这项工作得到了国家自然科学基金（第82070502号，31972909,32171099），四川省科技计划（23NSFSC0576,2022YFS0607）的资助。作者感谢浙江大学的徐清波在细胞培养方面的帮助，并感谢西南医科大学流式细胞术平台的科学和技术帮助。

本研究获得批准，并按照《中国实验动物管理使用指南》对动物进行处理。本研究严格遵守动物实验的伦理要求，并经动物伦理委员会批准（批准文号：SWMU2020664）。本研究使用8周龄的健康C57BL / 6小鼠，体重在18-20g之间。这些动物被安置在西南医科大学（SWMU）的实验动物中心。
1. 来自主动脉和肠系膜动脉的外膜组织阻滞培养
<ol><li>常驻 CD34+ VW-SCs 隔离<ol><li>用3%异氟烷吸入麻醉两只小鼠（遵循机构批准的方案）。通过捏脚趾确认充分麻醉。用医用胶带将小鼠置于仰卧位，并喷洒 70% 乙醇消毒剂以消毒小鼠的皮毛。使用无菌眼科剪刀和镊子打开胸腔和腹腔，露出心脏和肠道。解剖分离的主动脉和肠系膜动脉后，将主动脉和肠系膜床固定在含有硅弹性体的培养皿中，并填充有生理盐溶液。用另一套消毒的剪刀和镊子，迅速解剖主动脉和肠系膜动脉周围的脂肪。</li>
		<li>将解剖的组织转移到含有1%青霉素 - 链霉素 - 两性霉素B溶液的6cm培养皿中（参见 材料表）。冲洗两次后，将动脉转移到立体显微镜下的组织培养罩中。</li>
		<li>纵向切开动脉，用细镊子剥去主动脉的外层和肠系膜动脉的第一分支。将外层转移到含有 0.1-0.2 mL 培养基的 3.5 cm 培养皿中（参见 材料表）。将外层切成小块（约 1 mm3）。</li>
		<li>将组织块转移到明胶包被的 T25 烧瓶中，确保在烧瓶底部均匀分布。在组织块之间保持适度的距离，以促进细胞爬行和生长。将烧瓶垂直置于培养箱（37°C，5%CO2）中3小时，以使组织块粘附在烧瓶底部。</li>
		<li>3小时后，加入5mL DMEM高葡萄糖生长培养基以浸没组织块。水平放置 T25 烧瓶。每隔 3 天通过显微镜监测组织块周围的细胞迁移。 注：培养基应含有 20% 胎牛血清、0.2% 白血病抑制因子 （LIF）、0.2 mM β-巯基乙醇和 1% 青霉素/链霉素（参见材料表）。胎牛血清支持细胞增殖，而 LIF 和β-巯基乙醇阻碍细胞分化 7,8。</li>
		<li>当 T25 烧瓶中的细胞达到约 80% 汇合时，将它们传代并培养到一个或两个 T75 烧瓶中。在五次传代中传代培养细胞，以确保正常生长和健康。</li>
	</ol></li>
	<li>常驻CD34+ 干细胞的磁性分离<ol><li>当一个或两个 T75 烧瓶中的细胞达到 80% 汇合时，用胰蛋白酶解离细胞，并将它们重悬于 1 mL 分选缓冲液（2 mM EDTA 和 0.5% FBS）中。确定细胞数（107 / T75烧瓶）并将细胞悬浮液以300× g 离心5分钟（在室温下）。</li>
		<li>完全弃去上清液，并将细胞沉淀重悬于每 107 个总细胞的 98 μL 分选缓冲液中。加入 2 μL CD34 抗体（参见 材料表）。充分混合，在 4°C 下在黑暗中孵育 30 分钟，偶尔摇晃。</li>
		<li>通过加入 2 mL 分选缓冲液洗涤细胞，并在室温下以 300 x g 离心 10 分钟。</li>
		<li>小心完全地取出上清液，并将细胞重悬于 80 μL 分选缓冲液中。</li>
		<li>将 20 μL 抗 FITC MicroBeads（每 107 个细胞，参见 材料表）加入缓冲液中。通过重复移液充分混合，并在4°C下在黑暗中间歇性摇晃孵育15分钟。</li>
		<li>通过加入 2 mL 缓冲液洗涤细胞两次，并以 300 x g 离心 5 分钟以重新沉淀细胞。完全弃去上清液，将细胞重悬于 1 mL 缓冲液中。</li>
		<li>将一根 MS 柱放置在适当分离器的磁场中，确保将收集管（例如，15 mL 离心管）放置在 MS 柱下方以收集分离的组分。（见 材料表）。用 500 μL 缓冲液冲洗色谱柱，并等待其完全流过。</li>
		<li>将细胞悬浮液加载到色谱柱中，并将含有未标记细胞的流出物收集到 15 mL 管中。</li>
		<li>从分离器中取出色谱柱，并将其放在新的 15 mL 离心管上。将 500 μL 缓冲液引入色谱柱中，然后通过将柱塞推入色谱柱，立即冲洗出磁性标记的细胞。</li>
		<li>为了提高CD34+ 细胞的纯度，可以使用第二根MS色谱柱对洗脱的部分进行富集。重复上述分离过程。 通过 流式细胞术评估分选细胞的纯度。 注：通常获得 10%-20% 的 CD34+ 细胞，约 70%-80% 的阴性细胞，以及约 10% 的损失，由于染色和离心程序。</li>
		<li>为了进一步确认血管壁CD34+ 干细胞的纯度，通过流式细胞术鉴定分选的细胞。本研究表明纯度超过90%。</li>
	</ol></li>
</ol>2. 免疫荧光细胞鉴定
