Name: isolation of extracellular vesicles from murine bronchoalveolar lavage fluid using an ultrafiltration centrifugation technique
Uploaded: 2018-11-09T13:30:00
Description: Watch this Scientific Journal Video about Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique at JoVE.com

这项工作得到了 nhlbi龙湾国家卫生研究院颁发 hl103868 (至 p. c.) 和 hl137076 (至 p. c.)、美国心脏协会援助奖 (至 p. c.) 和塞缪尔·奥辛综合癌症研究协会 (pc) 的支持。我们要对塞达西奈医疗中心的施密特心脏研究所表示高度赞赏, 该研究所为我们提供了一台用于电动车纳米粒子跟踪分析的纳米视觉机器。

动物的利用和所有动物程序得到了塞达西奈医疗中心 (csmc) 机构动物护理和使用委员会 (iacuc) 的批准。
1. 小鼠支气管肺泡灌洗液 (valf) 的采集和制备
<ol><li>balf 系列<ol>
<li>用氯胺酮 (300 mgkg) 和 xylazine (30 mg kg) 的鸡尾酒对小鼠进行安乐死, 然后通过腹膜内途径进行颈椎脱位。</li>
		<li>将 22 g 血管造影剂插入气管。将含有1毫升 (ml) 冰凉的杜尔贝科磷酸盐缓冲液盐水 (dpbs) 的胰岛素?...

<ol>
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L., Khosroheidari, M., Kanchi Ravi, R., DiStefano, J. 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在过去的几十年里, 科学家们已经解开了 ev 在细胞稳态中的意义。更重要的是, ev 通过生物活性货物分子 1、21、22、26、27调节相邻和远的细胞, 在许多疾病过程中发挥着重要作用,28,29,30. 这一领域?...

外质体是 ev 的最小子集, 直径为 50–200 nm, 在各种信令过程1、2、3、4、5中具有多种生物功能。它们主要通过通过脂类、蛋白质和核酸等货物分子促进细胞间的交流来控制细胞和组织的稳态.电动汽车研究的一个关键步骤是隔离过程。有或没有密度梯度离心 (dgc) 的差动超离心 (uc) 被认为是黄金标准法, 但这种方法存在很大的局限性, 包括 ev 回收率低和可扩展性低 10,11,12、将其最佳利用限制在较大体积的样品中, 如细胞培养上清液或高外体细胞生产样品。其他方法的优点和缺点, 如超滤或色谱仪排除尺寸、珠子或柱的免疫亲和力分离以及微流体, 都有很好的描述, 现代补充程序已经发展到克服并最大限度地减少每种方法中的技术限制 11、12、13、14、15。其他研究表明, 在过滤单元中使用纳米多孔膜的超滤离心 (ufc) 是一种替代技术, 可提供与 uc 方法16、17、18 相当的纯度。这种技术可以被认为是一种替代的隔离方法。
支气管肺泡灌洗液 (balf) 含有在不同呼吸条件下具有多种生物功能的ev19、20、21、22.研究 bal 衍生的 ev 带来了一些挑战, 由于在人类的支气管镜检查程序的侵入性, 以及有限的大量灌洗液回收。在小动物, 如小鼠, 只有几毫升可以恢复在正常的肺条件下, 更不知道在发炎或纤维化的肺 23。因此, 收集足够数量的 balf, 用于通过差动超离心进行电动汽车隔离, 用于下游应用可能是不可行的。然而, 分离正确的 ev 种群是研究电动汽车生物学功能的关键因素。在完善的电动汽车隔离方法中, 效率和功效之间的微妙平衡仍然是一个挑战。
在目前的研究中, 我们证明了一种离心超滤方法, 利用 100 kda 分子量截止 (mwco) 纳米膜过滤单元, 适用于小体积生物样品, 如 balf。该技术简单、高效, 具有较高的纯度和可扩展性, 可支持对 balf 衍生 ev 的研究。

