这项工作得到了加拿大卫生研究院 (148808 和 148792) 的支持, SE 得到了心脏和中风基金会的支持, 来自安大略省级办事处的人员奖, 3月的硬币, 泰德罗杰斯心脏研究中心和彼得蒙克。心脏中心。XCC 持有卫生研究院班廷奖学金。LA 举行心脏 & 中风/理查德 Lewar 奖学金奖。

伦理声明: 本议定书已由大学卫生网 (加拿大多伦多) 动物保育委员会审查和批准, 并符合加拿大动物保育理事会的规定。
1. 缓冲准备
<ol><li>准备 HBB 缓冲液 (汉克的平衡盐溶液 (HBSS), 2% 热灭活牛血清, 0.2% 牛血清白蛋白)。添加10毫升的热灭活牛血清和1克牛血清白蛋白到500毫升的 HBSS。过滤器消毒通过0.2 µm 过滤器和存储在4摄氏度。</li>
	<li>制备血液学缓冲剂 (磷酸缓冲盐水 (PBS), 2% 热灭活牛血清, 1 毫米乙二胺乙酸 (EDTA, pH 值 8.0))。添加10毫升热灭活牛血清和1毫升0.5 米 EDTA 到500毫升的 PBS。过滤器消毒通过0.2 µm 过滤器和存储在4摄氏度。 注: 解决方案可存储在摄氏4摄氏度, 长达1月。</li>
</ol>2. 循环白细胞的标签
<ol><li>灌装制备抗小鼠 CD45 注射液, 1 µg 的抗体稀释在无菌磷酸盐缓冲盐水 (PBS) 中, 最终注射量为200µL/小鼠。将体积装入28.5G 胰岛素注射器中, 远离光线。 注: 使用两种不同的 fluorochrome 标记的 anti-CD45 抗体, 以区分血管内 (染色通过静脉注射管理) 从心内染色 (染色后消化; 见步骤 4)。</li>
	<li>
预热小鼠 (8–12周), 通过将尾部暴露在一盏红色的热灯上5分钟。 注意: 不要使动物过热。把灯保持在安全的距离 (> 30 厘米)。<ol>
<li>使用适当的设备来抑制胸骨位置上的动物。用拇指和无名指将尾部的底部与食指、中指和尾部的中远端区域固定, 以非显性手暴露和稳定尾部。 注意: 不要在尾部的尖端施加太多的力, 因为皮肤可以被拔掉。插入针与斜面朝上的静脉。始终保持针平行的静脉在任何时候, 因为它是容易穿刺静脉。 注: 血液进入注射器可能会发生, 是一个良好的注射迹象。将针弯曲到90°角度可以促进静脉的管理。与您的安全办公室确认, 弯曲的针头可能代表危险。</li>
		<li>静脉注射200µL 静脉入尾脉。液体应该容易地流动并且临时地清除静脉。 注意: 如果遇到阻力, 不要强迫流体。这是针错位的迹象。</li>
		<li>允许抗体在人道 euthanizing 老鼠之前循环5分钟。</li>
	</ol>
</li>
</ol>3. 心脏隔绝和消化
<ol><li>
弄死小鼠使用 CO2或由其他体制上接受的安乐死做法。
	<ol>
<li>对于共2安乐死, 将鼠标放置在 co2腔内, 并将填充速率设置为每分钟室容积的 20% (即,如果分庭为10升, 则填写2升/分钟)。运行2分钟和监控鼠标, 直到呼吸停止。</li>
		<li>关闭 CO2 , 并将鼠标留在另外的1-2 分钟内. 确保在打开房间前停止呼吸, 并在开始处理鼠标前进行脚趾捏以确认鼠标已死。</li>
	</ol>
</li>
	<li>用70% 乙醇喷洒老鼠。抬起腹部的皮肤, 并解剖鼠标沿腹中线开始在臀部的水平到胸骨。打开腹部并移动肝脏以暴露隔膜。</li>
	<li>
切开隔膜, 然后在中线两侧的肋骨, 小心避免刺穿肺部和心脏。
	<ol>
<li>通过剥离肋骨来暴露心脏, 然后先切开左心房, 然后右心房。 注意: 在切开心房时要小心, 确保没有动脉或静脉受伤, 因为这样可以降低器官中的血液冲洗效率。</li>
		<li>灌注的心脏与15–20毫升的冷 pbs 使用60毫升注射器装有21克针和冷 pbs。用镊子轻轻地抓住心脏的底座, 将针插入靠近顶端的左心室。在右心室重复这个过程。适当的注射会导致白肺和苍白的肝脏。 注: 应在安乐死后5分钟内进行灌注, 以避免血液凝固, 这可能会污染器官与循环细胞的利益。</li>
	</ol>
</li>
	<li>
用镊子握住心脏的底座, 轻轻地拉起心脏, 然后通过切断主要的肺动脉和静脉、主动脉和心包袋来分离心脏。