<ol><li>在预涂有0.04%明胶的盖玻片（直径8mm）上接种细胞。</li>
	<li>当细胞达到约60%-70%汇合时，用PBS冲洗两次，并用4%多聚甲醛固定细胞10分钟。</li>
	<li>用 PBS 洗涤细胞 3 分钟（3 次），并在室温下用 0.2% Triton X-100（在 PBS 中）透化细胞 5 分钟。</li>
	<li>用 PBS 洗涤细胞 3 分钟（3 次），并用 5% 驴血清（在 PBS 中）阻断抗体的非特异性结合 1 小时。</li>
	<li>用镊子将每个盖玻片在其边缘握住，然后将其排到一张无绒擦拭纸上，以去除阻塞缓冲液。</li>
	<li>将细胞在100倍稀释的一抗（CD34，Flk-1，c-kit，Sca-1，Ki67，参见 材料表）中在1%BSA（PBS中）中在4°C下孵育过夜。</li>
	<li>倒出溶液并用 PBS 洗涤细胞 3 分钟（3 次）。将细胞与 Alexa Fluor 488 标记的二抗（在 1% BSA 中以 1：200 的比例）（参见 材料表）在室温下在黑暗中孵育 1 小时。</li>
	<li>通过将 DAPI 储备溶液在 PBS 中稀释至 0.5 μg/mL 并将细胞孵育 5 分钟来进行 DAPI 复染。</li>
	<li>用PBS冲洗并用抗淬灭试剂密封盖玻片。在激光扫描共聚焦显微镜下捕获图像。</li>
</ol>3. CD34+ VW-SCs的诱导分化和表征
<ol><li>分别以适当的密度（40%-50%汇合度）将CD34 + 干细胞接种在盖玻片和6孔板上。</li>
	<li>通过在内皮细胞培养基 EBM-2 中培养细胞（参见材料表）1 周，诱导 CD34+ 细胞的内皮细胞定向分化。</li>
	<li>通过在成纤维细胞培养基FM-2（参见材料表）中培养细胞3天，诱导成纤维细胞定向从CD34 +细胞分化。</li>
	<li>在488nm的激光激发和40x物镜下，将未分化和分化的细胞进行免疫荧光染色（如步骤2中所述）。检测内皮细胞标志物（CD31，VWF）和成纤维细胞标志物（PDGFRα，波形蛋白）的表达（参见 材料表）。</li>
	<li>用胰蛋白酶解离细胞，并通过离心（300× g，5分钟，室温）获得细胞沉淀。用 100 μL 分选缓冲液重悬细胞。将细胞悬浮液与纯化的抗小鼠CD16 / CD32单克隆抗体（1μg）（参见 材料表）在4°C下预孵育5分钟。</li>
	<li>在小鼠 Fc Block 存在下，将 2 μL PE 大鼠抗小鼠 CD34 （1：50）、CD31 （PECAM-1） 单克隆抗体 APC（2 μL，1：50）和 CD140a （PDGFRa） 单克隆抗体 FITC （2 μL，1：50） 直接加入预孵育细胞中，并在 4 °C 下进一步孵育 15 分钟。</li>
	<li>通过加入 2 mL 缓冲液洗涤细胞两次，并以 300 x g 离心 5 分钟（室温）以重新沉淀细胞。完全弃去上清液，将细胞重悬于 300 μL 缓冲液中。将装有染色细胞的流管放入准备进行流式细胞术的冰箱中。</li>
</ol>4. 血管CD34+干细胞中细胞内Ca2+信号的检测
<ol><li>在实验前10小时将细胞接种在盖玻片上，密度高达70%汇合。</li>
	<li>取出盖玻片，用任何凡士林将它们固定在定制室的底部。</li>
	<li>用PBS洗涤盖玻片一次，在Tyrode溶液（NaCl 137mM，KCl 5.4mM，MgCl2 1.2mM，葡萄糖10mM，HEPES 10mM，CaCl2 2.4mM）中用Fura-2 / AM（5μM）替换（参见 材料表），并在黑暗中孵育细胞30分钟。</li>
	<li>用Tyrode溶液处理10分钟以去除染料，以允许fura-2 / AM去酯化。</li>
	<li>将腔室安装在配备有包含单色仪和CCD相机的宽场成像系统的倒置荧光显微镜的载物台上（参见 材料表）。</li>
	<li>打开 主窗口，进入 “抓取设置 ”对话框和相机页面，然后按 实时 按钮进行 实时 图像显示。</li>
	<li>选择荧光图像类型，将 340 nm/380 nm 循环重复时间、图像尺寸和相机曝光时间设置为 380 nm 和 340 nm，以实现最佳图像亮度。</li>
	<li>拍摄单个快照，并绘制几条手绘线，用不同的颜色圈出单元格，并预定义感兴趣区域 （ROI），将背景指示为 ROI 0。</li>
	<li>在工作区视图中重新编辑已嵌入的协议，并创建新的工作区。</li>
	<li>执行协议，“Fluorescence 340 nm div Fluorescence 380 nm （Live Kinetic）”将显示在线动力学图。</li>
	<li>在数值视图中，将看到一个电子表格，显示灰度值列和相应的时间信息。使用剪贴板导出电子表格数据。</li>
	<li>计算 F340 nm/F380 nm，表明减去背景荧光 （ROI 0） 后细胞内 Ca2+ 变化。</li>
</ol>