<table>
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 centrifugal filters Ultracel-100K</td>
			<td>Sigma-Millipore, St. Louis, MO</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>Corning Cellgro, Manassas, VA</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Millipore, St. Louis, MO</td>
			<td>EMD8550</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Research Products International, Prospect, IL</td>
			<td>75277-39-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Corning Cellgro, Manassas, VA</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Millipore, St. Louis, MO</td>
			<td>S3014-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep</td>
			<td>Sigma-Millipore, St. Louis, MO</td>
			<td>MKCD9753</td>
			<td>Density Gradient Medium</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>VetOne, Boise, ID</td>
			<td>13985-702-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn Animal Health, Lake Forest, IL</td>
			<td>59399-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1 mL</td>
			<td>BD Syringe, Franklin Lakes, NJ</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiocatheter 20G</td>
			<td>BD Syringe, Franklin Lakes, NJ</td>
			<td>381703</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes 15 mL</td>
			<td>VWR, Radnor, PA</td>
			<td>89039-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes 50 mL</td>
			<td>Corning Cellgro, Manassas, VA</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>Bicinchonic acid (BCA) protein assay</td>
			<td>Pierce, Thermo Fischer Scientific, Rockford, IL</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse TSG101 Antibody</td>
			<td>AbCam, Cambridge, MA</td>
			<td>AB125011</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse PE-CD63 Antibody</td>
			<td>Biolegend, San Diego, CA</td>
			<td>143904</td>
			<td></td>
		</tr>
		<tr>
			<td>CD81</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD9</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked antibody</td>
			<td>Cell Signaling Technology, Danvers, MA</td>
			<td>7074S</td>
			<td></td>
		</tr>
		<tr>
			<td>4x LDS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Reducing agent (Bolt)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Lysis buffer (Bolt)</td>
			<td>Cell Signaling Technology, Danvers, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bolt 4-12% Bis-Tris Plus acrylamide gel</td>
			<td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td>
			<td>NW04120</td>
			<td></td>
		</tr>
		<tr>
			<td>iBlot 2 Nitrocellulose mini stacks</td>
			<td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td>
			<td>IB23002</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemiluminescent HRP antibody detection reagent HyGLO</td>
			<td>Denville Scientific, Holliston, MA</td>
			<td>E2400</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes 17 mL</td>
			<td>Beckman Coulter, Pasadena, CA</td>
			<td>337986</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes 38.5 mL</td>
			<td>Beckman Coulter, Pasadena, CA</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning SFCA Syringe Filters 0.2 &#181;m pore</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>09-754-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter, Pasadena, CA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanosight (NS300)</td>
			<td>Malvern, Worcestershire, UK</td>
			<td>-</td>
			<td>To measure particle size distribution and particle concentration</td>
		</tr>
		<tr>
			<td>MACSQuant Analyzer 10 flow cytometer</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>iBlot Transfer Apparatus</td>
			<td>Thermo Fischer Scientific, Waltham, MA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad ChemiDoc MP Imaging System</td>
			<td>Bio-Rad, Hercules, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo v. 10</td>
			<td></td>
			<td></td>
			<td>Analysis software</td>
		</tr>
	</tbody>
</table>

isolation of extracellular vesicles from murine bronchoalveolar lavage fluid using an ultrafiltration centrifugation technique

细胞外囊泡 (ev) 是新发现的亚细胞成分, 在生理和病理状态下的许多生物信号功能中起着重要作用。由于每种技术固有的限制, ev 的隔离仍然是这一领域的主要挑战。采用密度梯度离心法的微分超离心法是一种常用的方法, 被认为是电动汽车隔离的黄金标准程序。然而, 此过程是耗时的, 劳动密集型的, 一般会导致低可扩展性, 这可能不适合小体积样品, 如支气管肺泡灌洗液。证明了超滤离心分离方法简单、时间高效、省力, 但具有较高的回收率和纯度。我们建议这种隔离方法可以是一种替代方法, 适用于电动汽车隔离, 特别是小批量生物样品。

我们在同一天使用 ufc 和 uc-dgc 隔离方法从鼠标 balf 执行 ev 隔离。ufc 方法大约需要 2.5-3小时, 而 uc-dgc 技术需要8小时的处理时间。这不包括缓冲区和试剂制备时间。应当指出, 其他一些任务可以在长离心期内执行。然而, 整个过程持续了近一整天的 uc-dgc 隔离技术。
与 uc-dgc ev (17.67±7.8 nm,图 1b) 相比, ufc 方?...

Watch this Scientific Journal Video about Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique at JoVE.com

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

在这里, 我们描述了两个细胞外囊泡分离方案, 超滤离心和超离心与密度梯度离心, 从小鼠支气管肺泡灌洗液样品中分离细胞外囊泡。用两种方法对小鼠支气管肺泡灌洗液中的细胞外囊泡进行了定量和表征。

