这项工作得到了希腊研究与创新基金会 （00376）、美国国立卫生研究院、美国国家癌症研究所 （NCI） [Grant R15CA231339]、德克萨斯理工大学健康科学中心 （TTUHSC） 药学院科学办公室赠款（至 CMM））和路易斯安那大学门罗分校药学院启动资金的支持， 美国国立卫生研究院 （NIH） 通过美国国家普通医学研究所 [Grant P20 GM103424-21] 和路易斯安那州董事会的研究竞争力子计划 （RCS） 通过董事会支持基金 （LEQSF（2021-24）-RD-A-23）（给 GM）。常用的 TTUHSC 设备是通过德克萨斯州癌症预防研究所 （CPRIT） RP190524 和 RP200572 的赠款获得的。资助者在研究设计、写作决定或手稿准备方面没有任何作用。 图1A 中的图形摘要是使用 BioRender.com 创建的。

动物研究是根据德克萨斯理工大学健康科学中心 （TTUHSC） 的机构动物护理和使用委员会 （IACUC） 批准的协议进行的，用于 TTUHSC 的实验，以及根据指令 2010/63 在希腊西部地区兽医管理局进行的实验在帕特雷大学的实验。在处理动物粪便时，认真遵守废物处理规定。
1.小鼠真皮的分离
<ol><li>第1天 注意：在第 1 天;准备好以下材料和设备：麻醉室、动物毛发夹、异氟醚、细镊子、0.2 μm 注射器过滤器。<ol><li>在含有 1x 抗生素 - 抗真菌溶液的 DMEM 中制备 2 mg/mL 分散酶溶液，并对其进行过滤灭菌。 注：培养基或PBS的使用可以用HBSS代替，因为它已在其他报道的方案中成功使用12。1x抗生素 - 抗真菌溶液可以通过添加青霉素（100IU / mL），链霉素（100μg/ mL）和两性霉素（2.5μg/ mL）来替代。</li>
		<li>通过将小鼠置于具有异氟烷给药（0.5-2%异氟烷，流量：1L / min）的麻醉室中来麻醉小鼠。小鼠停止移动后，将其从笼子中移出，并继续麻醉调整具有相似异氟烷流量的鼻锥。用脚趾捏住动物以确认足够的麻醉水平，并不断监测小鼠以确保保持深度麻醉（没有运动）。使用理发器沿中线纵向夹约 9 cm2 的背毛和腹毛（分别为小鼠背部和腹部）。让小鼠从麻醉中恢复过来，然后将其放回笼子里。 注意：可以改用脱毛（脱毛）霜。确保在要隔离的皮肤区域完全去除毛发，同时保持皮肤的完整性。尝试夹尽可能延长的皮肤区域。为确保脱毛的潜在炎症得到解决，必须在第二天进行细胞分离。为了增加分离细胞的数量，每组聚集2至4只小鼠。</li>
	</ol></li>
	<li>第2天 注意：准备好以下材料和设备：安乐死室、生物安全柜、CO2 培养箱、乙醇、细剪刀、细镊子、钝镊子、100 毫米组织培养板、分散酶、DMEM、1x 抗生素 - 抗真菌溶液。<ol><li>通过CO2 给药对小鼠实施安乐死，并将其置于仰卧位置，露出夹紧的区域。喷洒 70% 乙醇作为消毒剂。 注意：从此步骤开始，所有步骤都必须在无菌条件下（在生物安全柜中）执行。</li>
		<li>将无菌 100 mm 组织培养板放在冰上。</li>
		<li>在腹部做一个 0.5 英寸的浅切口，并沿中线纵向切至颈部和下腹部。进行横向切割，以便能够剥开皮肤。 注意：切口大小和分离的皮肤区域取决于小鼠的大小。</li>
		<li>使用钝镊子，小心地将皮肤与周围组织分开。这样做绕过小鼠的整个躯干，在颈部和下腹部横向切开皮肤。</li>
		<li>小心地将皮肤放入 100 毫米组织培养板中，真皮面朝下，尽可能保持皮肤拉伸。盖上盘子，把它们放在冰上。完成组中所有小鼠的分离。</li>
		<li>从所有小鼠中提取皮肤后，在室温下孵育板10分钟以使外植体变平。</li>
		<li>向板中加入6mL Dispase溶液，确保皮肤完全浸入水中，并在4°C下轻度搅拌孵育过夜。 注意：也可以进行胰蛋白酶消化而不是分散酶12，然后进行 Liberase 而不是 II 型胶原酶处理（下图）。</li>
	</ol></li>
</ol>2. 从真皮中分离细胞
