此外，合并F-肌动蛋白和磷酸化-PAK1通道以验证鉴定的隧道纳米管是F-肌动蛋白和磷酸化-PAK1共染色结构。要量化隧穿纳米管，请手动计算总细胞数和识别的隧穿纳米管，并将数字表示为百分比。为了从Z堆栈图像中区分F-肌动蛋白和β III微管蛋白正隧道纳米管（如悬停导管），合并F-肌动蛋白和β III微管蛋白通道，然后分析合并图像的Z堆栈。寻找专门F-肌动蛋白染色的隧道纳米管，这些纳米管在Z等于3时隐约可见，在Z等于6和Z等于9时突出。同样，识别 F-肌动蛋白和 β III 微管蛋白。双正隧穿纳米管，如悬停导管，Z 等于 6 和 Z 等于 9。从 Z 堆栈的下部识别其他 F-肌动蛋白和 β III 微管蛋白染色的非悬停突起。使用斐济的线工具测量隧道纳米管的直径。通过单击“分析”验证测量比例，然后设置比例，以便自动设置与 czi 图像的距离（以像素为单位）。测量XY平面上隧穿纳米管的直径。使用斐济的体积查看器插件，允许 3D 重新切片和启用阈值的 3D 可视化，将 Z 堆栈图像拆分为单独的通道。然后裁剪单通道Z堆栈图像以使用3D重建视图一次突出显示一个或两个隧道纳米管或神经突。在XY平面上钉一个隧道纳米管或神经突，并标记XZ和YZ通道横截面。观察XZ平面底部，XZ和YZ平面的神经突，以及上Z堆栈中的隧穿纳米管。选择单个隧道纳米管或神经突体以在 XZ 平面中重建 3D 体积视图。在3D重建中，观察Z平面底部的神经突和隧穿纳米管显示为悬停结构，连接两个细胞而不接触底部Z平面。分析具有F-肌动蛋白和磷酸化PAK1的免疫染色细胞的共聚焦Z堆栈图像以鉴定隧道纳米管。此外，分析DIC图像以验证F-肌动蛋白和磷酸化PAK1染色隧道纳米管结构是细胞之间的膜导管。用F-肌动蛋白和β III微管蛋白对细胞进行双重免疫染色，隧道纳米管样F-肌动蛋白和隧道纳米管样F-肌动蛋白和β III微管蛋白双阳性膜导管与仅F-肌动蛋白阳性隧道纳米管区分开来。通过构建3D体积视图图像，将磷酸化PAK1和F-肌动蛋白共表达的隧道纳米管与其他神经突起区分开来。使用正确的固定剂很重要，因为样品的不完全固定可能导致靶蛋白的快速蛋白水解降解和特异性免疫反应性的降低。

Research

JoVE Journal

Biology

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

使用微量的生物规格的纳米分光光度计核酸和蛋白的浓度测定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

科研

生物学

Rapid Analysis and Exploration of Fluorescence Microscopy Images

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

心脏压力容量环分析小鼠使用电导导管

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

Colorimetric Detection of Bacteria Using Litmus Test

细菌利用试金石比色法检测

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

自动定量分析细胞计数程序使用ImageJ插件

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

从巨 Unilamellar 泡中提取膜纳米管

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench

Volume Segmentation and Analysis of Biological Materials Using SuRVoS Super-region Volume Segmentation Workbench

利用 SuRVoS (超区域体积分割) 工作台进行生物材料的体积分割与分析

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

用于SARS-CoV-2检测和定量的两步逆转录液滴数字PCR方案

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

使用磁悬浮定量细胞密度和抗原特性

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

使用单细胞荧光成像对菌际竞争进行量化

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...