Name: isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation
Uploaded: 2017-04-03T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

我们要感谢宾夕法尼亚州立大学千禧科学综合体的流式细胞仪核心设施。

所有实验性协议 (1、1.2 和1.3 节) 均由宾夕法尼亚州立大学动物保育和使用委员会 (IACUC) 批准。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组织</td>
			<td>离解缓冲器准备</td>
			<td>最终卷</td>
			<td>存储</td>
		</tr>
<tr>
<td>白脂肪组织 (笏)</td>
			<td>
离解缓冲: 2.5% HEPES, 10 毫克/毫升牛血清白蛋白 (BSA), 3 毫克/毫升 (0.3%)Dulbecco 氏改良鹰培养...

<ol>
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饮食诱导代谢紊乱模型, 模仿疾病, 如动脉粥样硬化, 简单的脂肪, 脂肪性肝炎和2型糖尿病广泛使用, 以更好地了解潜在的分子机制的疾病进展。胶原酶依赖性消化经常被用于游离组织, 从细胞外基质 (ECM)16,27中释放出癌细胞。酶, 如胶原蛋白酶扰乱胶原蛋白, 为相邻细胞提供结构支持。组织的结构组成决定了组织基质的刚度 (抵抗变形), 哪些粗胶原酶产品?...

小鼠模型已被广泛用于研究人类疾病的动态。从患病状态的小鼠中正确分离组织驻留细胞可以提供一个平台, 以了解分子和细胞对疾病发病机制的贡献1。肥胖是一种极其重要的疾病。随着胰岛素抵抗和2型糖尿病、心血管疾病和脂肪性肝病的发生, 肥胖的发病率在全球范围内持续上升2,3。通过减少脂肪组织产生的改变信号的体力活动而进一步扭曲了过量的养分消耗, 从而改变了其他外围组织的细胞环境, 如主动脉和肝脏4。这种扰乱代谢稳态导致慢性低等级系统性炎症5。
经典活化的巨噬细胞的主动脉和肝脏, 以及他们的招募白脂肪组织 (笏) 已被证明不仅启动失调的代谢信号, 但也持续炎症6,7。巨噬细胞表型和功能性异质性与肥胖相关疾病的发病机制有密切关系7。巨噬细胞极化的动态可塑性允许这些单元表现出一系列激活的表型, 以协调炎症的进展和分辨率8。虽然经典激活 (M1) 巨噬细胞是牵连炎症的传播, 或者激活 (M2) 巨噬细胞已与分辨率和组织修复相关的9,10。
当机体经历新陈代谢的压力时, 白色脂肪组织异常堆积。膨胀的脂肪组织吸引并保留了深刻改变正常脂肪细胞功能的炎性神经细胞, 以促进胰岛素抵抗、高血糖和最终2型糖尿病、胰岛素抵抗或高血糖11, 12。在平行, 白色脂肪组织改造反应的炎症信号的渗透经典活化 (M1) 脂肪组织巨噬细胞 (atm)13,14。这多器官施加一连串的信号, 出轨正常功能的其他机构, 如主动脉和肝脏4。
肝脏是一个新陈代谢的发电站, 适应的刺激来自附近的失调笏15。肝巨噬细胞或枯否体细胞, 在反应代谢变化, 分泌炎症细胞因子, 改变实质和实质细胞表型和促进组织重塑。肝脂堆积、炎症、细胞外基质沉积、坏死及最终功能丧失伴随着非酒精性脂肪肝引起的广泛肝损害的炎症反应16,17,18。
当身体承受慢性代谢压力19时, 大动脉在动脉壁内积聚脂质, 同时与受损的笏和肝功能相同。动脉脂质积累引发了活化的内皮细胞分泌的趋化因子, 并随后招募单核20。一旦被吸收, 单核细胞就会增殖, 分化, 摄取脂蛋白, 成为泡沫。动脉粥样硬化的启动和持续的炎活动的招募和组织居民脂质巨噬细胞。这些巨噬细胞屈服于动脉粥样硬化微环境中的胞外和胞内压力信号, 然后参与凋亡信号级联。由于这些泡沫细胞死亡, 他们贡献了他们的脂质填充内容的坏死核心病变, 然后导致斑块破裂, 心肌梗塞, 中风。
整体而言, 巨噬细胞表型的异质性协调在失调组织中观察到的炎症性改变引起的肥胖, 如笏、肝和主动脉8,21。特征的招募和组织驻留巨噬细胞可以提供洞察的潜在分子目标, 操纵巨噬细胞表型1。为了有效地描述由肥胖引起的炎症组织的巨噬细胞, 可以通过酶消化得到单细胞悬浮液。这种离解协议必须有效地在充分降解结缔组织, 同时尽量减少免疫细胞死亡和提供最佳的细胞产量。酶的混合物取决于组织的类型和它的结构构成。弹性组织, 如主动脉, 需要更强的酶活性, 比肝脏和笏, 以实现组织离解。从单细胞悬浮, 组织驻留巨噬细胞可以明确的特征或孤立的进一步的下游分析, 如转录剖面。
在这里, 一个组织特异性的协议描述, 使用胶原酶相关的组织消化和多色流式细胞仪有效分离和表征组织驻留巨噬细胞从传统的饮食诱发肥胖,动脉粥样硬化, 简单的脂肪和脂肪小鼠模型。同时染色细胞表面标记抗体抗白细胞-(CD45 和/或 CD11b) 和巨噬细胞 (F4/80) 特异抗原通常用于识别巨噬细胞的数量22。荧光活化细胞分类 () 是一种强有力的策略, 用来对这些被识别的种群进行高纯度的分类。然后利用下游分子分析 (如定量实时聚合酶链反应)23对表型特定基因图谱进行排序。虽然标准流式细胞仪和流 cytometry-based 细胞排序是强大的工具, 区分巨噬细胞在一个巨大的异构电池悬浮, 前协议必须优化, 以确保成功的输出。在本研究中, 描述了有效分离和表征可行组织特异性巨噬细胞的协议;更重要的是, 这项研究提供了对经常出现的技术问题的重要洞察力, 以及预防和/或解决它们的主动和 trouble-shooting 战略。