<ol><li>第3天 注意：准备好以下材料和设备：离心机、CO2 培养箱、生物安全柜、剪刀、DMEM、1x 抗生素-抗真菌溶液、II 型胶原酶、PBS、50 mL 锥形管、100 mm 和 60 mm 组织培养板、DNAse I 溶液、I 型胶原、70 μm 和 40 μm 细胞过滤器、0.2 μm 注射器过滤器、LEC 培养基。<ol><li>制备 500 mL 1x PBS（1.37 M NaCl、27 mM KCl、100 mM Na2HPO4、18 mM KH2PO 4，pH 7.4）并在 15 psi 下高压灭菌 20 分钟或过滤器灭菌。或者，使用市售的无菌 PBS。</li>
		<li>在含有 1x 抗生素 - 抗真菌溶液的 DMEM 中制备 10 mL 2 mg/mL II 型胶原酶溶液，并对其进行过滤灭菌。 注意：在无菌罩下执行后续步骤，以减少样品污染的机会。</li>
		<li>使用移液管从培养皿中取出液体。</li>
		<li>使用镊子将真皮（皮肤底部）与表皮（顶部）分开，并去除任何明显的脂肪组织。将真皮转移到干净的组织培养板上。</li>
		<li>在干净的组织培养板中加入 1 mL DMEM 和 1x 抗生素 - 抗真菌溶液，并倾斜它，使液体积聚在板的一侧。</li>
		<li>将真皮切成 1-2 mm2 块，然后将它们转移到新的 50 mL 锥形管中。 注意：理想情况下，此步骤必须在 4-5 分钟内完成，因为预计更长的处理时间不会提供更高的产量或细胞存活率。</li>
		<li>加入 10 mL II 型胶原酶溶液。</li>
		<li>加入50μL（25单位/ mL溶液）DNAse I溶液（减少细胞聚集），并在37°C下连续搅拌消化真皮溶液2小时（使用旋转/振荡培养箱，转速为30-50rpm）。 注意：定期检查以确定大部分真皮是否已被消化。如果没有，如果可能，增加消化时间（最多额外 1 小时）和搅拌速度。</li>
		<li>在37°C下用2mL 10μg/ mLI型胶原蛋白在0.02M乙酸中包被60mm组织培养板>20分钟。 用无菌 PBS 洗涤 2 次。</li>
		<li>使用 70 μm 细胞过滤器过滤细胞悬浮液，并以 250 × g 离心 5 分钟。丢弃上清液。</li>
		<li>用 1x 抗生素 - 抗真菌溶液重悬于 10 mL DMEM 中。</li>
		<li>使用 40 μm 细胞过滤器过滤细胞悬浮液，并以 250 × g 离心 5 分钟。丢弃上清液。</li>
		<li>重悬于 1 mL 完全 LEC 培养基（含添加剂的内皮细胞生长基础培养基）中。</li>
		<li>将细胞铺板在胶原包被的 60 mm 组织培养板中，置于完全 LEC 培养基中。这将是细胞培养的第 0 天。</li>
		<li>每 2 天更换培养基，用 1x PBS 洗涤细胞两次并加入新鲜的 LEC 培养基。</li>
		<li>重复步骤 2.1.15，直到细胞汇合 80-90%。 注意：如果在细胞汇合 80-90% 之前进行纯化，则分离出的淋巴内皮细胞会更少。此过程大约需要 7 到 10 天。</li>
	</ol></li>
</ol>3. 带有 LYVE-1 抗体的磁珠涂层
注意：在纯化步骤前一天，请准备好以下材料和设备：4°C冰箱，转子，磁选仪，PBS，抗兔IgG磁珠，微量离心管，0.2μm注射器过滤器，磁珠涂层溶液，LYVE-1抗体。
<ol><li>制备磁珠包衣溶液：20 mL PBS 与 0.1% BSA 和 2 mM EDTA 并过滤灭菌。将其储存在4°C。 注意：不建议存放超过几天;使用前检查是否有沉淀物或污染。</li>
	<li>将 500 μL 抗兔 IgG 磁珠移液至微量离心管中，并加入 1 mL 冰冷的磁珠包被溶液。</li>
	<li>使用磁性分离器清洗珠子。</li>
	<li>用 1 mL 冰冷的磁珠涂层溶液重复洗涤 2 次。</li>
	<li>将珠子重悬于 500 μL 磁珠包被溶液中，并在微量离心管中制备 150 μL 的等分试样。</li>
	<li>向每个试管中加入 15 μg LYVE-1 抗体（从 1 mg/mL 抗体储备液浓度中加入 15 μL）。</li>
