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>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;L, 200 &#956;L, 10 &#956;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 &#215; 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&#160;</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&#160;</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&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Dil-labled low density lipoprotein</td>
			<td>ACMEC</td>
			<td>AC12038</td>
			<td>Definition of distribution of interfering components&#160;</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&#160;</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>Escherichia coli&#160;</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 &#956;L of preserved Escherichia coli into the medium. Cultivate at 37 &#176;C in the incubator shaker. Then centrifuge at 3, 000 &#215; g for 20 min at 4 &#176;C, 12, 000 &#215; g for 30 min at 4 &#176;C, filter the supernatant through 0.22 &#956;m membrane, and perform ultra-speed centrifugation at 160, 000 &#215; g for 70 min at 4 &#176;C. Pellet defined as crude BEVs from Escherichia coli was suspended in 1.2 mL PBS (Step 3, 4).&#160;&#160;&#160;&#160;</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&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Kimwipes&#8482; 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&#160;</td>
		</tr>
		<tr>
			<td>Low temperature freezer (-80 &#176;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&#160;</td>
			<td>Thermo Fisher&#160;</td>
			<td>Multiskan MK3</td>
			<td>Measure protein content of BEVs at Step 8.2</td>
		</tr>
		<tr>
			<td>Millipore filter 0.22 &#956;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&#8482; XPN-100</td>
			<td>Beckman Coulter</td>
			<td>A94469</td>
			<td>Ultracentrifugation for BEVs isolation at Step 4, 7</td>
		</tr>
		<tr>
			<td>OptiPrep&#8482;</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&#8211;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&#160;</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&#160;</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。

The gut is widely recognized as the organ harboring the most abundant microbial communities in the human body, with over 90% of bacteria involved in colonization and multiplication1,2. Extensive evidence has demonstrated that the gut microbiota modulates the gut microenvironment and simultaneously interacts with dysfunction in distant organs, primarily through an impaired intestinal barrier3,4. Mounting evidence indicates a correlation between the imbalance of gut microbiota and the progression of inflammatory bowel disease (IBD)5,6, as well as cognitive disorders through the gut-brain axis5,6,7,8. Bacterial extracellular vesicles (BEVs) produced by bacteria play significant roles in these pathological processes.
BEVs are nanoscale particles encapsulating bacterial derivatives, with diameters ranging from 20 to 400 nm. They have been demonstrated to facilitate interactions between bacteria and their host organisms9,10. Despite their invisibility, these particles have garnered increasing attention from researchers due to their prospective broad applications as diagnostic biomarkers, therapeutic targets, and drug delivery vehicles11. Human feces, often used as biospecimens for studying BEVs, predominantly sourced from gut bacteria, contain a complex mixture of water, bacteria, lipids, proteins, undigested food residue, and exfoliated epithelial cells among others. The intricate fecal composition poses challenges to the isolation and purity of BEVs, thereby impeding a comprehensive, objective, and realistic analysis of BEVs. Hence, effective strategies to minimize interference from contaminating components and enhance the yield of BEVs have emerged as critical issues warranting immediate attention.
Existing isolation strategies largely rely on techniques such as ultra-high-speed centrifugation (UC), density gradient centrifugation (DGC), and size exclusion chromatography (SEC)12,13,14,15,16,17. Currently, DGC is one of the most widely applied methods in the field of BEV separation, encompassing two sedimentation-floating modes, &#34;Top-down&#34; and &#34;Bottom-up&#34;, which are determined by the initial loading position of the sample. These methodologies differentiate extracellular vesicles (EVs) from other components based on size and density disparities, yielding variable purity and recovery rates. Prior research has indicated that single-approach strategies are insufficient for adequately separating EVs from soluble proteins in body fluid samples, such as lipoprotein in blood18 and Tamm-Horsfall protein in urine19. Additionally, the size distribution of eukaryotic extracellular vesicles (EEVs) often overlaps with that of BEVs, thereby necessitating further methodological enhancements to optimize BEV yield. Consequently, advancing the study of BEVs hinges on the development of effective separation and purification methodologies. Notably, Tulkens et al15 employed an orthogonal biophysical strategy to separate fecal BEVs from EEVs, in which the centrifugation time of a Bottom-up DGC mode was up to 18 h. In contrast, this study reduced it to 7 h, greatly saving the gradient-ultracentrifugation time and simplifying the process.
In the present study, we isolated and purified fecal BEVs employing two DGC modes under optimized buffer conditions, after enriching BEVs with a range of differential centrifugation speeds, from low to extremely high velocity. Evaluations based on morphology, particle size, and concentration indicated a commendable performance by this enhanced method. This study could serve as the foundation for future research, extending its applications to a broader domain, and offering insights into the heterogeneity of BEVs within the human body. It also contributes to the standardization of BEV separation and analysis techniques.

作者聚焦：加速细菌细胞外囊泡分离和异质性的研究 

使用密度梯度离心从人粪便中分离和纯化细菌胞外囊泡

Our study isolates, purifies, and characterize bacterial extracellular vesicles from physicists using density gradient centrifugation. We aim to address the separation of BEVs from contaminants and provide a reliable method for researchers. Our protocol reduces centrifugation time from 18 to 7 hours.Top down mode improves BEV separation and reduces contamination from liver proteins. Our method simplifies the separation of BEVs, setting a standard for future studies. It can be applied to other body fluids with appropriate modification, enhancing our understanding and facilitating the study of BEVs revealing their inherent heterogeneity within the human body.

