Name: isolation and characterization of extracellular vesicles produced by iron-limited mycobacteria
Uploaded: 2019-10-31T14:30:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria at JoVE.com

我们感谢拉斐尔·普拉多斯-罗萨莱斯分享了抗MEV抗血清和纳夫内特多格拉执行纳米粒子跟踪分析。

1. 制备铁耗定义介质
<ol><li>在塑料容器中溶解5克KH2 PO 4、5gL-芦笋、20 mL甘油和2克脱离子水中的脱离子水，制备1升最小介质（MM）。避免使用玻璃以防止铁污染。将 pHH 调节到 6.8，带 5 N NaOH，用水将体积调节到 1 L。</li>
	<li>加入 50 g 金属合合树脂 （MCR），在 4°C 下使用磁性搅拌棒轻轻搅拌 24 小时。通过带塑料接收器的 0.22 μm 过滤单元对 MCR 进行消毒和去除。为了加速过滤和防止?...

<ol>
	<li>Prados-Rosales, 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=Mycobacteria+release+active+membrane+vesicles+that+modulate+immune+responses+in+a+TLR2-dependent+manner+in+mice.">Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice.</a> Journal of Clinical Investigation. 121, 1471-1483 (2011).</li><li>Gupta, S., Rodriguez, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterial+extracellular+vesicles+and+host+pathogen+interactions.">Mycobacterial extracellular vesicles and host pathogen interactions.</a> Pathogens and Disease. 76 (4), (2018).</li><li>Athman, J. 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=Bacterial+Membrane+Vesicles+Mediate+the+Release+of+Mycobacterium+tuberculosis+Lipoglycans+and+Lipoproteins+from+Infected+Macrophages.">Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages.</a> Journal of Immunology. 195, 1044-1053 (2015).</li><li>Athman, J. 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=Mycobacterium+tuberculosis+Membrane+Vesicles+Inhibit+T+Cell+Activation.">Mycobacterium tuberculosis Membrane Vesicles Inhibit T Cell Activation.</a> Journal of Immunology. 198, 2028-2037 (2017).</li><li>Rath, P., 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=Genetic+regulation+of+vesiculogenesis+and+immunomodulation+in+Mycobacterium+tuberculosis.">Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis.</a> Proceedings of the National Academy of Science U.S.A. 110, E4790-E4797 (2013).</li><li>White, D. W., Elliott, S. R., Odean, E., Bemis, L. T., Tischler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+Pst/SenX3-RegX3+Regulates+Membrane+Vesicle+Production+Independently+of+ESX-5+Activity.">Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity.</a> mBio. 9, pii 00778 (2018).</li><li>Dauros Singorenko, P., 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=Isolation+of+membrane+vesicles+from+prokaryotes:+a+technical+and+biological+comparison+reveals+heterogeneity.">Isolation of membrane vesicles from prokaryotes: a technical and biological comparison reveals heterogeneity.</a> Journal of Extracellular Vesicles. 6, 1324731 (2017).</li><li>Prados-Rosales, R., Brown, L., Casadevall, A., Montalvo-Quiros, S., Luque-Garcia, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+membrane+vesicle-associated+proteins+in+Gram-positive+bacteria+and+mycobacteria.">Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria.</a> MethodsX. 1, 124-129 (2014).</li><li>Prados-Rosales, 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=Role+for+Mycobacterium+tuberculosis+membrane+vesicles+in+iron+acquistion.">Role for Mycobacterium tuberculosis membrane vesicles in iron acquistion.</a> Journal of Bacteriology. 196, 1250-1256 (2014).</li><li>Sanders, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Laboratory+Techniques:+Plating+Methods.">Aseptic Laboratory Techniques: Plating Methods.</a> Journal of Visualized Experiments. 63, e3064 (2012).</li><li>Harlow, E., Lane, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Antibodies. A laboratory manual. , (1988).</li><li>Lotvall, 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=Minimal+experimental+requirements+for+definition+of+extracellular+vesicles+and+their+functions:+a+position+statement+from+the+International+Society+for+Extracellular+Vesicles.">Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.</a> Journal of Extracellular Vesicles. 3, 26913 (2014).</li></ol>

多种纯化真核细胞衍生体外体的方法已经开发12。相比之下，关于纯化细菌衍生EV7的有效方法的信息有限。Mtb衍生的EV的有效分离需要考虑生长这种致病性分枝杆菌的内在困难。Mtb 的分部时间长（±24 小时），应在生物安全三级 （BSL-3） 条件下处理。因此，优化MEV隔离方法的效率非常重要。由于分枝杆菌释放糖脂和其他疏水性分子，聚合和容易污染粗MEV制剂到?...