	<li>在4°C下孵育过夜，持续搅拌。</li>
</ol>4.淋巴管内皮细胞纯化
注意：准备好以下材料和设备：CO2 培养箱、摇床、计时器、封口膜、DMEM、1x 抗生素 - 抗真菌溶液、BSA、PBS、磁珠包被溶液、胰蛋白酶-EDTA、100 mm 或 60 mm 组织培养板、0.2 μm 注射器过滤器、I 型胶原蛋白、乙酸、LEC 培养基。
<ol><li>用 1x 抗生素 - 抗真菌溶液和 0.1% BSA 制备 50 mL DMEM，并对其进行过滤灭菌。</li>
	<li>在37°C下用10μg/ mL I型胶原蛋白在0.02M乙酸中包被60mm组织培养板>20分钟。 用无菌 PBS 洗涤 2 次。</li>
	<li>使用磁性分离器用 1 mL 磁珠包被溶液洗涤预包被的珠子。</li>
	<li>将珠子重悬于含有 0.1% BSA 的 150 μL DMEM 中。 注意：珠子现在可以在4°C下储存长达1个月。</li>
	<li>用 PBS 洗涤细胞 2 次。</li>
	<li>向细胞中加入 3 mL 含 0.1% BSA（用于 60 mm 组织培养板）的 DMEM 和 50 μL 抗体偶联珠。 注意：如果来自两只或更多小鼠的细胞合并，请使用 100 mm 组织培养板并加入 6 mL 含有 0.1% BSA 的 DMEM。</li>
	<li>用石蜡膜密封培养皿。</li>
	<li>将培养皿放在振荡器上，在室温下以 10 RPM 搅拌，孵育正好 10 分钟。 注意：较长的孵育时间将导致非特异性结合。为确保珠子与整个板表面相互作用，请在板继续摇晃时每隔几分钟手动旋转板。</li>
	<li>丢弃培养基并用 PBS 洗涤细胞一次。快速检查细胞以验证板上是否存在 LEC 集落。</li>
	<li>胰蛋白酶消化细胞：使用1mL胰蛋白酶-EDTA，并在37°C下孵育3分钟。 检查细胞是否成功脱离细胞。</li>
	<li>用 2 mL 完全 LEC 培养基中和溶液，并将溶液转移到 5 mL 无菌聚丙烯管中。</li>
	<li>使用磁性分离器洗涤含有 0.1% BSA 的 4 x 3 mL DMEM 细胞以去除未结合的细胞。</li>
	<li>将剩余的珠子结合细胞重悬于完全 LEC 培养基中，并铺在先前胶原包被的 60 mm 组织培养板上。</li>
	<li>将细胞在37°C下孵育，并在每次两次PBS洗涤后每隔一天更换培养基。</li>
	<li>培养细胞直至它们达到汇合（通常需要 7 到 10 天）。</li>
	<li>此时，在显微镜下检查细胞的汇合度，并继续准备用于流式细胞术的细胞。如果确定 F4/80+ 百分比较高，则再次使用 LYVE-1（第 4 步：淋巴内皮细胞纯化）重复纯化步骤，理想情况下使用另一种淋巴标志物（即 podoplanin14）。 注：抗体靶向的蛋白质在磁珠纯化细胞群上的高表达已被证明15,16。然而，对于 LYVE-1，进行流式细胞术控制以识别其他 LYVE-1+ 细胞（主要是巨噬细胞）的百分比非常重要。LYVE-1 仅在与肿瘤和伤口相关的巨噬细胞亚群中表达，而在健康皮肤中，LYVE-1 的表达更局限于淋巴内皮细胞17。因此，F4/80 筛选不是强制性的，而是取决于实验环境。用于演示的小鼠不携带肿瘤或伤口，只是脱毛引起的潜在炎症，但我们将 F4/80 染色作为 LYVE-1+ 巨噬细胞百分比的基线。</li>
</ol>5. 流式细胞术
注意：准备好以下材料和设备：离心机、CO2 培养箱、计时器、血细胞计数器、EDTA 溶液、1x 抗生素 - 抗真菌溶液、BSA、PBS、胰蛋白酶-EDTA、聚丙烯管、0.2 μm 注射器过滤器、70% 乙醇、F4/80 FITC 偶联抗体。
<ol><li>制备含有 0.1% BSA 的 PBS 并对其进行过滤灭菌。</li>
	<li>在PBS中制备1mM EDTA溶液。</li>