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

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。

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

1.7K 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 

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.

科研

生物学

Bacterial Extracellular Vesicles (BEVs) Enrichment from Human Faeces Using Differential Centrifugation Speeds

To begin, take the prepared human fecal samples. Cool the high speed refrigerated centrifuge to 4 degrees Celsius. Use PBS to adjust the weight of the sample tubes to 0.1 grams, and centrifuge them at 3, 000 G for 20 minutes at 4 degrees Celsius.Using a plastic pipette, transfer the supernatant into two 50 milliliter tubes, leaving 1 milliliter behind. Then, centrifuge the supernatant at 12, 000 G for 30 minutes at 4 degrees Celsius. Using a 20 milliliter syringe, aspirate the supernatant, and remove the needle from the syringe.Filter the liquid through 0.22 micrometer filters into 50 milliliter tubes. Next, wipe the bucket with 75%alcohol to eliminate any remaining contamination. Transfer approximately 30 milliliters of the filtered fecal supernatant into the ultracentrifuge tube.Fill the tube with approximately 8 milliliters of PBS, leaving a 3 millimeter gap from the tube's opening. Place the two ultracentrifuge tubes in opposing ultracentrifugation buckets, and equalize the weight of the two buckets using PBS. Load all buckets onto the rotor, irrespective of whether they are loaded with tubes.Place the samples in the centrifuge, initiate the vacuum and spin them at 160, 000 G for 70 minutes at 4 degrees Celsius. As soon as the centrifugation completes, release the chamber vacuum and open the door of the ultra centrifuge. Then, promptly remove the rotor.Transfer the buckets from the rotor to the rack, and pick up the tubes using a nipper. Discard the supernatant, and save the visible brown pellets. Add 1 milliliter of pre-chilled PBS to resuspend the pellets completely.Fill the tube with approximately 37 milliliters of PBS, leaving a 3 millimeter gap from the opening. After performing another round of ultracentrifugation, discard the supernatant and leave the tube inverted for 5 minutes. Remove any remaining solution on the inner wall with wipers.Using a 1, 000 microliter pipette, add 1.2 milliliters of pre-chilled PBS to resuspend pellets, and mix them by pipetting. Transfer 1.2 milliliters of the PBS BEV solution from one ultracentrifuge tube to a clean 1.5 milliliter micro tube.

使用差异离心速度从人类粪便中富集细菌细胞外囊泡 （BEV）

Establishment of a Density Gradient Centrifugation System to Isolate and Purify Bacterial Extracellular Vesicles (BEVs) from Human Faeces

To begin, take 500 microliters of the prepared PBS/BEV solution. Combine it with three milliliters of PBS and mix gently using a 1, 000 microliter pipette. Place a 31 milliliter ultracentrifuge tube on a foam plate with holes, and label the tube with a downward arrow.Then hold a 1, 000 microliter pipette vertically and add three milliliters of the 50%iodixanol solution to the bottom of the tube. Place a tube holder slightly above the level of the foam plate below the tube opening, and tilt the tube to an angle of 70 degrees. Using a 1, 000 microliter pipette, add three milliliters of 40%iodixanol solution on top of the 50%iodixanol solution.Then layer three milliliters of 20%iodixanol solution on the 40%iodixanol solution. Using a 1, 000 microliter pipette, add three milliliters of a 10%iodixanol solution on top of the prepared 20%iodixanol solution. Next, add 3.5 milliliters of the PBS/BEV solution on top of the 10%iodixanol solution, and gently return the tubes to an upright position.Now, position a 31 milliliter ultracentrifuge tube on a foam plate with holes, and label it with an upward arrow. Using a 1, 000 microliter pipette, vertically add 2.5 milliliters of the 60%iodixanol stock solution into the bottom of the tube, and combine it with 500 microliters of PBS/BEV solution to get three milliliters of 50%iodixanol BEV solution. Mix the solution thoroughly by pipetting.Then after layering 40, 20 and 10%iodixanol solution in sequence, use a 1, 000 microliter pipette to add 3.5 milliliters of PBS on top of the 10%iodixanol solution. Adjust the weight of the two tubes with PBS to a total weight within plus minus 0.005 grams. Next, position the tubes in the buckets and centrifuge them at 160, 000 G for seven hours at four degrees Celsius.Using a pipetter, collect the fractions obtained from a gradient ultracentrifugation mode sequentially from top to bottom against the sidewall into 10 separate 38.5 milliliter ultracentrifuge tubes. Then ultracentrifuge the fractions at 160, 000 G for 70 minutes at four degrees Celsius. Remove the supernatant, and invert the ultracentrifuge tubes for five minutes.Next, resuspend the pellets in 200 microliters of pre-chilled PBS using a 200 microliter pipette. Transfer the PBS/BEV solution into a clean 1.5 milliliter microtube. Label the fractions from F1 to F10 as obtained from one gradient ultracentrifugation mode, and proceed for analysis.