利用超滤离心技术分离小鼠支气管肺泡灌洗液中的细胞外囊

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

此方法可以帮助解决与使用超滤离心法衍生的支气管离泡液的效率、回收率和纯度有关的问题。我们直接将该技术与超离心和密度梯度纯化技术进行比较。该技术的主要优点是简单、高效，适用于小体积样品，如穆林支气管洗液。更重要的是，与超离心隔离方法相比，它可提供高纯度和更高的可扩展性。演示这个程序的将是诺曼·加勒特三世先生，我实验室的副研究员。首先，将22测表血管导管插入安乐死小鼠的气管。将胰岛素注射器与一毫升冰冷无菌 DPBS 连接，将一毫升注入两个肺。慢慢提取注射器柱塞，以检索支气管洗液或BAL液体。将BAL液体放入冰上锥形管中。之后，从25只小鼠中拉出BAL流体，将其分成两组相等。在4摄氏度下将BAL流体在400倍G下离心5分钟。收集上一代，在4摄氏度下以15000倍G离心10分钟。然后收集上一液，并立即执行 EV 隔离步骤。首先，通过0.2微米无菌注射器过滤器过滤超级纳兰特。在此之后，用无菌 DPBS 将离心过滤单元平衡 10 分钟。然后在摄氏4度下将离心装置在15，000倍G下离心10分钟。用 15 毫升 BAL 液体填充过滤器。在3000G下离心30分钟，温度为4摄氏度。在此之后，通过反复轻轻移液，用14毫升无菌DMB洗涤。在4摄氏度的3000 G下将过滤装置离心30分钟，取出DPBS并浓缩了止动物。要收集浓缩的BAL流体衍生EV，请将移液器插入过滤单元的底部，并使用侧扫侧扫除运动提取样品。然后，将BAL流体衍生出EV，并将其储存在零下80摄氏度。或者，从将之前获得的上一个上一个液位转移到超离心管开始。在摄氏4度时，在1万G下离心30分钟。然后收集上一代和离心机。丢弃上一级，在 DPBS 的 200 微升中重新悬浮 EV 颗粒。在此之后，将 EV 悬架与 300 微升 50%碘素醇工作溶液混合。并转移到15毫升的超离心管。然后根据文本协议添加缓冲液，以创建浮力密度梯度，并在 100，000 G 下将其离心 3 小时 50 分钟。收集 15% 和 25% 的碘糖醇馏分，并在 DPBS 中稀释它们，使体积达到 15 毫升。将分数转移到一个新的超离心管中，并在摄氏4度的10万G下离心60分钟。丢弃上一杯，在 50 微升无菌 DPBS 中重新悬浮 EV 颗粒。要开始纳米颗粒跟踪分析，请将EV样品稀释在一毫升的DPBS中。将样品装入胰岛素注射器。在此之后，将注射器连接到注射器泵，并测量颗粒数和大小。将摄像机级别设置为 14，将检测阈值设置为 1。接下来，使用板读卡器通过色度检测量化EV样品中的蛋白质含量。用印迹加载缓冲液和DTT溶解每个样品中等量的EV蛋白。然后在70摄氏度加热样品10分钟。将样品装入双流加丙烯酰胺凝胶中，并运行电泳35分钟。然后使用干转法将蛋白质转移到硝基纤维素膜上。在此之后，用5%的脱脂牛奶阻断膜60分钟，轻轻摇动。在四摄氏度下用EV表面蛋白标记的抗体孵育膜，并温和摇动过夜。在室温下用HRP链接抗体孵育膜60分钟，形成膜。用TBST缓冲液清洗膜10分钟三次。最后，在进行成像之前，利用化学发光HRP抗体检测试剂开发膜。为了开始流动细胞学，在49微升PEB染色缓冲液中稀释BAL流体衍生EV。然后在每个样品中加入三个抗体，然后在黑暗中摇动60分钟，在4摄氏度下孵育样品。最后，用40微升膜过滤PEB染色缓冲液稀释样品，进行流式细胞测定分析。在此协议中，EV 使用 UFC 和 UC-DGC 方法与小鼠 BAL 流体隔离。与 UC-DGC-EV 的尺寸和均匀尺寸分布不同，从 UFC 方法分离出的从正常小鼠中提取的 EV 的 BAL 流体显示尺寸更小、尺寸更均匀。与 UC-DGC 技术相比，UFC 技术的总粒子计数也显著增加。以UFC-EV毫克为单位的蛋白质总回收率也高于UC-DGC-EV，表明UFC技术更加时间和精力高效。最后，通过流式细胞仪对BAL流体衍生EV进行进一步描述。UFC-EV 和 UC-DGC-EV 均表示 CD63、CD9 和 CD81。在尝试此过程时，重要的是要记住用 DPBS 对超滤单元膜进行补水。一旦膜被水合，装置需要保持湿润，直到使用。从过滤单元中检索细胞外囊泡节可能具有挑战性。确保移液器尖端被一边扫到一边，以便从过滤器单元中恢复大部分 EV。该技术开发后，为细胞外囊泡分离领域的研究人员探索小动物正常或病理条件下发泡体外囊体的生物功能铺平了道路。

Research

JoVE Journal

Immunology and Infection

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

科研

免疫与感染

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Isolation of Murine Lymph Node Stromal Cells

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation and Activation of Murine Lymphocytes

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

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 ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

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 ...

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment