本研究得到了 国家杰出青年科学基金（82025024）、国家自然科学基金重点项目（82230080）; 国家重点研发计划（2021YFA1300604）;国家自然科学基金（81871735、82272438、82002245）;广东省杰出青年自然科学基金（2023B1515020058）; 广东省自然科学基金（2021A1515011639）; 山东省自然科学基金国家重大基础研究发展计划（ZR2020ZD11）; 博士后科学基金（2022M720059）;南方医科大学南方医院优秀青年发展计划（2022J001）。

南方医科大学南方医院伦理委员会批准了这项研究，该研究是在参与者知情同意的情况下进行的。本文采用的所有方法均符合国际人类微生物组标准（IHMS：http://www.microbiome-standards.org/）提供的标准操作指南。所有随后的液体处理程序都必须在生物安全柜或超洁净工作台内进行。
1. 粪便样本的采集和分装
<ol><li>分发粪便采样器、密封袋和冰盒，并向参与者提供有关如何获取和保存样本的全面说明。</li>
	<li>指示每个参与者使用提供的采样器收集他们的粪便样本，并在 24 小时窗口内将其以 4 °C 的温度运送到实验室。</li>
	<li>在实验室收到后，使用无菌勺将少于 3.5 g 的粪便分装到预先称重的 50 mL 离心管中。在试管上记下粪便样本的重量，以便进行后续的预处理程序。 暂停点：如果无法立即处理样品，请在适当记号后将样品分配在-80°C下长期储存。</li>
</ol>2. 粪便样品制备
<ol><li>在4°C下冷却500mL磷酸盐缓冲盐水（PBS）。 使用 50 mL 注射器通过 0.22 μm 聚醚砜 （PES） 过滤器将预冷的 PBS 过滤到 10 个 50 mL 离心管中。</li>
	<li>将两个 50 mL 试管放在冰上，每个试管含有 3.5 g 粪便样品。向每个试管中加入 35 mL PBS。 注意：计算粪便溶解所需的 PBS 量，确保最大样品浓度为 10% （w/v）。如果处理其他样品，请根据需要使用更多试管。</li>
	<li>在4°C下以300rpm摇动样品2小时，或在4°C下放置至少8小时，直到粪便完全悬浮可见。</li>
</ol>3.差速离心
<ol><li>将高速冷冻离心机冷却至4°C。</li>
	<li>用PBS调整含有步骤2.3中制备的样品的两个试管的重量，使其总重量达到0.1g。</li>
	<li>将样品在4°C下以3,000× g 离心20分钟。</li>
	<li>小心地将上清液移液到两个干净的 50 mL 离心管中，在沉淀上方留下约 1 mL。在此过程中，使用一次性塑料巴斯德移液器。</li>
	<li>用PBS调整两个试管的重量，使其总重量在0.1g±以内。</li>
	<li>将转移的上清液在4°C下以12,000× g 离心30分钟。</li>
	<li>使用20 mL注射器吸出上清液，取出注射器针头，并通过0.22μm过滤器将其过滤到50 mL管中。此时，上清液体积应约为 30 mL。 注意：如果在12,000× g 离心后仍然可见大量杂质，则必须在4°C下以12,000× g 重复离心步骤30分钟。 如果不这样做，可能会因过滤过程中的孔隙堵塞而导致 BEV 的大量损失。</li>
</ol>4.超高速离心
<ol><li>用 75% （v/v） 酒精擦拭转子和铲斗以消除任何残留污染，以清洁转子和铲斗。</li>
	<li>将 38.5 mL 超速离心管置于管架中。 注意： 根据样品体积选择合适的超速离心管。避免使用紫外线辐射和不合适的化学试剂对离心管进行灭菌，如产品手册所示。</li>
	<li>将步骤3.7中过滤的粪便上清液约30mL转移到超速离心管中。</li>
	<li>用大约 8 mL 的 PBS 填充超速离心管，从管的开口处留出 3 mm 的间隙。</li>
	<li>将两个超速离心管与样品一起放置在相对的超速离心桶中，例如，桶1对应于4（1-4），2-5,3-6。</li>
	<li>用PBS调整两个桶的重量，使总重量在0.005克±以内。</li>
	<li>将所有铲斗安装到转子上，无论管子是否加载。</li>
	<li>启动真空并将样品在4°C下以160,000× g 离心70分钟。</li>
	<li>释放腔室真空，并在仪器主页上显示就绪后打开门。</li>
	<li>从超速离心机中取出转子。</li>
	<li>将铲斗从转子移到机架上，然后使用钳子取回管子。</li>
	<li>丢弃上清液，其底部将含有可见的棕色颗粒。</li>
	<li>使用含有1mL预冷（4°C）PBS的1,000μL移液管反复上下移液，直至完全悬浮，以重悬沉淀。用大约 37 mL 的 PBS 填充试管，从开口处留出 3 mm 的间隙。</li>
	<li>按照步骤4.5-4.11在4°C下以160,000× g 进行70分钟的另一次超速离心，以从管壁上除去部分附着的污染组分。</li>
	<li>弃去上清液，将两个超速离心管倒置5分钟，并用刮水器清除内壁上的任何残留溶液。 注意： 清洁内壁可最大限度地减少粘附在超速离心管壁上的残留污染物的干扰。选择不会影响BEV分析的刮水器，特别是在粒径方面。</li>
	<li>使用1,000μL移液管用1.2mL预冷（4°C）PBS上下移液，将沉淀重悬于每个试管中。</li>
	<li>将 1.2 mL 的 PBS/BEV 溶液从一个超速离心管转移到干净的 1.5 mL 微管中。 暂停点：从一个超速离心管收集的 PBS/BEV 溶液可以进行步骤 6。将另一管中的溶液储存在-80°C。</li>
</ol>5. 密度梯度离心的溶液制备
<ol><li>0.02 M HEPES缓冲液的制备<ol><li>将 0.477 g HEPES 粉末和 0.8 g NaCl 与 90 mL 高压灭菌去离子水混合。</li>
		<li>通过加入 1 M 氢氧化钠 （NaOH） 将 pH 值调节至 7.2。用去离子水使体积达到 100 mL，并通过 0.22 μm PES 膜过滤溶液。 注意：氢氧化钠是一种强苛性碱。操作人员必须在通风柜中小心处理和准备它。</li>
	</ol></li>
	<li>密度梯度缓冲液的制备<ol><li>根据密度梯度离心的超速离心管数量计算每个密度梯度缓冲液所需的体积（步骤6）（在本方案中，60%、50%、40%、20%和10%梯度溶液的体积分别为2.5mL、3mL、6mL、6mL和6mL）。</li>
		<li>为了制备50%（w / v）碘克沙醇工作溶液，将0.02M HEPES缓冲液和60%（w / v）碘克沙醇储备溶液以1：5（0.5mL：2.5mL）的体积比混合。 注意：使用一次性 20 mL 注射器抽出碘克沙醇储备溶液并避免引入空气。</li>
		<li>为了制备不同浓度的碘克沙醇缓冲液，根据表1所示的比例，使用1,000μL移液管将步骤5.2.2中的50%碘克沙醇工作溶液与步骤 5.1中获得的HEPES缓冲液混合。 注意： 在超洁净工作台上关灯执行步骤 5。将打开的碘克沙醇储备溶液储存在4°C的冰箱中，以防止细菌生长。保持碘克沙醇溶液和HEPES缓冲液避光。</li>
	</ol></li>
</ol>6. 密度梯度离心系统的建立
<ol><li>自上而下的 DGC 模式<ol><li>将从步骤 4 中分离的 500 μL PBS/BEV 溶液与 3 mL PBS 混合。使用 1,000 μL 移液管轻轻混合溶液，以获得 3.5 mL PBS/BEV 溶液。</li>
		<li>将 31 mL 超速离心管放在带孔的泡沫板上，并将其标记为“↓”。</li>
		<li>使用 1,000 μL 移液管将 3 mL 的 50% 碘克沙醇溶液垂直添加到管底部。</li>
		<li>将管子倾斜 70° 角，并将管架或其他支架放置在泡沫板水平略高于泡沫板水平、管子开口下方的位置。</li>
		<li>使用 1,000 μL 移液管在 50% 碘克沙醇溶液上加入 3 mL 40% 碘克沙醇溶液。</li>
		<li>使用1,000 μL移液管在40%碘克沙醇溶液上加入3 mL 20%碘克沙醇溶液。</li>
		<li>使用 1,000 μL 移液管在 20% 碘克沙醇溶液之上加入 3 mL 10% 碘克沙醇溶液。</li>
		<li>使用1,000 μL移液管在10%碘克沙醇溶液的顶部加入步骤6.1.1中的3.5 mL PBS/BEV溶液。</li>
		<li>轻轻地将管子放回直立位置。 注意：移液后，分层应可见。</li>
	</ol></li>
	<li>自下而上的DGC模式<ol><li>将 31 mL 超速离心管放在带孔的泡沫板上，并将其标记为“↑”。</li>
		<li>将 2.5 mL 的 60% 碘克沙醇储备溶液垂直加入管底部。使用 1,000 μL 移液管将其与从步骤 4 中分离的 500 μL PBS/BEV 溶液轻轻混合，以获得 3 mL 50% 碘克沙醇/BEV 溶液。</li>
		<li>将管子倾斜 70° 角，并将管架或其他支架放置在泡沫板水平略高于泡沫板水平、管子开口下方的位置。</li>
		<li>使用 1,000 μL 移液管在 50% 碘克沙醇/BEV 溶液上加入 3 mL 40% 碘克沙醇溶液。</li>
		<li>使用1000 μL移液管在40%碘克沙醇溶液上加入3 mL的20%碘克沙醇溶液。</li>
		<li>使用1000 μL移液管在20%碘克沙醇溶液上加入3 mL的10%碘克沙醇溶液。</li>
		<li>使用 1,000 μL 移液管在 10% 碘克沙醇溶液上加入 3.5 mL PBS。</li>
		<li>轻轻地将管子放回直立位置。</li>
		<li>此时，在步骤4.17中获得的悬浮液中剩余200 μL，随后可以作为名为“UC”的组进行分析。 注意：在步骤 6 中转移溶液时，始终将移液器尖端靠在垂直于管轴线的超速离心管壁上。</li>
	</ol></li>
</ol>7.密度梯度离心和馏分收集
<ol><li>用PBS调整两个试管的重量，使总重量在0.005g±以内。</li>
	<li>根据步骤4.5将试管放入桶中，并在4°C下以160,000× g 离心7小时。</li>
	<li>使用移液器（1000μL）从上到下依次靠在侧壁上，按以下顺序收集馏分：3 mL，2 mL，1 mL，1 mL，1 mL，1 mL，1 mL，1 mL，1.5 mL和3 mL进入38.5 mL超速离心管中。 注意： 将视线保持在与液体表面相同的水平。</li>
	<li>根据步骤4，对步骤7.3中获得的每种馏分在4°C下以160,000× g 进行超速离心70分钟。</li>
	<li>除去上清液，将超速离心管倒置5分钟。</li>
	<li>使用200μL移液管将沉淀重悬于200μL预冷（4°C）PBS中。</li>
	<li>将 PBS/BEV 溶液转移到干净的 1.5 mL 微管中。</li>
	<li>标记从 F1 到 F10 的分数，并根据步骤 8 对其进行分析。 暂停点：如果在步骤7.8中获得的样品不能立即处理，请将其储存在-80°C。</li>
</ol>8. 对收集的馏分进行表征和定量分析
<ol><li>使用带有空白对照的微孔板检测器确定每个部分的吸光度值（OD 340nm）以计算其相应的密度。 注意：用PBS替换PBS / BEV溶液（步骤6.1.1和步骤6.2.2）以建立空白对照。<ol><li>制备0%、5%、10%、12.5%和20%碘克沙醇溶液各100μL，以50%储备溶液（步骤5.2.2）为基础，分别对应于1.0058g/mL、1.0318g/mL、1.058g/mL、1.0708g/mL和1.111g/mL的密度。</li>
		<li>加入 50 μL 空白对照（在步骤 7.3 中制备）和50 μL 每种标准溶液（在步骤 8.1.1 中制备）以复制 96 孔板的孔中。</li>
		<li>将波长设置为 340 nm，测量端点光密度，并计算每个部分的密度。</li>
		<li>根据 BEV 的密度将 F4-F9 确定为 BEV 分数的范围。</li>
	</ol></li>