分枝杆菌细胞外囊泡（MEVs）是膜结合的纳米颗粒，60~300nm大小，由快速生长和缓慢生长的分枝杆菌1自然释放。致病性分枝杆菌释放的MEVs通过免疫活性蛋白、脂质和糖脂以浓缩和保护方式分泌的宿主相互作用的机制2，3，4。为了描述MEV的特征并了解其生物发生和功能，严格而高效的囊泡纯化和验证方法至关重要。到目前为止，MEV已被分离出在富含铁的介质1，5，6，7，8的分枝杆菌的培养中。
然而，以前的工作表明，铁限制极大地刺激了Mtb的囊泡释放，可能通过mycobactin捕获铁，一种在MEVs9中分泌的侧磷酸盐。虽然已经描述了从高铁培养的Mtb培养的MEV分离的程序，但没有报告从低铁培养物获得MEV的有效方法。因此，此方法的目标是分离、纯化和量化从低铁培养物中获得的 MEV，以便它们可用于生化和功能测定以及分析分枝杆菌中囊泡生产的遗传决定因素。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amicon stirred cell Model 108</td>
			<td>EMD Milipore</td>
			<td>UFSC40001</td>
			<td>Cell Ultrafiltration system</td>
		</tr>
		<tr>
			<td>BD Polypropilene 225 ml conical tubes</td>
			<td>Fisher</td>
			<td>05-538-61</td>
			<td>Conical centrifuge tubes</td>
		</tr>
		<tr>
			<td>Biomax 100-kDa cut-off ultrafiltration membrane</td>
			<td>EMD Milipore</td>
			<td>PBHK07610</td>
			<td>Ultrafiltration membrane</td>
		</tr>
		<tr>
			<td>Chelex-100 resin</td>
			<td>Bio-Rad</td>
			<td>142-2842</td>
			<td>Metal chelating resin</td>
		</tr>
		<tr>
			<td>Middlebrook 7H10 Agar</td>
			<td>BD Difco</td>
			<td>262710</td>
			<td>Mycobacterial Agar plates</td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 Broth</td>
			<td>BD Difco</td>
			<td>271310</td>
			<td>Mycobacterial broth medium</td>
		</tr>
		<tr>
			<td>Nitro cellulose blotting membrane</td>
			<td>GE Healthcare</td>
			<td>10600001</td>
			<td>Blotting Membrane</td>
		</tr>
		<tr>
			<td>Optiprep</td>
			<td>Sigma</td>
			<td>D1556</td>
			<td>Iodixanol</td>
		</tr>
		<tr>
			<td>Polycarbonate ultra centrifugation tubes 25 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>355618</td>
			<td>Polycarbonate ultra centrifugation tubes 25 x 89 mm</td>
		</tr>
		<tr>
			<td>Polypropylene thin walled centrifuge tube 13x15 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td>Polypropylene thin walled centrifuge tube 13x15 mm</td>
		</tr>
		<tr>
			<td>Protein Assay dye</td>
			<td>BioRad</td>
			<td>5000006</td>
			<td>Bradford Protein Staining</td>
		</tr>
		<tr>
			<td>SYPRO Ruby</td>
			<td>Molecular Probes</td>
			<td>S12000</td>
			<td>Ultrasensitive protein stain</td>
		</tr>
		<tr>
			<td>TMA-DPH</td>
			<td>Molecular Probes</td>
			<td>T204</td>
			<td>1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene p-Toluenesulfonate</td>
		</tr>
		<tr>
			<td>Vacuum filtration flasks</td>
			<td>CellPro</td>
			<td>V50022</td>
			<td>Filter Unit</td>
		</tr>
	</tbody>
</table>

isolation and characterization of extracellular vesicles produced by iron-limited mycobacteria

分枝杆菌，包括人类结核病的病原体结核分枝杆菌（Mtb），自然释放含有免疫活性分子的细胞外囊泡（EV）。关于囊泡生物成因的分子机制、囊泡的含量及其在病原体-宿主界面中的作用的知识非常有限。解决这些问题需要严格的EV隔离、纯化和验证程序。以前，当M. 结核病受到铁限制时，囊泡的产生会增强，Mtb 在宿主环境中遇到这种情况。这里介绍了一个完整和详细的协议，从缺铁的分枝杆菌分离和纯化EV。采用定量和定性方法验证纯化EV。

MEV通过在密度梯度中的差分沉淀得到纯化（图1，图2）。在所述条件下，MEV主要以梯度分数3（F3）分离，对应于25%的碘二醇。这一结论基于对蛋白质、膜脂的检测、完整MEV的微观可视化、纳米颗粒大小分布以及抗病毒抗血清的正反应（图2，图3）。向菌落形成单位（CCFUs）规范化的蛋?...