从血管壁分离和磁性分离小鼠 CD34+ 干细胞

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A., 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=Angiotensin+II,+ATP+and+high+extracellular+potassium+induced+intracellular+calcium+responses+in+primary+rat+brain+endothelial+cell+cultures.">Angiotensin II, ATP and high extracellular potassium induced intracellular calcium responses in primary rat brain endothelial cell cultures.</a> Cell Biochem Funct. 39 (5), 688-698 (2021).</li><li>Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+as+a+tool+to+investigate+sarcoplasmic+reticulum+and+intracellular+calcium+dynamics+in+human+skeletal+muscles.">Caffeine as a tool to investigate sarcoplasmic reticulum and intracellular calcium dynamics in human skeletal muscles.</a> J Muscle Res Cell Motil. 42 (2), 281-289 (2021).</li></ol>

作者没有要披露的利益冲突。

本研究提供了一种从小鼠主动脉和肠系膜动脉中获取功能性CD34+ VW-SCs的快速便捷的方法。该方法得到的CD34+ VW-SCs具有增殖活性和多向分化特性。三磷酸肌醇 1,4,5-三磷酸受体 （IP3Rs）、兰尼定受体 （RyRs） 和储存操作的钙通道介导 Ca2+ 释放和进入 CD34+ VW-SCs。该技术的建立将为进一步研究CD34+ VW-SCs参与心血管疾病结构和功能重塑的机制奠定基础。
...