	<li>使用二辛可宁酸测定法（BCA）测定BEV组分的蛋白质浓度。<ol><li>在制造商提供的PBS中将该物质稀释十倍，以制备0.5μg/ μL标准蛋白溶液。</li>
		<li>根据试剂说明加入不同体积的标准蛋白溶液（在步骤8.2.1中制备），并用PBS补充96孔板的每个孔至总体积为20μL。</li>
		<li>将步骤 7.8 中制备的样品和步骤 6.2.9 中定义的 UC 后剩余的样品加入每个孔中。</li>
		<li>以 50：1 的比例混合试剂 A 和 B，并向每个孔转移 200 μL。</li>
		<li>将板在37°C水浴中孵育30分钟。</li>
		<li>使用酶标仪测量吸光度值，计算蛋白质浓度（μg/ μL），并根据从步骤8.2.2中稀释的标准溶液的值生成的标准曲线（x：光密度;y：蛋白质浓度）确定样品的蛋白质含量（μg）。</li>
	</ol></li>
	<li>使用透射电子显微镜（TEM）表征步骤8.1.4中定义的BEV部分，以验证BEV的存在。以下所有程序均应在冰上进行。<ol><li>将步骤7.8中分离出的10μL每个分离的F4-F9级分和步骤6.2.9中定义的UC后留下的样品施加到Formvar / Carbon支持的铜网格上20分钟，并用滤纸吸干。</li>
		<li>用 100 μL PBS 冲洗样品 3 次，每次 1 分钟，并用滤纸吸干。</li>
		<li>用 100 μL 1% （w/v） 戊二醛固定样品 5 分钟，并用滤纸吸干。</li>
		<li>用 100 μL PBS 洗涤网格 10 次，每次 2 分钟，并用滤纸吸干。</li>
		<li>用 50 μL 1.5% （w/v） 醋酸铀酰染色网格 10 分钟，并用滤纸吸干。 注意：乙酸铀酰具有放射性，与皮肤接触或吸入时毒性极强。在通风柜中处理含有醋酸铀酰的溶液，并遵循适当的安全措施。</li>
		<li>将网格转移到1%（w / v）甲基纤维素液滴上5分钟，并用滤纸吸干。</li>
		<li>将风干的网格存放在黑暗、无尘的环境中，直到观察。</li>
		<li>捕获图像并使用 TEM 进行分析。</li>
	</ol></li>
	<li>使用纳米颗粒跟踪分析（NTA）来表征步骤8.1.4中定义的BEV组分，以计算颗粒的数量。<ol><li>使用 110 nm 聚苯乙烯颗粒校准系统。</li>
		<li>使用PBS清洗样品池。</li>
		<li>将步骤7.8中获得的不同馏分的样品和步骤6.2.9中定义的UC后剩余的样品稀释至105-10 9 颗粒/mL的浓度范围内，优化浓度为107 颗粒/mL。</li>
		<li>在11个位置进行记录和分析，进行三次重复，将温度保持在23-30°C左右。</li>
	</ol></li>
	<li>通过考马斯亮蓝染色 （CBBS） 和蛋白质印迹 （WB） 分析样品的蛋白质含量。<ol><li>如果需要定量，使用PBS和5×上样缓冲液将步骤7.8中获得的不同馏分中的BEVs样品和步骤6.2.9中定义的UC后剩余的样品稀释至终浓度为0.5或1μg/ μL，确保步骤8.5.2中每个孔的20μL（10μg或20μg）的足够样品上样。将样品在95°C煮沸10分钟。</li>
		<li>将预制的聚丙烯酰胺凝胶组装到电泳槽中，并将样品转移到孔中。</li>
		<li>使用以下参数进行垂直电泳：160 V，50分钟。</li>
		<li>将凝胶在考马斯亮蓝色溶液中染色1小时，用去离子水冲洗直至蓝色背景变浅，并用相机捕捉图像。</li>
		<li>使用以下参数对其他凝胶进行蛋白质印迹程序：400 mA，20 分钟。</li>
		<li>在室温下用5%（w / v）BSA溶液封闭印迹膜1小时。</li>
		<li>在TBST缓冲液中洗涤膜三次，每次5分钟。</li>
		<li>用一抗（BEV标记物：LPS，OmpA，LTA;EEV 标志物：CD63、CD9、TSG-101、Syntenin、整合素 β1;其他污染标志物：Flagellin，Calnexin）在4°C下8-12小时。</li>
		<li>在TBST缓冲液中洗涤膜三次，每次10分钟。</li>
		<li>在室温下将膜与二抗孵育1小时。</li>
		<li>执行步骤 8.5.9。</li>
		<li>使用化学发光装置进行印迹。</li>
		<li>用来自大肠杆菌的BEV代替PBS / BEV溶液（步骤6.1.1和步骤6.2.2）以建立阳性对照（有关分离粗大肠杆菌-BEV的程序，请参见材料表），并进行上述所有实验以表征BEV。 暂停点：根据上述粪便 BEV 表征结果，确定 F6-F8 作为富含 BEV 的组分的分布，并利用此缩小范围进行后续分析。</li>
	</ol></li>
	<li>根据颗粒和蛋白质计算回收率，以评估两种DGC模式的效率。以下结果以百分比表示。<ol><li>计算自上而下模式下粒子的回收率：步骤6.2.9中定义的UC后F6-F8/粒子的总粒子。</li>
		<li>计算自下而上模式下颗粒的回收率：步骤6.2.9中定义的UC后F6-F8/颗粒的总颗粒。</li>
		<li>在自上而下模式下计算蛋白质的回收率：F6-F8 （μg） 中的总蛋白质含量/步骤 6.2.9 中定义的 UC 后的蛋白质含量 （μg）。</li>
		<li>计算自下而上模式下蛋白质的回收率：步骤6.2.9（μg）中定义的UC后F6-F8（μg）中的总蛋白质含量（μg）中的蛋白质含量。</li>
	</ol></li>
	<li>计算颗粒/蛋白质比率以评估传统上定义的 UC 和两种 DGC 模式的纯度，以下结果均以百分比表示。<ol><li>步骤6.2.9中定义的UC后的颗粒/蛋白质比率：UC后的颗粒/UC后的蛋白质含量（μg）。</li>
		<li>自上而下模式下的颗粒/蛋白质比率：F6-F8 中的总颗粒/F6-F8 中的蛋白质含量 （μg）。</li>
		<li>自下而上模式下的颗粒/蛋白质比率：F6-F8 中的总颗粒/F6-F8 中的蛋白质含量 （μg）。</li>
	</ol></li>
	<li>选择最合适的离心模式（在方案中选择了自上而下模式）进行粪便BEV纯化和未来分析。</li>
</ol>