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria at JoVE.com

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

结核分枝杆菌显示，由于低铁条件，细胞外囊泡的产量增加并释放。这项工作详细介绍了产生低铁条件的协议，以及针对缺铁而释放的分菌外囊泡的纯化和表征方法。

铁限分枝杆菌产生的细胞外囊泡的分离和特征

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

该方法允许在严格的低铁条件下培养结核分枝杆菌，从低铁培养物中净化膜囊泡。由于铁限制已知可以刺激膜囊泡的产生，因此该协议产生高丰度的纯膜囊泡，可用于进一步生化或功能测定。当我们演示使用分枝杆菌，非病毒菌株的协议，该协议可以遵循在BSL-2设施使用结核分枝杆菌，致命的菌株。首先，在塑料容器中溶解5克磷酸磷酸盐、5克L-芦笋、20毫升甘油和2克葡萄糖，在900毫升的去维化水中，准备1升最小介质。使用五个正常氢氧化钠将 pH 调节到 6.8，用去离子水将体积调节为 1 升。然后加入50克金属溷合树脂，在4摄氏度下使用磁搅拌棒轻轻搅拌24小时。第二天，停止搅拌，让树脂沉淀物约30分钟。通过 22 微米过滤单元（带塑料接收器）进行过滤，对溶液中剩余的树脂进行消毒和去除。现在，补充最低介质每升氯化锌0.5毫克，每升硫酸镁40毫克，每升硫酸锰0.1毫克。在两毫升分枝杆菌中接种单一聚落的分枝杆菌，辅以 ADN 浓缩。在37摄氏度下与搅拌孵育。当有可见的生长时，提取培养的两毫升，并在分光光度计上测量OD 540。当 OD 540 达到 0.8 时停止孵育，这表示对数阶段较晚。将 200 微升的晚期对数培养剂传播到分枝杆菌油板上，并辅以 0.2 甘油、0.5%Tween 80 和 ADN。至少接种五个板。在37摄氏度下孵育板。一周后，细菌的生长是可见的汇合层。然后在低铁最小介质中湿润无菌棉签。使用此拭子从阿加板中收集细菌。在管中用100毫升的低铁最小介质中接种细菌。收集足够的细菌，准备一个悬挂与OD 540之一。用低铁最小介质将悬浮液稀释10倍至1升，并分成两个无菌塑料瓶。接下来，将培养的两毫升转移到五毫升培养管。按体积tyloxapol添加10微升10%体积。在37摄氏度下孵育培养14天。将培养器转移到五个 225 毫升锥形离心管和离心机，温度为 2，850 次 g，在 20 摄氏度下为 7 分钟。用 50 毫升移液器收集培养物，并通过 0.22 微米过滤器进行过滤器消毒。为了分离 MEV，将培养物过滤转移到放置在 4 摄氏度的搅拌细胞超滤系统中，并通过 100 千吨截止膜以低于 50 PSI 的压力过滤浓缩物。然后在4摄氏度下将浓度培养物以15，000倍g的过滤器离心15分钟，并在聚碳酸酯超离心管中收集上流液。在摄氏4度下将上一代在10万次g下离心两小时。之后，通过温和的移液，去除上清液，将无菌颗粒重新在一毫升无菌PBS中。在聚丙烯薄壁超离心管底部将 0.5 毫升颗粒悬浮液与 1.5 毫升 60%碘异醇溶液混合。覆盖此 MVE 碘沙醇 45%悬浮液与一毫升 40%35%30%25%和 20% 碘桑醇溶液和一毫升 PBS 的顶部。在4摄氏度下以10万次g的离心机，18小时。接下来，使用一毫升汉密尔顿注射器收集从顶部开始的一毫升密度梯度分数。用PBS将每个收集的分数稀释到20毫升，在4摄氏度下以10万倍g的离心机稀释两小时。取出0.5毫升PBS中的上上液和重新暂停。将管子存放在摄氏四度。为了进行膜脂质分析，将每个梯度分数的10微升与荧光膜探针TMA-DPH结合，最终浓度为每毫升1微克，在96孔黑板的每个孔中每毫升PBS中含量为1微克。在33摄氏度下孵育板20分钟。测量荧光在360纳米的激发和430纳米的排放。在该协议中，MEV通过密度梯度的差分沉积进行纯化。在所述条件下，MEV主要以梯度分数三分离，对应于25%的碘桑醇。囊泡相关蛋白质集中在点印分析显示的分数三中。负染色也证实了第三部分中存在 MEV，纳米粒子分析表明，MEV的大小约为 100 纳米。蛋白质和脂质浓度正常化为菌落形成单位，相对于高铁条件，低铁的 MEV 产量增加了约八倍。点印迹分析表明，与铁足够的 MTB 培养物不同，来自铁有限 MTB 培养物的 MEV 相关标记的强信号。按照说明制备最小介质，以确保铁有限的条件，并防止膜囊泡与脂蛋白聚合物污染。以这种方式纯化的分枝杆菌囊泡可用于生化特性和功能分析。

Research

JoVE Journal

Immunology and Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

科研

免疫与感染

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征：两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

流量Virometry来分析病毒颗粒的抗原谱和外囊泡

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

中性粒细胞衍生微粒的分离与鉴定功能研究

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

成年日本血吸虫细胞外囊泡的分离与鉴定

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...