	<ol>
<li>用干净的无绒纸巾轻轻地握住拇指和食指之间的心脏, 用镊子将主要动脉和静脉的心房和残余物拉出来, 将心脏清洁。 注: 确保心房全部被移除, 没有其他组织, 如肺或血管, 因为这些组织有自己的免疫细胞。特别是肺有一个大的免疫细胞的人口, 可以掩盖的小群体的人在心肌。</li>
	</ol>
</li>
	<li>
把心脏放在一个5厘米的塑料盘子里, 用50µL 的冷 PBS。
	<ol>
<li>用弯曲的解剖剪刀切碎心脏, 直到没有可见的碎片, 它有平滑的一致性。 注意: 在切的过程中尽量保持心脏在盘子的一个包含区域, 以减少组织损失。</li>
		<li>用弯曲钳将 thetrituratedheart 组织移植到2毫升离心管中, 800 µL 冷 Dulbecco 的改性鹰培养基 (DMEM)。</li>
	</ol>
</li>
	<li>
添加 450 u/毫升 ofcollagenase i, 60 u/毫升的透明质酸酶类型的 i S, 和 60 U/毫升的 DNase i 对每个样品。
	<ol>
<li>在37摄氏度的摇摆振动筛上孵化样品, 在 50 rpm 45–60分钟。</li>
	</ol>
</li>
	<li>
在消化之后, 简要地漩涡样品 (二十年代) 和立刻放置在冰保持样品在4°c。
	<ol>
<li>使用1毫升 pipettor, 吸管的样品向上和向下, 直到组织样本是均匀的, 并没有堵塞的吸管尖端。 注: 定义特定次数的吸管可能会增加重现性。如果心脏没有得到最佳的消化, 部分心肌可能会阻碍吸管。轻轻研磨的吸管尖端对离心管将有助于融汇更大的组织。</li>
	</ol>
</li>
	<li>
预湿40µm 细胞过滤器放置在50毫升圆锥管与1毫升的冷 HBB。
	<ol>
<li>将匀质样品添加到过滤器中, 通过缓慢浇注12–14毫升的冷 HBB 来洗涤。</li>
		<li>将经过过滤的样品转移到标签为15毫升的锥形管上, 保持在4摄氏度。</li>
	</ol>
</li>
	<li>
离心机样品在 400 x g 5 分钟在4摄氏度。吸出上清。
	<ol>
<li>溶解红细胞通过增加1毫升的氯化铵-钾 (ACK) 裂解缓冲到每个样品。并用重悬样品涡流, 室温孵育5分钟。</li>
		<li>一旦溶解完成, 添加5–8毫升的冷汉克的平衡盐溶液 (HBSS), 以防止溶血。</li>
	</ol>
</li>
	<li>离心机样品在 400 x gfor 5 分钟在4°c。吸出上清。</li>
	<li>在每个样品中添加1毫升的流化器缓冲器, 并将其转移到标签为1.5 毫升的离心管。 注意: 该协议可以在这里暂停多达2小时, 样品存储在4摄氏度。</li>
</ol>4. 流式细胞仪染色及分析
<ol><li>
离心机样品在 400 x g 5 分钟在4摄氏度。
	<ol>
<li>为了减少非特异性抗体的结合, 稀释 1:100 anti-CD16/CD32 和单核细胞阻滞剂 (1:20) (见材料表) 核算每样的50µL。室温孵育15分钟。</li>
		<li>准备抗体稀释 (每种抗体的 1:250) 核算每个样品的50µL。 注: 可将活性染料纳入抗体鸡尾酒中, 以排除死细胞。或者, 可以使用流分析的小尺寸排除 (图 1C和D)。</li>
		<li>没有洗涤, 添加50µL 抗体主混合到每个样本和混合。</li>
		<li>在黑暗中孵化4摄氏度的样品, 30 分钟。</li>
	</ol>
</li>
	<li>在每样样品上加600µL, 以 400 x克为5分钟, 在4摄氏度处冲洗和离心样品。</li>
	<li>在300µL 中吸取上清和并用重悬颗粒。在黑暗中保持样品在4摄氏度。 注: 一些活性染料, 如 DAPI, 需要在运行流式细胞术前10分钟内染色。</li>
	<li>通过流式细胞术 (见图1和 2) 在未来3小时内进行分析。如果需要较长的时间, 建议使用低浓度多聚甲醛 (PBS 0.5–1%) 进行短期固定, 以保持染色。 注: 强烈建议在启动流式细胞仪分析前, 通过40µm 细胞过滤器筛选样品, 以防止射流技术的流量。</li>
</ol>