用于微生物群多组学的增强细菌细胞外囊泡分离

<ol>
	<li>Costello, E. K., 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+community+variation+in+human+body+habitats+across+space+and+time.">Bacterial community variation in human body habitats across space and time.</a> Science. 326 (5960), 1694-1697 (2009).</li><li>Greenhalgh, K., Meyer, K. M., Aagaard, K. M., Wilmes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+gut+microbiome+in+health:+establishment+and+resilience+of+microbiota+over+a+lifetime.">The human gut microbiome in health: establishment and resilience of microbiota over a lifetime.</a> Environmental Microbiology. 18 (7), 2103-2116 (2016).</li><li>de Vos, W. M., Tilg, H., Van Hul, M., Cani, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microbiome+and+health:+mechanistic+insights.">Gut microbiome and health: mechanistic insights.</a> Gut. 71 (5), 1020-1032 (2022).</li><li>Zhou, P., Yang, D., Sun, D., Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microbiome:+New+biomarkers+in+early+screening+of+colorectal+cancer.">Gut microbiome: New biomarkers in early screening of colorectal cancer.</a> Journal of Clinical Laboratory Analysis. 36 (5), 24359 (2022).</li><li>Paik, D., 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=Human+gut+bacteria+produce+&#932;(&#919;)17-modulating+bile+acid+metabolites.">Human gut bacteria produce &#932;(&#919;)17-modulating bile acid metabolites.</a> Nature. 603 (7903), 907-912 (2022).</li><li>Parada Venegas, D., 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=Short+chain+fatty+acids+(SCFAs)-mediated+gut+epithelial+and+immune+regulation+and+its+relevance+for+inflammatory+bowel+diseases.">Short chain fatty acids (SCFAs)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases.</a> Frontiers in Immunology. 10, 277 (2019).</li><li>Jiang, C., Li, G., Huang, P., Liu, Z., Zhao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Gut+microbiota+and+Alzheimer's+disease.">The Gut microbiota and Alzheimer's disease.</a> Journal of Alzheimer's Disease. 58 (1), 1-15 (2017).</li><li>Morais, L. H., Schreiber, H. L. t., Mazmanian, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut+microbiota-brain+axis+in+behaviour+and+brain+disorders.">The gut microbiota-brain axis in behaviour and brain disorders.</a> Nature Reviews Microbiology. 19 (4), 241-255 (2021).</li><li>Kim, J. H., Lee, J., Park, J., Gho, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gram-negative+and+Gram-positive+bacterial+extracellular+vesicles.">Gram-negative and Gram-positive bacterial extracellular vesicles.</a> Seminars in Cell and Developmental Biology. 40, 97-104 (2015).</li><li>Toyofuku, M., Nomura, N., Eberl, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+and+origins+of+bacterial+membrane+vesicles.">Types and origins of bacterial membrane vesicles.</a> Nature Reviews Microbiology. 17 (1), 13-24 (2019).</li><li>Xie, J., Li, Q., Haesebrouck, F., Van Hoecke, L., Vandenbroucke, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tremendous+biomedical+potential+of+bacterial+extracellular+vesicles.">The tremendous biomedical potential of bacterial extracellular vesicles.</a> Trends in Biotechnology. 40 (10), 1173-1194 (2022).</li><li>Coumans, F. A. W., 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=Methodological+guidelines+to+study+extracellular+vesicles.">Methodological guidelines to study extracellular vesicles.</a> Circulation Research. 120 (10), 1632-1648 (2017).</li><li>Northrop-Albrecht, E. J., Taylor, W. R., Huang, B. Q., Kisiel, J. B., Lucien, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+extracellular+vesicle+isolation+methods+from+human+stool+supernatant.">Assessment of extracellular vesicle isolation methods from human stool supernatant.</a> Journal of Extracellular Vesicles. 11 (4), 12208 (2022).</li><li>Park, Y. 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=Microbial+changes+in+stool,+saliva,+serum,+and+urine+before+and+after+anti-TNF-&#945;+therapy+in+patients+with+inflammatory+bowel+diseases.">Microbial changes in stool, saliva, serum, and urine before and after anti-TNF-&#945; therapy in patients with inflammatory bowel diseases.</a> Scientific Reports. 12 (1), 6359 (2022).</li><li>Tulkens, J., De Wever, O., Hendrix, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+bacterial+extracellular+vesicles+in+human+body+fluids+by+orthogonal+biophysical+separation+and+biochemical+characterization.">Analyzing bacterial extracellular vesicles in human body fluids by orthogonal biophysical separation and biochemical characterization.</a> Nature Protocols. 15 (1), 40-67 (2020).</li><li>Tulkens, 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=Increased+levels+of+systemic+LPS-positive+bacterial+extracellular+vesicles+in+patients+with+intestinal+barrier+dysfunction.">Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction.</a> Gut. 69 (1), 191-193 (2020).</li><li>Kang, C. 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=Extracellular+vesicles+derived+from+gut+microbiota,+especially+Akkermansia+muciniphila,+protect+the+progression+of+dextran+sulfate+sodium-induced+colitis.">Extracellular vesicles derived from gut microbiota, especially Akkermansia muciniphila, protect the progression of dextran sulfate sodium-induced colitis.</a> PloS one. 8 (10), 76520 (2013).</li><li>Simonsen, 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=What+are+we+looking+at?+Extracellular+vesicles,+lipoproteins,+or+both.">What are we looking at? Extracellular vesicles, lipoproteins, or both.</a> Circulation Research. 121 (8), 920-922 (2017).</li><li>Correll, V. L., 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=Optimization+of+small+extracellular+vesicle+isolation+from+expressed+prostatic+secretions+in+urine+for+in-depth+proteomic+analysis.">Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis.</a> Journal of Extracellular Vesicles. 11 (2), 12184 (2022).</li><li>Liang, X., 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=Gut+bacterial+extracellular+vesicles:+important+players+in+regulating+intestinal+microenvironment.">Gut bacterial extracellular vesicles: important players in regulating intestinal microenvironment.</a> Gut Microbes. 14 (1), 2134689 (2022).</li><li>Alberti, G., 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=Extracellular+vesicles+derived+from+gut+microbiota+in+inflammatory+bowel+disease+and+colorectal+cancer.">Extracellular vesicles derived from gut microbiota in inflammatory bowel disease and colorectal cancer.</a> Biomedical papers of the Medical Faculty of the University Palacky, Olomouc, Czech Republic. 165 (3), 233-240 (2021).</li><li>D&#237;ez-Sainz, E., Milagro, F. I., Riezu-Boj, J. I., Lorente-Cebri&#225;n, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+gut+microbiota-derived+extracellular+vesicles+on+obesity+and+diabetes+and+their+potential+modulation+through+diet.">Effects of gut microbiota-derived extracellular vesicles on obesity and diabetes and their potential modulation through diet.</a> Journal of Physiology and Biochemistry. 78 (2), 485-499 (2022).</li><li>Lajqi, T., 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=Gut+microbiota-derived+small+extracellular+vesicles+endorse+memory-like+inflammatory+responses+in+murine+neutrophils.">Gut microbiota-derived small extracellular vesicles endorse memory-like inflammatory responses in murine neutrophils.</a> Biomedicines. 10 (2), 442 (2022).</li><li>Lee, K. 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=The+extracellular+vesicle+of+gut+microbial+Paenalcaligenes+hominis+is+a+risk+factor+for+vagus+nerve-mediated+cognitive+impairment.">The extracellular vesicle of gut microbial Paenalcaligenes hominis is a risk factor for vagus nerve-mediated cognitive impairment.</a> Microbiome. 8 (1), 107 (2020).</li><li>Villard, A., Boursier, J., Andriantsitohaina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+and+eukaryotic+extracellular+vesicles+and+nonalcoholic+fatty+liver+disease:+new+players+in+the+gut-liver+axis.">Bacterial and eukaryotic extracellular vesicles and nonalcoholic fatty liver disease: new players in the gut-liver axis.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 320 (4), G485-G495 (2021).</li><li>Wei, 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=Outer+membrane+vesicles+enhance+tau+phosphorylation+and+contribute+to+cognitive+impairment.">Outer membrane vesicles enhance tau phosphorylation and contribute to cognitive impairment.</a> Journal of Cellular Physiology. 235 (5), 4843-4855 (2020).</li><li>Bitto, N. J., Kaparakis-Liaskos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+bacterial+membrane+vesicle+production,+purification,+quantification,+and+examination+of+their+immunogenic+functions.">Methods of bacterial membrane vesicle production, purification, quantification, and examination of their immunogenic functions.</a> Methods in Molecular Biology. 2523, 43-61 (2022).</li><li>Stentz, R., Miquel-Clop&#233;s, A., Carding, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+isolation,+and+characterization+of+bioengineered+bacterial+extracellular+membrane+vesicles+derived+from+Bacteroides+thetaiotaomicron+and+their+use+in+vaccine+development.">Production, isolation, and characterization of bioengineered bacterial extracellular membrane vesicles derived from Bacteroides thetaiotaomicron and their use in vaccine development.</a> Methods in Molecular Biology. 2414, 171-190 (2022).</li><li>Zhang, Q., Jeppesen, D. K., Higginbotham, J. N., Franklin, J. L., Coffey, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+isolation+of+extracellular+vesicles+and+nanoparticles.">Comprehensive isolation of extracellular vesicles and nanoparticles.</a> Nature Protocols. 18 (5), 1462-1487 (2023).</li><li>Iwai, K., Minamisawa, T., Suga, K., Yajima, Y., Shiba, K. <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+human+salivary+extracellular+vesicles+by+iodixanol+density+gradient+ultracentrifugation+and+their+characterizations.">Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations.</a> Journal of Extracellular Vesicles. 5, 30829 (2016).</li><li>Vandeputte, D., 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=Stool+consistency+is+strongly+associated+with+gut+microbiota+richness+and+composition,+enterotypes+and+bacterial+growth+rates.">Stool consistency is strongly associated with gut microbiota richness and composition, enterotypes and bacterial growth rates.</a> Gut. 65 (1), 57-62 (2016).</li><li>Wen, 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=Bacterial+extracellular+vesicles:+A+position+paper+by+the+Microbial+Vesicles+Task+Force+of+the+Chinese+Society+of+Extracellular+Vesicles.">Bacterial extracellular vesicles: A position paper by the Microbial Vesicles Task Force of the Chinese Society of Extracellular Vesicles.</a> Interdisciplinary Medicine. 1, 12046 (2023).</li></ol>

作者声明没有利益冲突。

细菌细胞外囊泡 （BEV） 是细菌分泌的脂质双层纳米颗粒，携带丰富的蛋白质、脂质、核酸和其他生物活性分子，有助于介导细菌的功能效应20。源自肠道的BEV已被证实与炎症性肠病、克罗恩病和结直肠癌等疾病的发展有关，还会影响一般代谢并介导认知功能受损4,16,17,20,21,22,23,24,25,26 </su...

肠道被广泛认为是人体内微生物群落最丰富的器官，超过 90% 的细菌参与定植和繁殖 1,2。大量证据表明，肠道微生物群调节肠道微环境，同时与远处器官的功能障碍相互作用，主要是通过受损的肠道屏障 3,4。越来越多的证据表明，肠道菌群的失衡与炎症性肠病 （IBD） 的进展之间存在相关性5,6，以及通过肠脑轴5,6,7,8 的认知障碍。细菌产生的细菌细胞外囊泡 （BEV） 在这些病理过程中起着重要作用。
BEV 是封装细菌衍生物的纳米级颗粒，直径范围为 20 至 400 nm。它们已被证明可以促进细菌与其宿主生物之间的相互作用 9,10。尽管它们不可见，但由于它们作为诊断生物标志物、治疗靶标和药物递送载体的广泛应用，这些颗粒越来越受到研究人员的关注11。人类粪便通常用作研究BEV的生物样本，主要来自肠道细菌，含有水、细菌、脂质、蛋白质、未消化的食物残渣和脱落的上皮细胞等的复杂混合物。复杂的粪便组成对BEV的分离和纯度提出了挑战，从而阻碍了对BEV的全面、客观和现实的分析。因此，尽量减少污染成分的干扰和提高纯电动汽车产量的有效策略已成为需要立即关注的关键问题。
现有的分离策略主要依赖于超高速离心 （UC）、密度梯度离心 （DGC） 和体积排阻色谱 （SEC）12、13、14、15、16、17 等技术。目前，DGC是BEV分离领域应用最广泛的方法之一，包括“自上而下”和“自下而上”两种沉降-浮动模式，由样品的初始加载位置决定。这些方法根据大小和密度差异将细胞外囊泡 （EV） 与其他组分区分开来，从而产生可变的纯度和回收率。先前的研究表明，单一方法策略不足以将 EV 与体液样本中的可溶性蛋白质充分分离，例如血液中的脂蛋白18 和尿液中的 Tamm-Horsfall 蛋白19。此外，真核细胞外囊泡 （EEV） 的大小分布通常与 BEV 的尺寸分布重叠，因此需要进一步改进方法学以优化 BEV 产量。因此，推进BEV的研究取决于开发有效的分离和纯化方法。值得注意的是，Tulkens等人[15]采用正交生物物理策略将粪便BEV与EEV分开，其中自下而上DGC模式的离心时间长达18 h。相比之下，本研究将其缩短至7 h，大大节省了梯度超速离心时间并简化了过程。
在本研究中，我们在优化的缓冲条件下，在以从低到极高的速度从低到极高的一系列差速离心速度富集BEV后，分离和纯化了采用两种DGC模式的粪便BEV。基于形态、粒径和浓度的评估表明，这种增强方法的性能值得称赞。这项研究可以作为未来研究的基础，将其应用扩展到更广泛的领域，并为人体内BEV的异质性提供见解。它还有助于BEV分离和分析技术的标准化。

<table><tbody><tr><td>1 % (w/v) glutaraldehyde (prepared from 2.5 % stock solution in deionized water)</td><td>ACMEC</td><td>AP1126</td><td>Morphological observation for BEVs using TEM at Step 8.3.3</td></tr><tr><td>1 % (w/v) methylcellulose (prepared from original powder in deionized water)</td><td>Sigma-Aldrich</td><td>M7027</td><td>Morphological observation for BEVs using TEM at Step 8.3.6</td></tr><tr><td>1.5 % (w/v) uranyl acetate (prepared from original powder in deionized water)</td><td>Polysciences</td><td>21447-25</td><td>Morphological observation for BEVs using TEM at Step 8.3.5</td></tr><tr><td>1000 &amp;mu;L, 200 &amp;mu;L, 10 &amp;mu;L Pipette</td><td>KIRGEN</td><td>KG1313, KG1212, KG1011</td><td>Transfer the solution</td></tr><tr><td>5 % (w/v) bovine serum albumin solution (prepared from the original powder in TBST buffer)</td><td>Fdbio science</td><td>FD0030</td><td>Used in western blotting for blocking at Step 8.5.6</td></tr><tr><td>5 &amp;times; loading buffer</td><td>Fdbio science</td><td>FD006</td><td>Used in western blotting and Coomassie brilliant blue stain at Step 8.5.1</td></tr><tr><td>75 % (v/v) alcohol</td><td>LIRCON</td><td>LIRCON-500 mL</td><td>Surface disinfection</td></tr><tr><td>96-well plate</td><td>Rar</td><td>A8096</td><td>Measure the absorbance values&amp;nbsp;</td></tr><tr><td>Anti-Calnexin antibody</td><td>Abcam</td><td>ab92573</td><td>Western blotting (Primary Antibody)</td></tr><tr><td>Anti-CD63 antibody</td><td>Abcam</td><td>ab134045</td><td>Western blotting (Primary Antibody)</td></tr><tr><td>Anti-CD9 antibody</td><td>Abcam</td><td>ab236630</td><td>Western blotting (Primary Antibody)</td></tr><tr><td>Anti-Flagellin antibody</td><td>Sino Biological</td><td>40067-MM06</td><td>Western blotting (Primary Antibody)</td></tr><tr><td>Anti-Integrin beta 1 antibody</td><td>Abcam</td><td>ab30394</td><td>Western blotting (Primary Antibody)</td></tr><tr><td>Anti-LPS antibody</td><td>Thermo Fisher</td><td>MA1-83152</td><td>Western blotting (Primary Antibody)</td></tr><tr><td>Anti-LTA antibody</td><td>Thermo Fisher&amp;nbsp;</td><td>MA1-7402</td><td>Western blotting (Primary Antibody)</td></tr><tr><td>Anti-OmpA antibody</td><td>CUSABIO</td><td>CSB-PA359226ZA01EOD, https://www.cusabio.com/</td><td>Western blotting (Primary Antibody)</td></tr><tr><td>Anti-Syntenin antibody</td><td>Abcam</td><td>ab133267</td><td>Western blotting (Primary Antibody)</td></tr><tr><td>Anti-TSG101 antibody</td><td>Abcam</td><td>ab125011</td><td>Western blotting (Primary Antibody)</td></tr><tr><td>Autoclave</td><td>ZEALWAY</td><td>GR110DP</td><td>Sterilization for supplies and mediums used in the experiment</td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td>AL104</td><td>Balance the tube sample-loaded with PBS</td></tr><tr><td>Bicinchoninic acid assay&amp;nbsp;</td><td>Fdbio science</td><td>FD2001</td><td>Measure protein content of BEVs at Step 8.2</td></tr><tr><td>BioRender</td><td>BioRender</td><td>https://app.biorender.com</td><td>Make the schematic workflow of BEVs isolation and purification showed in Figure 1</td></tr><tr><td>Biosafety cabinet</td><td>Haier</td><td>HR1200- II B2</td><td>Peform the procedures about feces sample handling</td></tr><tr><td>Centrifuge 5810 R; Rotor F-34-6-38</td><td>Eppendorf</td><td>5805000092; 5804727002, adapter: 5804774000</td><td>Preprocess for BEVs (Step 3)</td></tr><tr><td>Chemiluminescence Apparatus</td><td>BIO-OI</td><td>OI600SE-MF</td><td>Used in western blotting for signal detection at Step 8.5.12</td></tr><tr><td>Cytation 5</td><td>BioTek</td><td>F01</td><td>Microplate detector for measuring the absorbance (Step 8.1) and fluorescence (Figure 6) values&amp;nbsp;</td></tr><tr><td>Dil-labled low density lipoprotein</td><td>ACMEC</td><td>AC12038</td><td>Definition of distribution of interfering components&amp;nbsp;</td></tr><tr><td>Electrophoresis equipment</td><td>Bio-rad</td><td>1658033</td><td>Used in western blotting for protein separation and transfer at Step 8.5.2, 8.5.3, 8.5.5</td></tr><tr><td>Enhanced Chemiluminescence kit HRP&amp;nbsp;</td><td>Fdbio science</td><td>FD8020</td><td>Used in western blotting for signal detection at Step 8.5.12</td></tr><tr><td>&lt;em&gt;Escherichia coli&amp;nbsp;&lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC8739</td><td>Isolate BEVs as a positive control. Protocol: Dissolve 25 g of the LB powder in 1 L deionized water, and autoclave. Transfer the 800 &amp;mu;L of preserved Escherichia coli into the medium. Cultivate at 37 &amp;deg;C in the incubator shaker. Then centrifuge at 3, 000 &amp;times; g for 20 min at 4 &amp;deg;C, 12, 000 &amp;times; g for 30 min at 4 &amp;deg;C, filter the supernatant through 0.22 &amp;mu;m membrane, and perform ultra-speed centrifugation at 160, 000 &amp;times; g for 70 min at 4 &amp;deg;C. Pellet defined as crude BEVs from Escherichia coli was suspended in 1.2 mL PBS (Step 3, 4).&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Falcon tubes 50 mL</td><td>KIRGEN</td><td>KG2811</td><td>Preprocess for BEVs (Step 3)</td></tr><tr><td>Feto Protein Staining Buffer</td><td>Absci</td><td>ab.001.50</td><td>Coomassie brilliant blue staining at Step 8.5.4</td></tr><tr><td>Filter paper</td><td>Biosharp</td><td>BS-TFP-070B</td><td>Morphological observation for BEVs using TEM at Step 8.3 (Blotting the solution)</td></tr><tr><td>Formvar/Carbon supported copper grids&amp;nbsp;</td><td>Sigma-Aldrich</td><td>TEM-FCF200CU50</td><td>Morphological observation for BEVs using TEM at Step 8.3</td></tr><tr><td>HEPES powder</td><td>Meilunbio</td><td>MB6078</td><td>Prepare iodixanol buffers with different concentrations for density gradient centrifugation</td></tr><tr><td>HRP AffiniPure Goat Anti-Mouse IgG (H+L)</td><td>Fdbio science</td><td>FDM007</td><td>Western blotting (Secondary Antibody)</td></tr><tr><td>HRP AffiniPure Goat Anti-Rabbit IgG (H+L)</td><td>Fdbio science</td><td>FDR007</td><td>Western blotting (Secondary Antibody)</td></tr><tr><td>Incubator shaker</td><td>Qiangwen</td><td>DHZ-L</td><td>Cultivate Escherichia coli&amp;nbsp;</td></tr><tr><td>Kimwipes&amp;trade; Delicate Task Wipes</td><td>Kimtech Science</td><td>34155</td><td>Wipe the inner wall of the ultracentrifuge tube at Step 4.15</td></tr><tr><td>LB broth</td><td>Hopebio</td><td>HB0128</td><td>Cultivate Escherichia coli&amp;nbsp;</td></tr><tr><td>Low temperature freezer (-80 &amp;deg;C)</td><td>Haier</td><td>DW-86L338J</td><td>Store the samples</td></tr><tr><td>Methanol</td><td>Alalddin</td><td>M116118</td><td>Used in western blotting for activating PVDF membrane at Step 8.5.5</td></tr><tr><td>Micro tubes 1.5 mL</td><td>KIRGEN</td><td>KG2211</td><td>Recover fractions after density gradient centrifugation</td></tr><tr><td>Micro tubes 2 mL</td><td>KIRGEN</td><td>KG2911</td><td>Recover fractions after density gradient centrifugation</td></tr><tr><td>Micro tubes 5 mL</td><td>BBI</td><td>F610888-0001</td><td>Recover fractions after density gradient centrifugation</td></tr><tr><td>Microplate reader&amp;nbsp;</td><td>Thermo Fisher&amp;nbsp;</td><td>Multiskan MK3</td><td>Measure protein content of BEVs at Step 8.2</td></tr><tr><td>Millipore filter 0.22 &amp;mu;m</td><td>Merck millipore</td><td>SLGP033RB</td><td>Filtration sterilization; Material: polyethersulfone, PES</td></tr><tr><td>NaCl</td><td>GHTECH</td><td>1.01307.040</td><td>Density gradient centrifugation solution</td></tr><tr><td>NaOH</td><td>GHTECH</td><td>1.01394.068</td><td>Density gradient centrifugation solution (pH adjustment)</td></tr><tr><td>Optima&amp;trade; XPN-100</td><td>Beckman Coulter</td><td>A94469</td><td>Ultracentrifugation for BEVs isolation at Step 4, 7</td></tr><tr><td>OptiPrep&amp;trade;</td><td>Serumwerk Bernburg AG</td><td>1893</td><td>Density gradient centrifugation stock solution</td></tr><tr><td>Orbital Shaker</td><td>Youning</td><td>CS-100</td><td>Dissolve feces at Step 2</td></tr><tr><td>Phosphate buffered saline</td><td>Procell</td><td>PB180327</td><td>Dissolve feces at Step 2</td></tr><tr><td>Pipettor</td><td>Eppendorf</td><td>3120000267, 3120000259</td><td>Transfer the solution</td></tr><tr><td>Plastic pasteur pipette</td><td>ABCbio</td><td>ABC217003-4</td><td>Remove supernatant in preprocessing at Step 3.4</td></tr><tr><td>Polyvinylidene difluoride (PVDF) membranes</td><td>Millipore</td><td>ISEQ00010, IPVH00010</td><td>Used in western blotting for protein transfer at Step 8.5.5</td></tr><tr><td>Prefabricated polyacrylamide gel, 4&amp;ndash;20% 15 Wells</td><td>ACE</td><td>F15420Gel</td><td>Used in western blotting for protein separation at Step 8.5.2, 8.5.3</td></tr><tr><td>Primary antibody diluent</td><td>Fdbio science</td><td>FD0040</td><td>Used in western blotting at Step 8.5.8</td></tr><tr><td>Protein ladder</td><td>Fdbio science</td><td>FD0672</td><td>Used in western blotting and Coomassie brilliant blue stain at Step 8.5</td></tr><tr><td>Rapid protein blotting solution</td><td>UBIO</td><td>UW0500</td><td>Used in western blotting for protein transfer at Step 8.5.5</td></tr><tr><td>Rotor SW 32 Ti Swinging-Bucket Rotor</td><td>Beckman Coulter</td><td>369650</td><td>Ultracentrifugation for BEVs isolation at Step 4, 7</td></tr><tr><td>Syringe 20 mL, 50 mL&amp;nbsp;</td><td>Jetway</td><td>ZSQ-20ML, YCXWJZSQ-50 mL</td><td>Transfer buffers amd remove supernatant in preprocessing</td></tr><tr><td>TBS powder</td><td>Fdbio science</td><td>FD1021</td><td>Used in western blotting at Step 8.5</td></tr><tr><td>Transmission electron microscope (TEM)</td><td>Hitachi&amp;nbsp;</td><td>H-7650</td><td>Morphological observation for BEVs at Step 8.3</td></tr><tr><td>Tween-20</td><td>Fdbio science</td><td>FD0020</td><td>Used in western blotting at Step 8.5</td></tr><tr><td>Ultracentrifuge tube</td><td>Beckman</td><td>326823, 355642</td><td>Ultracentrifugation for BEVs isolation at Step 4, 7</td></tr><tr><td>Ultra-clean bench</td><td>AIRTECH</td><td>SW-CJ-2FD</td><td>Peform the procedures about liquid handling</td></tr><tr><td>Water bath</td><td>Bluepard</td><td>CU600</td><td>Used for measuring protein content of BEVs at Step 8.2.5</td></tr><tr><td>ZetaView</td><td>Particle Metrix</td><td>S/N 21-734, Software ZetaView (version 8.05.14 SP7)</td><td>Nanoparticle tracking analysis (NTA) for measuring the particle size and concentrarion of BEVs at Step 8.4</td></tr></tbody></table>

isolation and purification of bacterial extracellular vesicles from human feces using density gradient centrifugation

细菌细胞外囊泡 （BEV） 是来源于细菌的纳米囊泡，在细菌-细菌和细菌-宿主通讯中发挥积极作用，转移从亲本细菌遗传的蛋白质、脂质和核酸等生物活性分子。源自肠道微生物群的BEV在胃肠道内有影响，可以到达远处的器官，从而对生理学和病理学产生重大影响。探索源自人类粪便的BEV的类型、数量和作用的理论研究对于了解肠道微生物群中BEV的分泌和功能至关重要。这些研究也需要改进当前的BEV分离和纯化策略。
本研究通过建立自上而下和自下而上两种密度梯度离心（DGC）模式，优化了BEV的分离纯化过程。BEV的富集分布在分数6至8（F6-F8）中确定。根据颗粒形态、大小、浓度和蛋白质含量评估该方法的有效性。计算颗粒和蛋白质回收率，分析特异性标记物的存在，比较两种DGC模式的回收率和纯度。结果表明，自上而下离心模式具有较低的污染水平，并实现了与自下而上模式相似的回收率和纯度。7 h的离心时间足以达到108 / mg的粪便BEV浓度。
除粪便外，该方法可应用于其他体液类型，并根据成分和粘度的差异进行适当修改。综上所述，该详细可靠的方案将有助于BEV的标准化分离和纯化，从而为后续的多组学分析和功能实验奠定基础。

确定富含BEV的馏分的分布 为了确定富含细菌胞外囊泡（BEV）的组分的分布，建立了一个空白对照以测量OD 340nm处的吸光度值，并根据测量值和碘克沙醇指南计算每个组分的密度（步骤8.1）。 表 2 显示了密度结果，表明 F4 至 F9 级分的密度在通常与细胞外囊泡相关的范围内。这一发现表明，大多数BEV在这些部分中被分离出来，导致将F4-F9定义为BEV的粗略范围。采用透射电?...

Watch this Scientific Journal Video about Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity at JoVE.com

作者聚焦：加速细菌细胞外囊泡分离和异质性的研究 

本研究描述了一种 通过 密度梯度离心 （DGC） 分离和纯化从人类粪便中富集的细菌细胞外囊泡 （BEV） 的方法，从形态、粒径和浓度方面鉴定了 BEV 的物理特性，并讨论了 DGC 方法在临床和科学研究中的潜在应用。

使用密度梯度离心从人粪便中分离和纯化细菌胞外囊泡

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host ...

我们的研究使用密度梯度离心从物理学家那里分离、纯化和表征细菌细胞外囊泡。我们的目标是解决BEV与污染物的分离问题，并为研究人员提供可靠的方法。我们的方案将离心时间从 18 小时缩短到 7 小时。自上而下模式改善了 BEV 分离并减少了肝脏蛋白的污染。我们的方法简化了BEV的分离，为未来的研究设定了标准。通过适当的修改，它可以应用于其他体液，增强我们对BEV的理解，并促进对BEV的研究，揭示它们在人体内固有的异质性。

isolation-purification-bacterial-extracellular-vesicles-from-human

Research

JoVE Journal

Biology

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

2.3K 次观看.  Southern Medical University. 我们的研究使用密度梯度离心从物理学家那里分离、纯化和表征细菌细胞外囊泡。我们的目标是解决BEV与污染物的分离问题，并为研究人员提供可靠的方法。我们的方案将离心时间从 18 小时缩短到 7 小时。自上而下模式改善了 BEV 分离并减少了肝脏蛋白的污染。我们的方法简化了BEV的分离，为未来的研究设定了标准。通过适当的修改，它可以应用于其他体液，增强我们?...

视频: 作者聚焦：加速细菌细胞外囊泡分离和异质性的研究 

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

科研

生物学

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

从0.22微米Sterivex过滤器和氯化铯密度梯度离心法的DNA提取

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

免疫与感染

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

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

从 大肠杆菌 和其他细菌中可扩展地分离和纯化细胞外囊泡

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

蓝藻细胞外囊泡的分离和表征

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Extracellular Vesicle Extraction: A Technique to Isolate Extracellular Vesicles from Whole Tissue Specimens Using Differential Centrifugation

Source: Hurwitz, S. N. et al. <a href="https://www.jove.com/v/59143/extraction-of-extracellular-vesicles-from-whole-tissue">Extraction of Extracellular Vesicles from Whole Tissue.</a> J. Vis. Exp. (2019)
This video describes the technique of extracting extracellular vesicles from fresh or frozen tissues. The isolated extracellular vesicles can be used for further analysis to uncover their significant roles in disease pathogenesis.

细胞外囊泡提取:一种使用差速离心法从整个组织标本中分离细胞外囊泡的技术

Source: Hurwitz, S. N. et al. Extraction of Extracellular Vesicles from Whole Tissue. J. Vis. Exp. (2019)
This video describes the technique of extracting ...

JoVE Encyclopedia of Experiments

癌症研究

Cancers of the Nervous System

In vitro studies

Purification of Subpopulations of Extracellular Vesicles: A Technique to Purify Subpopulations of Extracellular Vesicles Using Density Gradient Ultracentrifugation

Source: Urwitz, S. N. et al. <a href="https://www.jove.com/v/59143/extraction-of-extracellular-vesicles-from-whole-tissue">Extraction of Extracellular Vesicles from Whole Tissue.</a> J. Vis. Exp. (2019)
This video demonstrates the density gradient ultracentrifugation-based purification method to isolate subpopulations of extracellular vesicles suitable for characterization studies.

细胞外囊泡亚群的纯化:一种使用密度梯度超速离心纯化细胞外囊泡亚群的技术

Source: Urwitz, S. N. et al. Extraction of Extracellular Vesicles from Whole Tissue. J. Vis. Exp. (2019)
This video demonstrates the density gradient ...

使用密度梯度离心从人粪便中分离和纯化细菌胞外囊泡

副本

摘要

探索更多视频

此视频中的章节

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

从0.22微米Sterivex过滤器和氯化铯密度梯度离心法的DNA提取

kinesin的分离和纯化果蝇胚胎

高产细胞外囊泡制剂的纯化远离病毒

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

从大肠杆菌和其他细菌中可扩展地分离和纯化细胞外囊泡

蓝藻细胞外囊泡的分离和表征

细胞外囊泡提取:一种使用差速离心法从整个组织标本中分离细胞外囊泡的技术

细胞外囊泡亚群的纯化:一种使用密度梯度超速离心纯化细胞外囊泡亚群的技术

使用密度梯度离心从人粪便中分离和纯化细菌胞外囊泡

副本

摘要

探索更多视频

此视频中的章节

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

从0.22微米Sterivex过滤器和氯化铯密度梯度离心法的DNA提取

kinesin的分离和纯化果蝇胚胎

高产细胞外囊泡制剂的纯化远离病毒

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

从 大肠杆菌 和其他细菌中可扩展地分离和纯化细胞外囊泡

蓝藻细胞外囊泡的分离和表征

细胞外囊泡提取:一种使用差速离心法从整个组织标本中分离细胞外囊泡的技术

细胞外囊泡亚群的纯化:一种使用密度梯度超速离心纯化细胞外囊泡亚群的技术

从大肠杆菌和其他细菌中可扩展地分离和纯化细胞外囊泡