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>
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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 border="0" cellpadding="0" cellspacing="0" style="width:815px;" width="814">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Phosphate buffered saline</td>
			<td style="width:123px;">Wisent</td>
			<td style="width:185px;">311-010-CL</td>
			<td style="width:167px;">1x PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">21Gx 1 1/2 (0.8mmx40mm) PrecisionGlide Needle</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#8217;s Balanced Salt Solution</td>
			<td>Wisent</td>
			<td>311-511-CL</td>
			<td>1x HBSS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-50G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum</td>
			<td>Sigma</td>
			<td>B9433</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.5M Ethylenediaminetetraacetic acid</td>
			<td>BioShop</td>
			<td>EDT111</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vacuum filter , 0.2 &#181;m Filtropur V50</td>
			<td>Sarstedt</td>
			<td>83.1823.001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">28G 1/2 1 cc insulin syringe</td>
			<td>BD</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">60 mL syringe</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;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 height="40">
			<td height="40" style="height:40px;">Collagenase I</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>from Clostridium histolyticum</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hyaluronidase type I-S</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNase-I</td>
			<td>Sigma</td>
			<td>D4513</td>
			<td>from bovine pancreas</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell strainer, 40 &#181;m Nylon</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ammonium-Chloride- Potassium (ACK) lysis buffer</td>
			<td>Lonza</td>
			<td>10-546E</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa Fluor 700 anti-mouse/human CD11b</td>
			<td>Biolegend</td>
			<td>101222</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC/Cy7 anti-mouse Ly-6c</td>
			<td>Biolegend</td>
			<td>128025</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC anti-mouse CD103</td>
			<td>Biolegend</td>
			<td>121414</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Brilliant Violet 605 anti-mouse CD11c</td>
			<td>Biolegend</td>
			<td>117334</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE anti-mouse Ly-6G</td>
			<td>Biolegend</td>
			<td>127607</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pacific Blue anti-mouse I-Ab</td>
			<td>Biolegend</td>
			<td>116422</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-mouse CD64 (FcgRI)</td>
			<td>Biolegend</td>
			<td>139315</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE/Cy7 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103113</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP/Cy5.5 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103132</td>
			<td>1:40 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruStain fcX (anti-mouse CD16/32)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">True-Stain Monocyte Blocker</td>
			<td>Biolegend</td>
			<td>426101</td>
			<td>1:20 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo V10</td>
			<td>TreeStar Inc</td>
			<td></td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

12.4K 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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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 ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪组织免疫细胞的分离

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...