Name: isolation and characterization of extracellular vesicles from adult schistosoma japonicum
Uploaded: 2018-05-22T13:00:00.000+00:00
Duration: 7 min 37 s
Description: Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

本研究部分或全部由中国国家自然科学基金 (31472187 和 31672550) 和中国科学院农业科技创新项目资助。

所有动物实验均获中国科学院上海兽医研究所地方伦理委员会批准 (许可证编号: SHVRIAU-14-0101)。
1.血吸虫的体外区域性
注意:血吸虫表示生物危害。在处理蠕虫、血吸虫悬浮液或其他相关生物材料时, 工人应始终佩戴乳胶手套。新西兰兔感染了2000尾蚴通过腹部暴露。血吸虫在肝脏阶段从感染了日本血吸虫尾蚴的家兔身上采集肝脏和肠系膜静脉, 感染后28天。在以前发布的18小鼠报告研究中引用了蠕虫收集过程。
<ol><li>在500毫升的烧杯中收集寄生虫。用200毫升预热 (37 °c) PBS (pH 7.4) 将其彻底洗净并轻轻地3x。</li>
	<li>分别为每90毫米培养皿200个蠕虫对。然后用50毫升预热的 (37 °c) 的 RPMI-1640 培养基, 用 100 U 的青霉素和100毫克/毫升的链霉素, 彻底和温和地洗涤寄生虫2x。</li>
	<li>在40毫升的预热 RPMI-1640 培养基中保持寄生虫, 37 摄氏度以下 5% CO2在5毫米培养皿 2 h 的密度为 ~ 90 蠕虫对/毫升。 备注:RPMI 1640 培养基不应含有血清, 否则将引入外源性胞外囊。</li>
</ol>2. 预处理的条件介质
<ol><li>收集培养基, 然后离心培养基在4°c 在 2000 x g 30 分钟, 以消除鸡蛋和蠕虫盖碎片。</li>
	<li>将上清液转移到新鲜的管子上, 然后用0.22 µm 注射器过滤器过滤溶液。</li>
	<li>收集过滤后的解决方案在一个透析袋 (分子量切断: 3.5kD), 然后透析解决方案使用 8% PEG8000 溶液在4摄氏度过夜。 备注:这一步对丰富 SjEVs 提高 SjEVs 分离率具有重要意义。</li>
</ol>3. SjEVs 的分离
<ol><li>将透析溶液转移到新管, 然后添加0.5 卷的总外切隔离试剂。</li>
	<li>通过涡流或吹打向上和向下混合解决方案, 以确保解决方案的均匀性。</li>
	<li>孵化混合物在2°c 到8°c 过夜。</li>
	<li>离心机混合物在 1.5万 x g 为1小时在2°c 到8°c。</li>
	<li>吸入并丢弃上清。 备注:电动车包含在管底部的颗粒中 (在大多数情况下不可见)。</li>
	<li>并用重悬2毫升 PBS 中的电动车颗粒, 然后将 evs 解决方案存储在-80 摄氏度, 直到进一步分析。</li>
</ol>4. 西方印迹对电动汽车的表征
<ol><li>测定 SjEVs 样品的蛋白质浓度,例如,通过鉴定分析。</li>
	<li>在22.5 µL 马国贤缓冲区中准备 10-20 µg 的 EVs。然后增加7.5 µL 4x 装载缓冲和热量5分钟在95°c。</li>