血管壁在血管发育、稳态调节和血管疾病的进展中起着关键作用。近年来，许多研究揭示了动脉中存在各种干细胞谱系。2004年，徐清波教授课题组首次报道了成体血管壁外围存在血管干/祖细胞，表达CD34、Sca-1、c-kit和Flk-11。这些血管干细胞表现出多向分化和增殖潜力。在正常情况下，它们保持相对静止;然而，当被特定因素激活时，它们可以分化成平滑肌细胞、内皮细胞和成纤维细胞。或者，它们可以通过旁分泌效应调节血管周围基质和微血管的形成，以促进受伤血管的修复或重塑 2,3,4,5,6。最近，发现血管壁中的常驻CD34+干细胞在股动脉导丝损伤后的内皮细胞再生中起作用2。因此，CD34+ VW-SCs的分离和定量以及CD34+干细胞基本生物学特性的检验对于进一步研究CD34+ VW-SCs调控过程中的信号通路至关重要。
目前有多种细胞分离方法可用，包括基于细胞培养特性或细胞物理特性的技术，例如密度梯度离心法，该方法导致分选的细胞含有许多非靶细胞和相对较低的纯度7,8,9,10,11,12 .另一种常用的技术是荧光/磁性辅助细胞分选。荧光激活细胞分选 （FACS） 是一个技术要求高的复杂系统，它相对昂贵、耗时，并且可能会影响分选细胞的活性13,14。然而，磁激活细胞分选（MACS）更高效、更方便，具有高回收率和细胞活性，对下游应用的影响较小8.因此，在该方案中，我们应用MACS纯化CD34 + VW-SCs，并通过流式细胞术进一步鉴定细胞。建立基于MACS的分离方法用于研究血管壁干细胞将是非常宝贵的。首先，它允许实验性遗传和细胞生物学研究。其次，通过对血管壁驻留干细胞进行高效分离和培养，可以系统地评估和筛选调节干细胞功能的信号因子。第三，鉴定干细胞中的关键表型为评估细胞状态提供了重要的“质量控制”。因此，鉴定纯化方法可能可用于与源自血管的类似干细胞的类似应用。
本报告详细演示了一种稳定可靠的 CD34+ VW-SC 培养方法，包括通过流式细胞术、免疫荧光染色和 Ca2+ 信号转导测量进行的细胞鉴定和功能评估。本研究为进一步深入研究CD34+ VW-SCs在生理和病理条件下的功能及其调控机制提供了基础。

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immunomagnetic isolation of the vascular wall-resident cd34+ stem cells from mice

驻留 CD34+ 血管壁驻留干细胞和祖细胞 （VW-SCs） 因其在调节血管损伤和修复中的关键作用而日益得到认可。建立一种稳定有效的方法培养功能性小鼠CD34+ VW-SCs对于进一步研究这些细胞在各种生理和病理条件下的增殖、迁移和分化机制至关重要。所述方法结合磁珠筛选和流式细胞术纯化原代培养的驻留 CD34+ VW-SCs。然后通过免疫荧光染色和 Ca2+ 成像对纯化的细胞进行功能鉴定。简而言之，来自鼠主动脉和肠系膜动脉外膜的血管细胞是通过组织阻滞附着获得的，然后进行传代培养，直到达到至少 1 × 107 的细胞计数。随后，使用磁珠分选和流式细胞术纯化CD34 + VW-SCs。CD34+ VW-SCs 的鉴定涉及细胞免疫荧光染色，而功能多能性是通过将细胞暴露于特定培养基进行定向分化来确定的。此外，在暴露于 ATP、咖啡因或 thapsigargin （TG） 的 Fura-2/AM 负载细胞中，使用商用成像工作站评估功能性内部 Ca2+ 释放和外部 Ca2+ 进入。该方法为分离、培养和鉴定血管壁驻留CD34+ 干细胞提供了一种稳定高效的技术，为VW-SCs调控机制的 体外 研究和靶向药物的筛选提供了机会。