	<li>胰蛋白酶消化细胞：将细胞与1mL 1mM EDTA溶液在37°C下孵育5分钟。 注意：用EDTA溶液孵育是一种较温和的细胞分离方法，可以更好地保留跨膜蛋白，是首选，但需要更长的孵育期。或者，胰蛋白酶-EDTA溶液可以在37°C下孵育1分钟，但这会影响流式细胞术应用的抗体亲和力。</li>
	<li>用 2 mL 完全 LEC 培养基中和溶液，并将溶液转移到 5 mL 无菌聚丙烯管中。</li>
	<li>将样品以 250 × g 离心 5 分钟。</li>
	<li>用PBS清洗。</li>
	<li>重复离心和PBS洗涤。</li>
	<li>将细胞重悬于含有0.1%BSA的PBS中，并使用血细胞计数器或其他方法对其进行计数。将 ~1 × 106 个细胞分装到聚丙烯管中，最终体积为 100 μl。从此步骤开始，在4°C下执行所有步骤。 注意：细胞可以在染色前固定，也可以在活细胞染色时进行染色。对于流式细胞术来说，固定可能不是最优的，因为一些抗体在固定后可能无法检测到抗原;因此，我们建议在上一步后立即运行流式细胞术，并跳过固定步骤。如果无法做到这一点，在流式细胞术实验之前进行固定将允许流式细胞术步骤的延迟。如果要进行固定，应事先评估抗体在固定细胞环境中的性能。</li>
	<li>进行固定（可选步骤;如果细胞不被固定，则进行免疫染色）：将细胞重悬于冰冷的70%乙醇中，并在冰上轻轻混合10分钟。 注意：乙醇固定可以透化细胞并导致GFP的释放;然而，基于多聚甲醛的固定会破坏用于流式细胞术的染料，理想情况下可以避免。</li>
	<li>在室温下孵育15分钟。</li>
	<li>在 1x PBS 中以 250 × g 离心 2 次洗涤细胞 5 分钟。</li>
	<li>继续下一步或将细胞重悬于0.5-1mL 1x PBS中，并储存在4°C。</li>
</ol>6. 免疫染色
注意：如果可能，轻柔地处理细胞并避免大量移液，以保持细胞膜的完整性并最大限度地减少细胞死亡，确保细胞不会形成团块。从分离的内皮细胞中，制备以下组：未染色细胞和具有另一种内皮细胞标志物（即 VEGFR3、podoplanin）、F4/80 和/或两者的染色细胞。包括市售的小鼠淋巴内皮细胞和巨噬细胞（如果有）作为阳性对照。出于演示目的，并且由于内源性 tdTomato 荧光 （PE+），我们使用了 FITC 偶联的 F4/80 染色。
<ol><li>根据推荐或优化的稀释度，用 0.1% BSA 稀释 PBS 中的一抗。将 0.5 μg（1 μL 抗体溶液）F4/80 FITC 偶联抗体加入 100 μL 细胞悬液中。</li>
	<li>加入稀释的荧光偶联抗体后重悬细胞。通过轻弹聚丙烯管进行混合，以确保没有结块并且所有细胞都正确包被。</li>
	<li>在室温（固定细胞）下孵育1小时或在黑暗中在冰（活细胞）上孵育30分钟。</li>
	<li>通过在4°C（对于活细胞）或室温（对于固定细胞）下以250× g 离心细胞5分钟，在黑暗中使用含有0.1%BSA的PBS洗涤细胞2次。</li>
	<li>对于非荧光偶联抗体<ol><li>在孵育缓冲液中稀释荧光染料偶联的二抗。对于 Alexa Flour 抗体，使用 1：500 稀释度。</li>
		<li>将二抗加入 100 μL 细胞悬浮液中。</li>
		<li>在室温（固定细胞）或冰（活细胞）下孵育30分钟。</li>
		<li>通过在4°C（对于活细胞）或室温（对于固定细胞）下在孵育缓冲液中以250× g 离心5分钟洗涤两次。</li>
	</ol></li>
	<li>将细胞重悬于 0.5 mL 1x PBS 中，并使用流式细胞仪进行分析。</li>
	<li>按照以下门控策略步骤来鉴定表达 LYVE-1 的细胞内的巨噬细胞群：<ol><li>考虑前向散射和侧向散射以区分细胞群。</li>
		<li>考虑对细胞大小、面积和高度进行前向散射，以区分单细胞（单峰）。</li>
		<li>考虑F4 / 80（FITC）表达和RFP（PE）表达以识别表达tdTomato（PE）的分离小鼠细胞中的巨噬细胞（F4 / 80 +）（由于在特定小鼠中存在荧光tdTomato报告基因，如下所述）。</li>
	</ol></li>
</ol>