<ol><li>Marboe, C. C., Fenoglio, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathology+and+natural+history+of+human+myocarditis%5BTitle%5D)+AND+%22Pathology+and+Immunopathology+Research%22%5BJournal%5D)">Pathology and natural history of human myocarditis.</a> Pathology and Immunopathology Research. 7 (4), 226-239 (1988).</li>
<li>Epelman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+and+adult-derived+resident+cardiac+macrophages+are+maintained+through+distinct+mechanisms+at+steady+state+and+during+inflammation%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.</a> Immunity. 40 (1), 91-104 (2014).</li>
<li>Pinto, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+abundant+tissue+macrophage+population+in+the+adult+murine+heart+with+a+distinct+alternatively-activated+macrophage+profile%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.</a> PLoS One. 7 (5), 36814(2012).</li>
<li>Lavine, K. J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/25349429">Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16029-16034 (2014).</li>
<li>Fujiu, K., Wang, J., Nagai, R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/24675722 ">Cardioprotective function of cardiac macrophages.</a> Cardiovascular Research. 102 (2), 232-239 (2014).</li>
<li>Hulsmans, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophages+Facilitate+Electrical+Conduction+in+the+Heart%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Macrophages Facilitate Electrical Conduction in the Heart.</a> Cell. 169 (3), 510-522 (2017).</li>
<li>Nahrendorf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+healing+myocardium+sequentially+mobilizes+two+monocyte+subsets+with+divergent+and+complementary+functions%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.</a> Journal of Experimental Medicine. 204 (12), 3037-3047 (2007).</li>
<li>Clemente-Casares, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+CD103%28%2B%29+Conventional+Dendritic+Cell+Surveillance+System+Prevents+Development+of+Overt+Heart+Failure+during+Subclinical+Viral+Myocarditis%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">A CD103(+) Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis.</a> Immunity. 47 (5), 974-989 (2017).</li>
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</ol>

作者没有什么可透露的。

心肌炎症, 或心肌炎, 是大多数心血管疾病的特征。然而, 在非疾病状态下, 心肌并不缺乏自身的免疫成分。在稳态状态下, 许多免疫细胞驻留在心肌中, 发挥着维护和保护的重要作用。如果没有本议定书所提出的方法, 就不可能对这些不同的细胞群体进行描述。
一种结合的机械和酶消化的心肌允许隔离单个细胞悬浮, 可用于结合其他分析方法, 如流式细胞术或磁珠隔离。必须考?...