The vascular wall plays a pivotal role in vascular development, homeostatic regulation, and the progression of vascular diseases. In recent years, numerous studies have unveiled the presence of various stem cell lineages in arteries. In 2004, Professor Qingbo Xu&#39;s group first reported the existence of vascular stem/progenitor cells in the periphery of the adult vascular wall, expressing CD34, Sca-1, c-kit, and Flk-11. These vascular stem cells exhibit multidirectional differentiation and proliferation potential. Under normal conditions, they remain relatively quiescent; however, when activated by specific factors, they can differentiate into smooth muscle cells, endothelial cells, and fibroblasts. Alternatively, they can regulate the perivascular matrix and microvessel formation through paracrine effects to promote the repair or remodeling of injured vessels2,3,4,5,6. Recently, resident CD34+ stem cells in the vascular wall were found to play a role in endothelial cell regeneration after femoral artery guidewire injury2. Consequently, the isolation and quantification of CD34+ VW-SCs and the examination of the basic biological characteristics of CD34+ stem cells are crucial for further studying the signal pathways involved in the regulation of CD34+ VW-SCs.
Various methods for cell separation are currently available, including techniques based on cell culture characteristics or physical properties of cells such as density gradient centrifugation, which results in sorted cells containing many non-target cells and relatively low purity7,8,9,10,11,12. Another commonly used technique is fluorescence/magnetic-assisted cell sorting. Fluorescence-activated cell sorting (FACS) is a complex system with high technical requirements, and it is relatively expensive, time-consuming, and potentially affects the activity of sorted cells13,14. However, magnetic-activated cell sorting (MACS) is more efficient and convenient, with a high recovery rate and cell activity and less impact on downstream applications8. Therefore, in this protocol, we applied MACS to purify CD34+ VW-SCs and further identified the cells by flow cytometry. The establishment of MACS-based isolation methods for studying vascular wall stem cells would be invaluable. Firstly, it permits experimental genetic and cell biological studies. Secondly, efficient isolation and culture of vascular wall resident stem cells allow systematic assessment and screening of signaling factors regulating stem cell functions. Thirdly, identification of crucial phenotypes in stem cells provides important &#39;quality control&#39; in assessing cell status. Thus, identifying methods to purify could be useful for similar applications to analogous stem cells derived from vessels.
This report provides a detailed demonstration of a stable and reliable method for the culture of CD34+ VW-SCs, including cell identification and functional assessment performed by flow cytometry, immunofluorescence staining, and Ca2+ signaling measurement. This study provides a basis for further in-depth research on the function of CD34+ VW-SCs and their regulatory mechanisms in physiological and pathological conditions.

作者聚焦：使用磁珠筛选偶联流式细胞术增强血管壁驻留小鼠 CD34+ 干细胞的分离和表征

从小鼠中免疫磁性分离血管壁驻留的 CD34+ 干细胞

Resident vascular wall CD34+stem cells are increasingly recognized for their crucial role in regulating vascular remodeling post-injury. Establishing a stable and efficient method to culture functional CD34+cells is essential for further investigating the participating mechanisms under various physiological and pathological conditions. This technique combines magnetic bead screening and flow cytometry to purify the primary cultured resident CD34+stem cells.Then, the purified cells can be functionally identified through immunofluorescent staining and calcium imaging. Due to the phenotypical and functional heterogeneity of resident CD34+stem cells in the vascular nerve wall, it will be necessary to identify the specific subcellular type of resident vascular CD34+cells. We present a refined methodology for the culture, identification, and the functional assessment of vascular wall-resident CD34+stem cells.This novel approach encompasses magnetic sorting, flow cytometry, immunofluorescence staining, and calcium signaling measurement. By employing these techniques, our study establishes a solid foundation for the future comprehensive investigations into the function under regulation of recipient CD34+stem cells under both physiological and pathological conditions.

CD34+ VW-SCs的分离纯化 CD34+ VW-SCs的高纯度是通过组织附着和磁性微珠分选从小鼠主动脉和肠系膜动脉的外膜中获得的。CD34+ 细胞在血管壁中的百分比一般为10%-30%。流式细胞术证实，通过磁珠分选获得的CD34+ 细胞的纯度超过90%（图1A）。细胞免疫荧光染色显示，CD34+ VW-SCs主要表达CD34、c-kit、Flk-1和Ki-67，Sca-1的表达相对较低...

Watch this Scientific Journal Video about Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice at JoVE.com

本研究为血管壁驻留CD34+ 干细胞（CD34+ VW-SCs）的分离、培养和功能测定建立了一种稳定高效的方法。这种易于遵循且省时的分离方法可以被其他研究人员用来研究心血管疾病涉及的潜在机制。

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

常驻血管壁CD34+干细胞因其在调节损伤后血管重塑中的关键作用而日益得到认可。建立一种稳定、高效的功能性CD34+细胞培养方法对于进一步研究不同生理和病理条件下的参与机制至关重要。该技术结合了磁珠筛选和流式细胞术，以纯化原代培养的常驻 CD34+ 干细胞。然后，可以通过免疫荧光染色和钙成像对纯化的细胞进行功能鉴定。由于留留血管神经壁CD34+干细胞的表型性和功能异质性，因此有必要鉴定留留血管CD34+细胞的特定亚细胞类型。我们提出了一种用于血管壁驻留 CD34+ 干细胞的培养、鉴定和功能评估的改进方法。这种新方法包括磁性分选、流式细胞术、免疫荧光染色和钙信号转导测量。通过采用这些技术，我们的研究为未来全面研究受体CD34+干细胞在生理和病理条件下的调控功能奠定了坚实的基础。

immunomagnetic-isolation-vascular-wall-resident-cd34-stem-cells-from

Research

JoVE Journal

Medicine

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

413 次观看.  Southwest Medical University. 常驻血管壁CD34+干细胞因其在调节损伤后血管重塑中的关键作用而日益得到认可。建立一种稳定、高效的功能性CD34+细胞培养方法对于进一步研究不同生理和病理条件下的参与机制至关重要。该技术结合了磁珠筛选和流式细胞术，以纯化原代培养的常驻 CD34+ 干细胞。然后，可以通过免疫荧光染色和钙成像对纯化的细胞进行功能鉴定。由于留留血管神经壁CD34+干细胞的表型...

视频: 作者聚焦：使用磁珠筛选偶联流式细胞术增强血管壁驻留小鼠 CD34+ 干细胞的分离和表征

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

科研

医学

Murine Aortal and Mesenteric Arteries Isolation and Culture to Separate CD34+ Stem Cells for Cardiovascular Studies

To begin, place an anesthetized mouse on the operating table. Using sterile ophthalmic scissors and forceps, open the thoracic and abdominal cavities. This will expose the heart and the intestines.Carefully dissect the mesenteric arteries and the aorta separately. Rinse the mesenteric arteries in PBS and transfer them to a 10-centimeter Petri dish containing 1%penicillin-streptomycin-amphotericin B solution. Similarly, rinse the aorta and transfer it to another Petri dish containing the same solution.Quickly remove all the fat around the aorta and the mesenteric arteries. After rinsing twice, place the arteries under a stereo microscope in a tissue culture hood. Carefully make a longitudinal cut along the arteries.Then using fine forceps, peel off the outer layer of the aorta and the first branch of the mesenteric arteries. Place the outer layer in a 3.5 centimeter Petri dish containing 0.1 to 0.2 milliliters of culture medium, and chop it into tiny pieces of about one cubic millimeter. Transfer the tissue pieces into a gelatin-coated T-25 flask.Place the flask vertically in an incubator at 37 degree Celsius with 5%carbon dioxide. After three hours, add five milliliters of DMEM high glucose growth medium to the flask to submerge all the tissue blocks.