从小鼠真皮淋巴毛细血管中分离内皮细胞

<ol>
	<li>Oliver, G., Kipnis, J., Randolph, G. J., Harvey, N. 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作者声明没有相互竞争的经济利益。

淋巴系统是机体稳态功能的重要调节器，最重要的功能是维持液体血浆、去除细胞代谢副产物和有毒分子、脂质吸收和免疫细胞运输 1,19。适当标记物的识别在淋巴管领域引发了新数据的激增，揭示了淋巴管系统的新功能，例如它们在某些器官的修复和再生能力中的作用 2,3。关于淋巴系统的知识越来越多，描绘了淋巴系统在各种病理条件下的作用，改变了以前建立的传统观点1.淋巴系统在许多疾病的结果中起着重要作用，例如癌症生长和转移20、Milroy 病（一种原发性淋巴水肿形式 21,22）和卡波西样淋巴管瘤病（一种以淋巴管扩张和互连为特征的罕见疾病23,24）.上述事实突出了淋巴系统在疾病病理学中的作用，并加强了进一步理解该系统的分子调控的必要性。
作为该过程的一部分，淋巴内皮细胞生理学的体外评估可以揭示功能缺陷，某些病理状况的特征，并且可以阐明这些病症背后的分子机制。已经发表了几种分离淋巴内皮细胞的方案，其中大多数使用荧光激活细胞分选7,25。该方法是一种无需荧光激活细胞分选即可分离淋巴内皮细胞的有效方法，但荧光激活细胞分选技术对淋巴内皮细胞群的纯度较差，这是基于磁珠的技术的主要局限性。为此，该方法可用于粗略鉴定淋巴内皮功能或评估条件性缺陷小鼠18 的蛋白质缺乏症，但不推荐用于 RNA 测序应用。基于荧光激活的细胞分选方案具有所选细胞纯度更高的优点，因为它们是通过对多种淋巴特异性标记物的共染色来选择的，并且由于省略了磁珠纯化之前长达数天的细胞生长步骤，因此它们的速度更快。基于荧光激活细胞分选的方案的另一个显著优势是不需要磁珠，使其更具成本效益。基于磁珠的分离方法的缺点是获得的产量降低，但在存在荧光激活细胞分选设备的情况下，它是首选的纯化方法。
我们之前发现，从成年小鼠皮肤中分离出的淋巴内皮细胞与LYVE-1+免疫细胞的污染小于10%18， 图3 中的当前示例显示了LYVE-1+巨噬细胞的~5%污染。LYVE-1 是一种经典的淋巴标志物，已成功与 CD31 和 CD34 联合用于基于磁珠的血液和淋巴内皮细胞纯化26。LYVE-1 和 CD31 组合提供高纯度的淋巴内皮细胞群。据报道，UEA-1 和 D2-40 抗体27 联合使用基于磁珠的替代选择。基于磁珠的皮肤淋巴内皮细胞分离技术，通过排除（阴性选择）靶向细胞并参与更多纯化步骤，已在小鼠胚胎中成功进行28，如果适用于成年小鼠，它肯定可以用于分离纯淋巴内皮群体用于相关的下游应用。然而，负选择的局限性是，并非所有与磁珠结合的细胞都可以被选中，正如我们也目睹了未选择的 LYVE-1+ 阳性细胞，这些细胞与较少的磁珠结合，因此不能充分结合到磁铁进行选择。
一种类似的基于磁珠的分离技术，用于从成年小鼠的肺部分离淋巴内皮细胞，是基于鬼臼拉宁的淋巴内皮细胞选择14。协议的结构非常相似，主要区别在于目标;然而，我们尚未进行比较分析以确定产量和纯度的差异。众所周知，不同的淋巴亚群可能会有所不同 9,27,29。在这个范围内，需要强调的是，并非所有的真皮淋巴内皮细胞都是LYVE-1+。LYVE-1 在同一组织、炎症组织（如肿瘤）中的表达存在差异，LYVE-1 在 CD8+ T 细胞浸润中起作用29 或在淋巴毛细血管中与淋巴收集血管9 中发挥作用。这导致了该方法的差异，对于淋巴内皮细胞亚型的淋巴内皮细胞分离具有特异性，主要来自淋巴毛细血管而不是集合血管9。因此，所提出的方法适用于主要在毛细血管中调节的现象，但也限制了可以分离的淋巴内皮细胞池。
基于磁珠的方法的一个局限性是通过流式细胞术实验来评估纯度的可选性。我们之前发现，从成年小鼠皮肤中分离出的淋巴内皮细胞与LYVE-1+免疫细胞的污染小于10%18， 图3 中的当前示例证实了这一发现，表明LYVE-1+巨噬细胞的污染率为~5%。对初始分离细胞群（未显示）进行 F4/80 染色的流式细胞术实验显示存在 ~3% 巨噬细胞，其中还包括 LYVE-1-巨噬细胞。因此，微球选择细胞群中百分比的增加表明了一种有效的纯化方法。鉴于健康小鼠皮肤中巨噬细胞的百分比低于存在肿瘤或炎症时的百分比17，这增加了该方法在健康小鼠中的可靠性。值得注意的是，有些未选择的细胞连接了一个或多个磁珠（未显示），这验证了要选择磁珠的细胞必须连接许多磁珠（图2），并且未选择一些LYVE-1表达水平较低的细胞（附着了磁珠）。
为了成功应用该方案，遵循淋巴内皮细胞与 LYVE-1 抗体偶联磁珠孵育的时间至关重要，因为增加孵育会导致非特异性结合。另一个关键因素是切碎皮肤的时机，以确保尽可能多的细胞存活。淋巴内皮细胞不是占主导地位的细胞群;因此，纯化后，预计获得的细胞数量有限。就一次可以处理的小鼠数量而言，我们已经处理了多达四只小鼠，第一只小鼠和最后一只小鼠之间的产量没有任何明显的差异。该方案可以应用于性别和不同年龄，尽管成年小鼠的年龄是内皮细胞复制潜力的决定因素。与人内皮细胞30类似，小鼠年龄的增加导致衰老增加31,32，因此，分离细胞的复制潜力有限。预计对于C57BL6背景的年轻（<3个月大）小鼠将提供更高的产量，我们已经使用了C57BL6背景，并且是本研究中的小鼠背景。总体而言，该协议提供了一种用于小鼠真皮淋巴内皮细胞分离的有效方法，可用于不优先考虑分离的淋巴内皮细胞群的绝对纯度的应用：纯化的细胞可用于体外实验或用于验证淋巴内皮特异性突变小鼠中目的基因的缺陷。这对于转基因小鼠或实验动物模型特别有用，在这些模型中，淋巴内皮细胞的快速和粗制纯化是首要任务，并且在荧光激活细胞分选仪不容易获得的研究所和设施中。