	<li>用12% 页凝胶分离 SjEVs 蛋白。</li>
	<li>将蛋白质转移到 PVDF 膜, 然后用 anti-CD63 抗体 (1:1, 000 稀释) 探针, 然后用抗兔 IgG (1:5, 000 稀释) 进行探测。</li>
	<li>利用 ECL 检测试剂进行膜开发, 通过化学发光成像系统检测信号。</li>
</ol>5. 电动汽车的透射电镜表征
<ol><li>添加2µL 的隔离 SjEVs 到200网格 formvar 涂层网格, 然后允许它在室温下干燥。</li>
	<li>用2% 磷钨酸染色1分钟, 然后使其在室温下干燥。</li>
	<li>将网格加载到透射电子显微镜的样品架上, 并暴露在 80 kV 电子束上以进行图像捕捉。</li>
</ol>6. SjEVs 标签与 PKH67 和细胞吸收试验
备注:所有后续步骤都是在室温下 (20-25 °c) 进行的, 除非另有说明。
<ol><li>将5µg SjEVs 溶液转化为锥形底聚丙烯管, 并通过添加 RPMI 1640 培养基将体积调整到12毫升。</li>
	<li>离心机 SjEVs 在 11万 x g 为90分钟在4°c 到颗粒 SjEVs。</li>
	<li>离心后, 小心地吸入上清液。 注意:PKH67 乙醇染料不应直接添加到 SjEV 颗粒, 因为这将导致异质染色和降低细胞活力。</li>
	<li>在 SjEV 颗粒中加入1毫升稀释剂 C, 制备 2x EV 悬浮液;为了确保完全分散, 并用重悬与温和的吹打。</li>
	<li>在稀释剂 c 染色前, 在聚丙烯离心管中加入2µL 的 PKH67 乙醇染料溶液, 将1毫升的稀释剂 c 添加到分散的混合井中, 制备出一种新鲜的2x 染料溶液。</li>
	<li>快速添加1毫升 2x EV 悬浮 (步骤 6.4) 到1毫升2x 染料溶液 (步骤 6.5), 并立即混合样品的吹打。</li>
	<li>室温下将混合物孵化4分钟。</li>
	<li>通过添加4毫升的血清, 然后孵育1分钟, 停止染色。</li>
	<li>添加 RPMI1640 介质, 以调整体积为12毫升, 然后离心机在 11万 x 克4°c 90 分钟。</li>
	<li>取出上清液并添加 RPMI1640 培养基并用重悬 PKH67 标记 SjEV 丸。</li>
	<li>离心机在 11万 x g 在4°c 90 分钟清洗一次, 然后添加 PBS 并用重悬 SjEVs 丸。</li>
</ol>7. SjEVs 细胞吸收试验
<ol><li>种子哺乳动物细胞, 如国家反恐中心克隆1469细胞或 HEK293T 细胞在6井板 (2×10 5 细胞每井) 和培养细胞过夜。</li>
	<li>添加5µg PKH67-labelled SjEVs, 然后培养细胞在37°c 为 2-4 h。</li>
	<li>孵化后, 取出培养基, 用 PBS 清洗细胞两次。</li>
	<li>用4% 甲醛溶液固定细胞15分钟, 再用 PBS 清洗两次。</li>
	<li>染色细胞细胞核与 4′-, 6-diamidino-2-苯基吲哚 (DAPI)。</li>
	<li>用荧光显微镜观察细胞。</li>
</ol>