不同形式的心肌应激或损伤, 包括缺血性 (缺血再灌注或心肌梗死) 和非缺血性 (高血压或心肌炎), 促进炎症细胞的招募与修复和保护, 但也致病性。早在 1891, 龙贝格首先描述了感染斑疹伤寒和猩红热1患者心肌细胞浸润的存在。然而, 对心脏免疫细胞的详细研究需要发展更先进的免疫分型技术。因此, 直到最近我们才开始了解到, 在稳定状态下, 具有重要维护作用的免疫细胞的不同种群驻留在心肌内。
组织学一直是, 而且仍然是最常用的方法来表征心脏免疫细胞在炎症。然而, 虽然组织学是一个有价值的诊断工具, 它在心脏免疫细胞研究中的应用有重要的局限性。驻留在心肌中的免疫细胞占总细胞的很小比例, 或多或少分布在一个比例巨大的空间中。同样, 几种形式的心脏炎症, 如病毒性心肌炎, 显示炎症的焦点模式。这意味着对心脏的免疫系统的准确分析往往需要更高的组织学取样, 增加成本, 以减少偏见。此外, 组织学在细胞识别中提供了非常有限的可用信息 (如参数数目)。随着越来越多的细胞子集需要使用多个表达标记 (包括表面标记, 转录因子或分泌分子), 流式细胞术已经建立了自己作为最强大的工具, 免疫分型,由于低成本和高吞吐量。
免疫分型技术的使用是密切依赖于开发高效的消化协议, 允许对大量细胞的单细胞分析。细胞提取的穷尽性协议的发展为心脏免疫学研究开辟了新的机遇之窗。通过消化、流式细胞术和转录组学的结合, 在心脏中的巨噬细胞和树突状体细胞的主要种群特征为2、3。最丰富的心脏免疫细胞在稳定状态是 CD64+MerTK+巨噬细胞, 可进一步分裂的基础上, 其表达 CCR2 或 CD11c2。CCR2+巨噬细胞起源于成人骨髓造血, 而大多数 CCR2 巨噬细胞, 可以表达高或低的 MHC II, 主要是产前起源。巨噬细胞执行关键功能, 如修复4和清除碎片5在伤害或支持电连接6稳定状态。在不同的炎症形式期间 Ly6C 的一个重要汇集 CD64int单核细胞发生, 以后区分成 Ly6Chi CCR2+巨噬细胞2,7.一个明显较小, 但重要的是, 居住在健康个体的心肌细胞的数量是由树突状细胞8,9组成。心脏常规 DCs (招揽) 的两个主要子集最近的特点是: cDC1 (CD103+ dcs) 和 cDC2 (CD11b+ dcs)。DCs 在抵御感染方面起着重要作用8 , 但也可以促进炎症期间的自我伤害, 特别是在心肌梗塞9期间。
在这里, 我们描述了一个简单的方法来隔离的存活的心脏免疫细胞的小鼠心肌。该方法结合酶和机械消化与细胞过滤过滤, 以获得一个单一的细胞悬浮, 可以分析, 或进一步纯化, 通过流式细胞仪分类或磁珠浓缩。准确测量外心肌细胞需要心脏灌注, 以消除可能的污染物从血液中的心脏微血管。此外, 我们提出了一个可选的步骤, 免疫细胞的血管内标记, 可用于进一步区分心肌细胞从血管内污染物, 根据 Galkina等10号议定书。最后, 我们提出了一个基本的流式细胞分析, 以确定主要巨噬细胞和 cDC 亚人口。

<table><tbody><tr><td>Phosphate buffered saline</td><td>Wisent</td><td>311-010-CL</td><td>1x PBS</td></tr><tr><td>21 G x 1 1/2 (0.8 mm x 40 mm) PrecisionGlide Needle</td><td>BD</td><td>305167</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Wisent</td><td>311-511-CL</td><td>1x HBSS</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A4503-50G</td><td></td></tr><tr><td>Bovine serum</td><td>Sigma</td><td>B9433</td><td></td></tr><tr><td>0.5 M Ethylenediaminetetraacetic acid</td><td>BioShop</td><td>EDT111</td><td></td></tr><tr><td>Vacuum filter , 0.2 &amp;micro;m Filtropur V50</td><td>Sarstedt</td><td>83.1823.001</td><td></td></tr><tr><td>28 G 1/2 1 cc insulin syringe</td><td>BD</td><td>329424</td><td></td></tr><tr><td>60 mL syringe</td><td>BD</td><td>309653</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Wisent</td><td>319-005-CL</td><td>1x DMEM with 4.5 g/L glucose and L-Glutamine and Sodium Pyruvate</td></tr><tr><td>Collagenase I</td><td>Sigma</td><td>C0130</td><td>from Clostridium histolyticum</td></tr><tr><td>Hyaluronidase type I-S</td><td>Sigma</td><td>H3506</td><td></td></tr><tr><td>DNase-I</td><td>Sigma</td><td>D4513</td><td>from bovine pancreas</td></tr><tr><td>Cell strainer, 40 &amp;micro;m Nylon</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Ammonium-Chloride- Potassium (ACK) lysis buffer</td><td>Lonza</td><td>10-546E</td><td></td></tr><tr><td>Alexa Fluor 700 anti-mouse/human CD11b</td><td>Biolegend</td><td>101222</td><td>1:250 dilution</td></tr><tr><td>APC/Cy7 anti-mouse Ly-6c</td><td>Biolegend</td><td>128025</td><td>1:250 dilution</td></tr><tr><td>APC anti-mouse CD103</td><td>Biolegend</td><td>121414</td><td>1:250 dilution</td></tr><tr><td>Brilliant Violet 605 anti-mouse CD11c</td><td>Biolegend</td><td>117334</td><td>1:250 dilution</td></tr><tr><td>PE anti-mouse Ly-6G</td><td>Biolegend</td><td>127607</td><td>1:250 dilution</td></tr><tr><td>Pacific Blue anti-mouse I-Ab</td><td>Biolegend</td><td>116422</td><td>1:250 dilution</td></tr><tr><td>FITC anti-mouse CD64 (FcgRI)</td><td>Biolegend</td><td>139315</td><td>1:250 dilution</td></tr><tr><td>PE/Cy7 anti-mouse CD45</td><td>Biolegend</td><td>103113</td><td>1:250 dilution</td></tr><tr><td>PerCP/Cy5.5 anti-mouse CD45</td><td>Biolegend</td><td>103132</td><td>1:40 dilution</td></tr><tr><td>TruStain fcX (anti-mouse CD16/32)</td><td>Biolegend</td><td>101320</td><td>1:100 dilution</td></tr><tr><td>True-Stain Monocyte Blocker</td><td>Biolegend</td><td>426101</td><td>1:20 dilution</td></tr><tr><td>FlowJo V10</td><td>TreeStar Inc</td><td></td><td>https://www.flowjo.com/solutions/flowjo</td></tr><tr><td>Mouse: Batf3-/-</td><td>The Jackson Laboratory</td><td>JAX: 013755</td><td></td></tr></tbody></table>

isolation and identification of extravascular immune cells of the heart

免疫系统是健康心脏的重要组成部分。心肌是一个丰富的不同免疫细胞亚群的家, 在稳定状态和不同形式的炎症期间功能划分。直到最近, 对心脏免疫细胞的研究需要使用显微镜或发育不良的消化协议, 这在严重炎症期间提供了足够的敏感性, 但无法自信地识别小但关键人群的稳定状态下的细胞。在这里, 我们讨论了一个简单的方法, 结合酶 (胶原酶, 透明质酸和 DNAse) 和机械消化的小鼠心脏之前的血管内管理荧光标记的抗体, 以区别较小但不可避免的血管内的细胞污染物。该方法通过流式细胞仪对细胞进行鉴定、分型和定量, 或通过荧光活化细胞分选或磁珠分离进一步纯化, 从而产生分离的活体细胞悬浮。转录分析或体外研究。我们包括一个分步流细胞分析的例子, 以区分关键的巨噬细胞和树突状体细胞群体的心脏。为中等大小实验 (10 心脏) 过程的完成要求 2–3 h。

到目前为止, 还没有一个好的方法可以隔离其他心脏细胞成分, 如心肌细胞。因此, 流式细胞术对心脏单细胞悬浮液的分析需要用 CD45 预浇来识别免疫细胞的数量, 其次是单细胞和小尺寸排斥门 (图 1a)。或者, 可以执行生存性染色, 以排除死细胞。小尺寸排除和活性染料染色表示几乎等同于排除死细胞的方法 (图 1<strong...

Watch this Scientific Journal Video about Isolation and Identification of Extravascular Immune Cells of the Heart at JoVE.com

Isolation and Identification of Extravascular Immune Cells of the Heart

心脏外免疫细胞的分离与鉴定

该协议提出了一种简便、有效的方法, 用于分离、鉴定和量化在小鼠心肌中的免疫细胞在稳态或炎症状态下的存在。该协议结合酶和机械消化的生成单细胞悬浮, 可以进一步分析的流式细胞术。

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

isolation-identification-extravascular-immune-cells

Research

JoVE Journal

Immunology and Infection

12.9K 次观看.  University Health Network (UHN). 该协议提出了一种简便、有效的方法, 用于分离、鉴定和量化在小鼠心肌中的免疫细胞在稳态或炎症状态下的存在。该协议结合酶和机械消化的生成单细胞悬浮, 可以进一步分析的流式细胞术。

视频: Isolation and Identification of Extravascular Immune Cells of the Heart

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

科研

免疫与感染

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

发育生物学

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

致心律失常心肌病患者心内膜心肌活检样间质基质细胞的分离与鉴定

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

从鼠心心室自由壁分离内膜细胞和冠状内皮细胞

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

从人类和小鼠心肌标本中分离巨噬细胞子集和细胞位细胞

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. However, the literature reports contrasting data on the prevalence of myocardial B-cells. B-cells have been reported to be both among the most prevalent immune cells in the rodent heart or to be present, but at a markedly lower prevalence than myeloid cells, or to be quite rare. Similarly, several groups have described that the number of myocardial B-cells increases after acute ischemic myocardial injury, but one group reported no changes in the number of B-cells of the injured myocardium. Implementation of a shared, reproducible method to assess the prevalence of myocardial B-cells is critical to harmonize observations from different research groups and thus promote the advancement of the study of B-cell myocardial interactions. Based on our experience, the seemingly contrasting observations reported in the literature likely stem from the fact that murine myocardial B-cells are mostly intravascular and connected to the microvascular endothelium. Therefore, the number of B-cells recovered from a murine heart is exquisitely sensitive to the perfusion conditions used to clean the organ and to the method of digestion used. Here we report an optimized protocol that accounts for these two critical variables in a specific way. This protocol empowers reproducible, flow cytometry-based analysis of the number of murine myocardial B-cells and allows researchers to distinguish extravascular vs. intravascular myocardial B-cells.

Here we report a protocol for the quantification and differentiation of myocardial B-lymphocytes based on their location in the intravascular or endothelial space using flow cytometry.

基于流式细胞术的心肌B细胞定量和分析

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

小鼠窦房和房室结常驻心脏巨噬细胞的分离和培养

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

医学

揭示小鼠肺静脉心肌结构

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial fibrillation (AF). PVs contain myocardial sleeves (MS) composed of cardiomyocytes. MS are implicated in the initiation and maintenance of AF, as they preserve similarities to the cardiac working myocardium, including the ability to generate ectopic electrical impulses. Rodents are widely used and may represent excellent animal models to study the pulmonary vein myocardium since cardiomyocytes are widely present all over the vessel wall. However, precise microdissection and preparation of murine PVs is challenging due to the small organ size and intricate anatomy.
We demonstrate a microscopy-guided microdissection protocol for isolating the murine left atrium (LA) together with the PVs. Immunofluorescence staining using cardiac Troponin-T (cTNT) and connexin 43 (Cx43) antibodies is performed to visualize the LA and PVs in full length. Imaging at 10x and 40x magnification provides a comprehensive view of the PV structure as well as detailed insights into the myocardial architecture, particularly highlighting the presence of connexin 43 within the MS.

作者聚焦：开发跨物种心房颤动研究的转化模型 

This protocol demonstrates microscopy-guided isolation and immunofluorescence staining of murine pulmonary veins. We prepare tissue samples containing the left atrium, pulmonary veins, and the corresponding lungs and stain them for cardiac Troponin T and Connexin 43.

小鼠肺静脉心肌套的显微解剖和免疫荧光染色

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial ...

心脏外免疫细胞的分离与鉴定

摘要

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