淋巴系统在人体生理学中起着举足轻重的作用。它被认为是一种重要的稳态因子，可促进重要功能，例如维持组织-浆液平衡、免疫监视和脂质吸收1，以及最近发现的功能，例如心脏的修复能力2 和骨骼和造血的再生能力3。尽管淋巴系统具有重要作用，但淋巴管作用的几个方面，以及控制某些生理参数和反应的分子机制仍然难以捉摸。此外，淋巴管缺损是某些病理生理状况的触发或恶化因素。众所周知的例子是原发性和继发性淋巴水肿，分别取决于疾病的遗传或非遗传起源，而淋巴系统对原发性肿瘤生长和转移性播散的作用至关重要，因为它可以作为转移的管道和免疫功能的调节剂1。
与血管系统相比，淋巴管的研究和知识滞后主要是由于淋巴系统与血管系统相比发现较晚，以及淋巴管特异性分子标志物的鉴定延迟。近几十年来，这种情况有了很大的改善，导致淋巴管在稳态和疾病条件下的传统情况发生了变化1。与血管生成类似，淋巴管生成是从预先存在的淋巴管形成新的淋巴管，在多种疾病的发展中起着关键作用4,5,6。然而，与血管生成不同，淋巴管生成的研究主要局限于体内模型，重点关注病理模型中的发育血管形成和结构缺陷。淋巴内皮细胞的分离对于体外研究很重要，这可以通过所提出的方案提供。
已经开发了几种用于从小鼠中分离淋巴内皮的方案，这些方案需要使用荧光激活的细胞分选7。细胞分选仪的优点是纯度更高，与基于微球的分离相反，后者的产量更高。该方案利用淋巴内皮透明质酸受体 1 （LYVE-1） 的表达，这是一种淋巴内皮标志物，对淋巴管内的细胞运输很重要 4,8。由于 LYVE-1 表达仅限于淋巴毛细血管而不是集合淋巴管，因此该技术适用于从淋巴毛细血管中特异性分离淋巴内皮细胞，这与其他淋巴标志物相反，例如在所有淋巴内皮细胞中均匀表达的 podoplanin9。对于该方案，排除了其他非跨膜的重要淋巴标志物，例如 Prox1。
由于生理结果是淋巴管生成，通常在淋巴毛细血管处启动，因此选择 LYVE-1 作为靶标，因为它在这些细胞中的选择性以及所选抗体的产量高。使用的酶是 II 型胶原酶，通常已知其在胶原解离方面比 I 型更有效10，以及分散酶（一种更广泛的蛋白酶，通常用于真皮-表皮分离11;然而，已经报道了其他用于组织分离的酶12,13 并且可以使用。该协议的目标是描述一种使用磁珠纯化从成年小鼠的淋巴毛细血管中高产量分离真皮淋巴内皮细胞的方法，特别是在荧光激活细胞分选不容易获得的地方。该方法适用于淋巴内皮细胞纯度可能会因下游应用而受到影响的应用。

<table><tbody><tr><td>0.2 &amp;mu;m Syringe Filters</td><td>Fisher Scientific</td><td>09-719C</td><td></td></tr><tr><td>100 mm Tissue Culture Dishes</td><td>Fisher Scientific</td><td>FB012924</td><td></td></tr><tr><td>60 mm Tissue Culture Dishes</td><td>Fisher Scientific</td><td>FB012921</td><td></td></tr><tr><td>Animal Hair Clipper</td><td>Wahl</td><td></td><td></td></tr><tr><td>Antibiotic-Antimycotic Solution 100x</td><td>Fisher Scientific</td><td>15240-062</td><td></td></tr><tr><td>Blunt Forceps</td><td>Fine Science Tools</td><td>11992-20</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A4919</td><td></td></tr><tr><td>Cell Strainer 40 &amp;mu;m</td><td>Fisher Scientific</td><td>542040</td><td></td></tr><tr><td>Cell Strainer 70 &amp;mu;m</td><td>Fisher Scientific</td><td>542070</td><td></td></tr><tr><td>CO2 Incubator</td><td></td><td></td><td></td></tr><tr><td>Collagen I, High Concentration Rat Tail</td><td>Corning</td><td>354249</td><td>dilute in 0.02 M Acetic Acid in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Collagenase Type II</td><td>Life Technologies</td><td>17101-015</td><td></td></tr><tr><td>Dispase</td><td>Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>DMEM</td><td>Fisher Scientific</td><td>11-995-073</td><td></td></tr><tr><td>DNAse I Solution (2,500 U/mL)</td><td>Thermo Scientific</td><td>90083</td><td></td></tr><tr><td>Dynal MPC-L Magnetic Particle Concentrator</td><td>Invitrogen</td><td>120-21D</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>3690</td><td></td></tr><tr><td>Endothelial Cell Growth Base Medium &amp;amp; Supplement (LEC medium)</td><td>R&amp;amp;D Systems</td><td>CCM027</td><td></td></tr><tr><td>Euthanasia chamber</td><td>Euthanex Corporation</td><td></td><td></td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Fine Scissors-Sharp</td><td>Fine Science Tools</td><td>14060-10</td><td></td></tr><tr><td>FITC anti-mouse F4/80 Antibody</td><td>Biolegend</td><td>123107</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG Magnetic Beads</td><td>New England Biolabs</td><td>S1432S</td><td></td></tr><tr><td>LARC-A E-Z Anesthesia Induction Chamber</td><td>Euthanex Corporation</td><td></td><td></td></tr><tr><td>MagnaBind Goat Anti-Rabbit IgG Beads</td><td>Thermo Scientific</td><td>21356</td><td></td></tr><tr><td>Paraformaldehyde Solution 4% in PBS</td><td>Fisher Scientific</td><td>AAJ19943K2</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Fisher Scientific</td><td>SH30256FS</td><td></td></tr><tr><td>Rabbit Anti-Mouse LYVE-1</td><td>ReliaTech GmbH</td><td>103-PA50</td><td></td></tr><tr><td>Rotating/Shaking Incubator</td><td></td><td></td><td></td></tr><tr><td>Round-Bottom Polypropylene Tubes</td><td>Corning</td><td>352063</td><td></td></tr><tr><td>Syringe Filters w 0.2 &amp;mu;m Pores</td><td>Fisher Scientific</td><td>09-719C</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Fisher Scientific</td><td>25-300-120</td><td></td></tr></tbody></table>