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作者没有什么可透露的。

最近对电动汽车的研究表明, 血吸虫电动车在宿主病原体相互作用中起着重要作用3,9,12,16。为了进一步解决它们的管理功能, 必须将 EVs 与血吸虫隔离开来。在这里, 我们描述了一种 SjEV 隔离的替代方法。此方法从 100 nm 到 400 nm, 在成年的日本血吸虫中产生 SjEVs 的宽范围大小。下面的细胞摄取检?...

胞外囊泡 (EVs) 是一种含有多种蛋白质、脂质和核酸的小膜状囊泡的种群。最近的研究表明, 电动车在细胞通讯中起着至关重要的作用, 并参与了许多生理过程的调节, 包括细胞发育、免疫调节、血管生成和细胞迁移2, 3,4,5。累积证据表明, 电动车、循环外来体及其 miRNA 货物代表某些疾病的潜在生物标志物 6.
一些原生动物如阴道毛滴虫、锥虫和利什曼原虫, 已被证明能够分泌电动车;此外, 还发现蠕虫将 EVs 分泌到活主机7中。寄生电动车已被证明涉及感染的维护, 致病性8和免疫调节9。最近在血吸虫的研究中,日本曼氏(曼氏) 10,11和日本血吸虫12已经表明, 成年血吸虫分泌外切样的水泡, 可能是参与特定生物过程的功能规范。
迄今为止, 已有几种方法用于分离胞外囊泡, 如离心、超滤13、聚合物基试剂的使用、大小排除色谱和免疫亲和层析隔离。这些不同的方法具有各自的优点和局限性14。一般认为, 离心是囊泡分离的金标准方法。但是, 此方法受到可能的 EV 聚合14的限制。
在血吸虫中, 已报告了两种 EV 隔离的方法: 包括离心11、12、10和使用商用 EV 隔离套件16等几个阶段 (鸡蛋16,血吸虫童虫11, 成人血吸虫10, 12, 17).鉴于微泡的尺寸范围从数以千纳米到上千奈米, 我们开发了一种替代方法, 结合使用台式离心机, 超滤, 和商用 EV 隔离套件, 以隔离电动车从成年的日本血吸虫。隔离的电动车通常拥有直径 100-400 nm, 并成功地内化的接收细胞。

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Total Exosome Isolation Reagent (from cell culture media)</td><td>ThermoFisher SCIENTIFIC</td><td>4478359</td><td>For isolating S. japonicum extracellular vesicles</td></tr><tr><td>PKH67</td><td>Sigma-aldrich</td><td>MINI67</td><td>For labeling Evs</td></tr><tr><td>RPMI 1640 Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11875119</td><td>For parasite culture</td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher SCIENTIFIC</td><td>15140122</td><td></td></tr><tr><td>0.22&amp;mu;m syringe filter</td><td>Merck</td><td>SLGP033RB</td><td></td></tr><tr><td>SnakeSkin Dialysis Tubing</td><td>Thermo SCIENTIFIC</td><td>68035</td><td></td></tr><tr><td>PEG8000</td><td>Sangon Biotech</td><td>A100159&amp;nbsp;</td><td></td></tr><tr><td>RIPA</td><td>Beyotime</td><td>P0013B</td><td></td></tr><tr><td>EMD Millipore&amp;trade; Immobilon&amp;trade; Western Chemiluminescent HRP Substrate (ECL)</td><td>Fisher scientific</td><td>WBKLS0100&amp;nbsp;</td><td></td></tr><tr><td>DAPI</td><td>Cell Signaling Technology</td><td>4083</td><td></td></tr><tr><td>BCA kit</td><td>Beyotime</td><td>P0010</td><td></td></tr><tr><td>CD63 antibodies</td><td>Sangon Biotech</td><td>D160973-0025</td><td></td></tr><tr><td>PVDF</td><td>Bio-Rad</td><td>1704273</td><td></td></tr><tr><td>Formvar/Carbon 400 mesh</td><td>Ted Pella</td><td>01754-F</td><td></td></tr><tr><td>Phosphotungstic Acid</td><td>Ted Pella</td><td>19402</td><td></td></tr><tr><td>anti-mouse IgG-HRP</td><td>CWBIO</td><td>CW0102</td><td></td></tr><tr><td>NCTC clone 1469 cells</td><td>ATCC</td><td>ATCC&amp;reg; CCL-9.1&amp;trade;</td><td></td></tr><tr><td>FBS</td><td>HyClone</td><td>SV30087.02</td><td></td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE chemoluminescance imaging system</td><td>GE</td><td>ImageQuant LAS4000mini</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Hitachi</td><td>H-7600</td><td></td></tr><tr><td>Milli-Q water</td><td>Milli-Q</td><td>Milli-Q Elix</td><td></td></tr><tr><td>Eppendor Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Centrifuge 5804R</td><td></td></tr><tr><td>Zetasizer Nano</td><td>Malvern</td><td>Zetasizer Nano ZS&amp;nbsp;</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima L-100 XP Ultracentrifuge</td><td></td></tr><tr><td>Incubator</td><td>ESCO</td><td>CCL-107B</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Zeiss Axin Observer Z1</td><td></td></tr></tbody></table>

isolation and characterization of extracellular vesicles from adult schistosoma japonicum