小鼠主动脉和肠系膜动脉分离和培养以分离 CD34+ 干细胞用于心血管研究

Magnetic Separation of Vascular Wall-Resident CD34+ Stem Cells Isolated from Murine Aortal and Mesenteric Arteries for Cardiovascular Studies

To begin, isolate and culture the outer layer of the aorta and the first branch of the mesenteric arteries isolated from the anesthetized mouse. Once the cells become 80%confluent, centrifuge the dissociated cell suspension at 300G for five minutes at room temperature, discard the supernatant completely and resuspend the cell pellet in 98 microliters of sorting buffer per 10 million total cells. After the addition of CD34 antibody, incubate the cells in the dark for 30 minutes at four degrees Celsius.To wash the cells, add two milliliters of sorting buffer to the cells, and centrifuge at 300G for 10 minutes. After discarding the supernatant, resuspend the cells in 80 microliters of sorting buffer. Then add 20 microliters of Anti-FITC MicroBeads to the cell suspension.After washing and centrifuging the cells as demonstrated previously, resuspend the cells in one milliliter of sorting buffer. Next, position a magnetic separation column within the magnetic field of a separator, and place a collection tube beneath the column. After rinsing with 500 microliters of buffer, load the cell suspension into the column and collect the flow through containing unlabeled cells in the collection tube.Remove the column from the separator and place it on a fresh 15 milliliter centrifuge tube. Introduce 500 microliters of sorting buffer into the column and push the plunger into the column to flush out the magnetically labeled cells.

从小鼠主动脉和肠系膜动脉中分离的血管壁驻留 CD34+ 干细胞的磁性分离用于心血管研究

Characterization of the Magnetically Isolated CD34+ Stem Cells for Cardiovascular Studies

To characterize the CD34+stem cells isolated from the murine aorta and mesenteric arteries, perform tissue block culture and obtain the cells by magnetic separation. To detect the endothelial cell in fibroblast cell marker expression after differentiation, observe the cells at 40x magnification with laser excitations of 488 nanometers. After trypsinizing the cells, pellet them by centrifugation and resuspend in 100 microliters of sorting buffer.Then incubate them with anti-mouse CD16/CD32 monoclonal antibodies at four degrees Celsius for five minutes. Then add two microliters of each desired monoclonal antibody for immunofluorescent staining and incubate again at 4 degrees Celsius for 10 minutes. Wash the cells twice with one milliliter of buffer.After centrifuging the cells as demonstrated, remove the supernatant and resuspend the cells in 300 microliters of buffer. Transfer the cells to the flow tubes and place them in an ice box. Run flow cytometry to check the percentage of differentiated cells.Flow cytometric evaluation confirmed the high purity of isolated CD34+vascular wall stem cells. Cellular immunofluorescent staining showed that the isolated CD34+stem cells predominantly expressed stem cell markers, CD34, c-kit, and Flk-1. Flow cytometry also confirmed that the isolated CD34+stem cells possessed the ability to differentiate into endothelial cells as well as fibroblasts in vitro.

用于心血管研究的磁性分离 CD34+ 干细胞的表征

Detection of Intracellular Calcium Signaling in the Magnetically Isolated Vascular Wall CD34+ Stem Cells

To begin, isolate the resident vascular wall CD34-positive stem cells from murine models. Obtain pure cells by magnetic activated cell sorting and characterize them. For detecting calcium signaling, obtain a coverslip pre-seeded with the cells and wash it once with PBS.Then incubate the cells with Fura 2-AM and Tyrode solution for 30 minutes in the dark. To remove the dye, incubate the cells with Tyrode solution for 10 minutes. Then mount the chamber on the stage of an inverted fluorescence microscope, open the main window and go to the grab settings dialogue and camera page.Press the live button for the live image display. To acquire images, draw several freehand lines to circle the cells with different colors and predefine the regions of interest. Fluorescence microscopy demonstrated that ATP administration increased the intracellular calcium levels in the isolated vascular wall CD34-positive stem cells.