murine dermal lymphatic endothelial cell isolation

淋巴系统参与免疫监视、脂质吸收和组织液平衡的调节。小鼠淋巴内皮细胞的分离是淋巴研究的一个重要过程，因为它允许对分离的细胞进行 体外 和生化实验。此外，Cre-lox技术的发展使无法全局靶向的组织特异性基因缺陷成为可能，从而可以精确确定它们在所研究组织中的作用。解剖某些基因在淋巴生理学和病理生理学中的作用需要使用淋巴特异性启动子，因此需要对靶基因的表达水平进行实验验证。
从野生型或转基因小鼠中有效分离淋巴内皮细胞的方法使得可以使用 离体 和 体外 测定来研究调节淋巴功能的机制和鉴定所研究蛋白质的表达水平。我们已经开发、标准化并提出了一种通过基于 LYVE-1 表达的磁珠纯化有效分离小鼠真皮淋巴内皮细胞 （DLECs） 的方案。概述的协议旨在为研究人员提供一种工具，以进一步理解和阐明淋巴内皮细胞功能的重要参与者，特别是在没有荧光激活细胞分选设备的设施中。

该方法用于鉴定小 GTP 酶 RhoA 的蛋白表达，该酶的编码基因在他莫昔芬诱导的淋巴内皮特异性启动子 Prox1-CreERT2 18 的作用下在两个等位基因中发生絮状并缺失。分离的 LEC 可以培养多达四代，之后它们会衰老。它们已被冷冻、解冻，并成功用血管生成素-2 刺激。由于细胞传代次数有限，我们尚未尝试多次冻融循环。从一只小鼠分离出的典型细胞产量提供了 60 mm 培养皿的 40-50% 汇合度（~1.5 × 106 个细胞），并且增殖缓慢，因为当我们进行基于 LYVE-1 的纯化时，培养皿在 5 天后达到汇合。
第一次纯化后，LYVE-1+ 细胞与 60 mm 培养皿（~4 × 105 个细胞）的汇合度为 15%。第二轮纯化是可选的，提供的数据和图像均为一轮纯化。有许多细胞未被选中，它们携带磁珠，似乎连接的磁珠数量不足以选择它们。如前所述，始终建议在分散酶消化的同时添加 DNAse，尽管我们没有观察到产量的显着增加。对于本手稿中的演示实验，我们使用了携带 tdTomato 荧光报告基因的小鼠，我们之前曾将其与 floxed 基因和不同的启动子16,18 结合使用。使用这些小鼠的目的是（红色;PE+）分离细胞的荧光视图。
图1A展示了组织处理的不同步骤，在用分散酶过夜处理（图1B）之前，小鼠的延伸皮肤图像，第二天从真皮中去除表皮（图1C），以及之后立即分离细胞的真皮切碎（图1D）).通过与磁珠孵育纯化 LYVE-1+ 细胞后，在低汇合度下，通过连接它们的多个珠子（图 2）很容易识别细胞，其中单细胞在分离后 24 小时显示（图 2A-C），在高汇合度下，7 天后（图 2D）。显示了从小鼠真皮中分离的混合细胞群的低放大图像（图2E）。
指示性流式细胞术数据来自可选分析，用于确定分离的淋巴内皮细胞群中 LYVE-1+ 巨噬细胞的百分比。 图 3 显示了流式细胞术设门策略，其中 5% 的选定 LYVE-1+ 细胞对 F4/80（巨噬细胞）呈阳性。该百分比从分离（未纯化）小鼠皮肤细胞（未显示）中F4 / 80 +细胞的3%增加。转基因小鼠的内源性红色荧光允许通过荧光进行互补监测。随着细胞的增殖，附着的磁珠密度降低，并且针对其他淋巴标志物（如 VEGFR3 或 podoplanin14 ）的可选纯化步骤可以提供更纯化的淋巴内皮细胞群。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65393/65393fig01.jpg"> 图 1：组织和细胞分离过程的不同步骤演示。 （A） 协议步骤的图形示意图。（B）将切除的皮肤置于带有分散酶溶液的组织培养板中，在4°C下孵育过夜。 （C）第二天表皮与下面的真皮分离。（D）真皮的组织处理，用于分离和分离组织细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65393/65393fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65393/65393fig02.jpg"> 图 2：纯化的 LYVE-1+ 细胞的代表性图像。 （A，B） 单个 LYVE-1+ 分离细胞的明场（顶部）和荧光（底部）图像，纯化后 24 小时，连接珠子;比例尺 = 31 和 25 μm。 （C） 纯化后 24 小时代表性细胞的明场（左）、荧光（中心）和合并（右）图像;比例尺 = 25 μm。 （D） 分离细胞在增殖时的明场（顶部）和荧光（底部）图像，纯化后 7 天具有高汇合度;比例尺 = 100 μm。 （E） 从真皮分离 5 天后混合细胞群的明场（左）和荧光（中）低放大图像;比例尺 = 100 μm。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65393/65393fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65393/65393fig03.jpg"> 图 3：流式细胞术设门策略和数据。 计算选定的LYVE-1+细胞内巨噬细胞群的百分比（蓝色：单细胞（单峰）;红色：细胞簇）。缩写：FSC-A = 前向散射峰面积;SSC-A = 侧散射峰面积;FSC-H = 前向散射峰高;RFP = 红色荧光蛋白;FITC = 异硫氰酸荧光素;PE = 藻红蛋白。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65393/65393fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Murine Dermal Lymphatic Endothelial Cell Isolation at JoVE.com