胞外囊泡 (EVs) 是由多种细胞释放到胞外微环境中的膜状囊泡。EVs 代表了异质泡的种群, 其大小范围介于40和 1000 nm 之间。积累的证据表明, 电动车在病原宿主相互作用中起着重要的调控作用。对血吸虫电动车的深入了解, 应能洞察血吸虫与宿主相互作用的机制, 从而有助于制定防治血吸虫病的新策略。在这里, 我们的目的是进一步研究电动车在血吸虫的功能, 提出了一个协议, 以隔离和特征的电动车从成年的日本血吸虫(S. 血吸虫)。使用离心结合商用外切隔离套件, 从体外培养基中分离出电动汽车。隔离的日本血吸虫EVs (SjEVs) 通常具有 100-400 nm 的直径, 其特征是透射电镜和西方印迹。PKH67 染料标记 SjEVs 的使用表明, SjEVs 是由受体细胞内部化的。总的来说, 我们的协议提供了一种从成人血吸虫隔离电动车的替代方法;隔离 SjEVs 可适用于功能分析。

为了用所描述的协议来量化孤立 SjEVs 的产量, 我们利用 SjEVs 蛋白质检测方法, 从28天成年血吸虫中获取分离的蛋白质浓度。如表 1所示, SjEV 蛋白浓度从208µg 到250µg 每100毫升培养基 (表 1) 不等。由马尔文纳米粒子分析确定的粒子大小表明, 隔离的 SjEVs 从 100 nm 到 400 nm 不等, 在大小上大约 220 nm (图 1)。通过透射电镜对 SjEVs 的进?...

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

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

在这里, 我们提出一个协议, 以描述细胞外囊泡 (电动车) 隔离在体外培养培养基从成年日本血吸虫。

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

isolation-characterization-extracellular-vesicles-from-adult

Research

JoVE Journal

Immunology and Infection

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

7.8K 次观看.  Ministry of Agriculture. 在这里, 我们提出一个协议, 以描述细胞外囊泡 (电动车) 隔离在体外培养培养基从成年日本血吸虫。

视频: Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

科研

免疫与感染

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

从使用鸡蛋<em&gt;血吸虫</em&gt;作为<em&gt;在体内</em&gt;蠕虫引起的肺部炎症模型

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

人血 Intramolluscan 期的体外分离与培养的研究血吸虫氏

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

发育生物学

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

肝癌组织来源于小细胞外囊泡的富集方法

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

癌症研究

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

用于从条件细胞培养基中分离细胞外囊泡的体积排阻色谱

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

神经科学

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

细粒棘球蚴小细胞外囊泡的分离和树突状细胞摄取

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

卡诺尔哈布炎细胞外囊泡的纯化与分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

生物学

<meta charset="utf8"></head><body>小鼠脾源性外泌体分离的改进技术

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

<meta charset="utf8"></head><body>小鼠脾脏组织来源的外泌体的分离和表征

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

小鼠脾脏组织来源的外泌体的分离和表征

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

成年日本血吸虫细胞外囊泡的分离与鉴定

摘要

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此视频中的章节

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

从使用鸡蛋

人血 Intramolluscan 期的体外分离与培养的研究血吸虫氏

肝癌组织来源于小细胞外囊泡的富集方法

用于从条件细胞培养基中分离细胞外囊泡的体积排阻色谱

细粒棘球蚴小细胞外囊泡的分离和树突状细胞摄取

卡诺尔哈布炎细胞外囊泡的纯化与分析

小鼠脾脏组织来源的外泌体的分离和表征

小鼠脾脏组织来源的外泌体的分离和表征

小鼠脾脏组织来源的外泌体的分离和表征