作者聚焦：基于磁珠的小鼠真皮淋巴内皮细胞分离

本文介绍了一种基于磁珠从真皮淋巴毛细血管中分离小鼠内皮细胞的方法。分离的淋巴内皮细胞可用于下游 体外 实验和蛋白质表达分析。

小鼠真皮淋巴内皮细胞分离

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

我们小组的研究重点是描述调节内皮细胞生理学的机制。该协议描述了一种基于磁珠的肾内皮细胞分离方法，特别是从荧光激活细胞分选不可用的设施中的热淋巴毛细血管中分离。该方案允许从转基因小鼠中分离淋巴内皮细胞，以在体外研究其生理学。它特别适用于淋巴内皮细胞的纯度可能会因下游应用而受到影响的情况。该过程提供了从淋巴毛细血管而不是其他集合血管获得的高产量淋巴内皮细胞。在没有细胞分选设施的情况下，这也是一个不错的选择。

murine-dermal-lymphatic-endothelial-cell-isolation

Nanyang Technological University

Research

JoVE Journal

Biology

Murine Dermal Lymphatic Endothelial Cell Isolation

1.3K 次观看.  Texas Tech University Health Sciences Center. 我们小组的研究重点是描述调节内皮细胞生理学的机制。该协议描述了一种基于磁珠的肾内皮细胞分离方法，特别是从荧光激活细胞分选不可用的设施中的热淋巴毛细血管中分离。该方案允许从转基因小鼠中分离淋巴内皮细胞，以在体外研究其生理学。它特别适用于淋巴内皮细胞的纯度可能会因下游应用而受到影响的情况。该过程提供了从淋巴毛细血管而不是其他集合?...

视频: 作者聚焦：基于磁珠的小鼠真皮淋巴内皮细胞分离

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

科研

发育生物学

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

免疫与感染

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable 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 of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

医学

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

小鼠原代肺内皮细胞的分离

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

使用磁性细胞选择建立体外 LLEC 培养物

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

作者聚焦：揭示软脑膜淋巴内皮细胞的细胞功能和潜在临床意义 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

软脑膜淋巴内皮细胞的收获和原代培养

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

小鼠真皮淋巴内皮细胞分离

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此视频中的章节

小鼠皮肤成纤维隔离用流式细胞仪

小鼠肺树突状细胞的分离

从人类胸腺髓样树突状细胞和上皮细胞的分离

鼠淋巴结基质细胞的分离

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活

人淋巴管内皮细胞中的多参数荧光激活细胞分选分离

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

小鼠原代肺内皮细胞的分离

软脑膜淋巴内皮细胞的收获和原代培养

小鼠真皮淋巴内皮细胞分离

副本

摘要

探索更多视频

此视频中的章节

小鼠皮肤成纤维隔离用流式细胞仪

小鼠肺树突状细胞的分离

从人类胸腺髓样树突状细胞和上皮细胞的分离

鼠淋巴结基质细胞的分离

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

小鼠原代肺内皮细胞的分离

软脑膜淋巴内皮细胞的收获和原代培养

人淋巴管内皮细胞中的多参数荧光激活细胞分选分离