<table border="0" cellpadding="0" cellspacing="0" width="940">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">26G x 5/8 in Needles</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">305115</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">23G x 0.75 in needle x 12 in. tubing Blood Collection Set</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">367297</td>
			<td style="width:349px;">Used for cannulation of subhepatic IVC during liver perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">21G 1 1/2 in. Needles</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">305156</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1mL syringe with rubber stops</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309659</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">10mL Syringes</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309604</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1mL Syringe</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309659</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">F4/80 PE</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">123110</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD11c PE/Cy7</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">117318</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD11b PE/Cy5</td>
			<td style="width:151px;">eBioscience</td>
			<td style="width:119px;">15-0112-81</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Anti-mouse CD16/32 Fc Block&#160;</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">101320</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD45 Pacific Blue</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">103126</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE Rat IgG2a</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400508</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE/Cy7 Armenian Hamster IgG</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400922</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE/Cy5 Rat IgG2b</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400610</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Pacific Blue Rat IgG2c</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400717</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)&#160;</td>
			<td style="width:151px;">Cellgro</td>
			<td style="width:119px;">15-017-CV</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1X Phosphate Buffered Saline (PBS)</td>
			<td style="width:151px;">Cellgro</td>
			<td style="width:119px;">21-031-CV</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">70 micron cell strainers</td>
			<td style="width:151px;">Corning, Inc.</td>
			<td style="width:119px;">352350</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">&#160;1.7 mL microcentrifuge tube&#160;</td>
			<td style="width:151px;">Denville</td>
			<td style="width:119px;">C2170</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Paraformaldehyde Aqueous Solution -16X</td>
			<td style="width:151px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">CAS #30525-89-4</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Micro Dissecting Scissors, 3.5 inch, Straight, 23mm, Sharp&#160;</td>
			<td>Stoelting</td>
			<td>52132-10P</td>
			<td style="width:349px;">Used for general dissecting purposes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Forceps, 4in, full curve, 0.8mm</td>
			<td>Stoelting</td>
			<td>52102-37P&#160;</td>
			<td style="width:349px;">Used for general dissecting purposes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Spring dissection scissors &#8211; 3 mm Cutting edge&#160;</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">15000-10</td>
			<td style="width:349px;">Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Curved 0.07 x 0.04 mm Tip Forceps&#160;</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">11297-10</td>
			<td style="width:349px;">Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hemostatic Forceps (Curved)</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">13021-12</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Heparin Sodium Salt</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">9041-08-1</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">35mm Cell Culture/Petri Dishes</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">12-565-90</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Polystyrene Petri Dishes (10 cm) w/lid</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">08-757-100D</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">15mL Conical Centrifuge Tubes (Polypropylene)</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-959-53A</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">50mL Conical Centrifuge Tubes&#160;(Polypropylene)</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">5mL Round-Bottom Polystyrene Tubes&#160;</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-959-5</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Fetal Bovine Serum</td>
			<td style="width:151px;">Gemini Bio-Products</td>
			<td style="width:119px;">100-106</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:321px;">Ethanol (Stock Ethyl Alcohol Denatured, Anyhydrous)</td>
			<td style="width:151px;">Millipore</td>
			<td style="width:119px;">EX0285-1</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Bovine Serum Albumin</td>
			<td style="width:151px;">Rockland</td>
			<td style="width:119px;">BSA-50</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HEPES</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenase Type II</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C6885</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenasse Type XI</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C7657</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hyaluronidase Type I</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3506</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">DNAse</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">DN25</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenase Type I</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0130</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">NaOH</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">1310-73-2&#160;</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:321px;">CaCl2</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">449709-10G</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">500mL beaker&#160;</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">02-540M</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">4 cm Hemostatic clamp</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:119px;">52120-40</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Toothed forceps</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:119px;">52104-33P</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">50 micron Disposable filters</td>
			<td style="width:151px;">Systemex</td>
			<td style="width:119px;">04-0042-2317</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Collagenase Type IV</td>
			<td style="width:151px;">ThermoFischer Scientific</td>
			<td style="width:119px;">17104019</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Ammonium Chloride Potassium (ACK)</td>
			<td style="width:151px;">ThermoFischer Scientific&#160;</td>
			<td style="width:119px;">A1049201</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Razors (0.22 mm (0.009&#34;))</td>
			<td style="width:151px;">VWR International</td>
			<td style="width:119px;">55411-050</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Texas Red Live/Dead stain</td>
			<td style="width:151px;"></td>
			<td style="width:119px;"></td>
			<td style="width:349px;">Red viability stain (in Figure 1A)</td>
		</tr>
	</tbody>
</table>

isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation

肥胖促进了一种慢性炎症状态, 主要由组织驻留的巨噬细胞以及单核细胞衍生的巨噬病介导。饮食诱发肥胖 (迪奥) 是研究巨噬细胞异质性作用的有价值的模型;然而, 足够的巨噬细胞隔离很难从发炎的组织中获得。在本协议中, 我们概述了从我们的研究获得一个合适的人群组织驻留巨噬细胞后18周高脂肪 (风) 或高脂肪/胆固醇的隔离步骤和必要的故障排除准则 (HFHCD) 饮食干预。本议定书的重点是三标志性组织研究的肥胖和动脉粥样硬化, 包括肝脏, 白脂肪组织 (笏), 和主动脉。我们强调流式细胞术的二元用法如何实现组织驻留巨噬细胞的分离和表征的新维度。本议定书的一个基本部分解决复杂的基础组织特定的酶消解和巨噬细胞分离, 以及随后的细胞表面抗体染色流细胞分析。这项协议解决了现有的复杂的荧光活化细胞分类 (资产管制), 并提出了这些复杂性的澄清, 以获得广泛的范围, 从适当的排序细胞群体的描述。可选的浓缩方法包括对细胞进行分类, 例如稠密的肝脏, 允许在与外地资产管理体系合作时具有灵活性和时间性。简言之, 这项协议帮助研究者评估了一项研究中大量炎症组织的巨噬细胞异质性, 并提供了有洞察力的诊断技巧, 以利于细胞的分离和鉴定。免疫细胞在迪奥介导的炎症。

当使用载脂蛋白 E 缺乏 (载脂蛋白 KO) C57BL/6 (BL6) 小鼠维持高脂肪高胆固醇饮食 (HCHFD 或 HCD) 18 周, 1 x 104 -2 x 104 CD45+F4/80+主巨噬细胞可以被隔离当两个样本汇集.肝解剖从 HFHCD 喂食的载脂蛋白高鼠, 产生大于 5 x 105排序的枯否细胞 (这取决于可用的排序时间)。当使用高脂肪饮食 (风) 喂养野生型 (重量级) C57BL/6 小鼠, 5 x 105到 1 x 10...

Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

该协议允许研究员分离和表征组织驻留巨噬细胞在各种标志发炎组织提取从饮食诱发的代谢紊乱模型。

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

The overall goal of this procedure is to effectively isolate and characterize macrophages from tissues commonly affected by diet-induced inflammation such as the liver. This method can help answer key questions in the fields of immunology and inflammation about how tissue-resident macrophage phenotype can dictate the progression of chronic inflammatory diseases. The main advantage of this is that the procedure optimizes the isolation steps needed for obtaining single-cell suspensions derived from tissues impacted by obesity-mediated low-grade chronic inflammation.Demonstrating the procedure will be Joselyn Allen, a graduate student from my laboratory. To isolate the liver, first place an ethanol-soaked mouse in the supine position on a polystyrene foam dissecting board and secure the fore and hind paws. Next, use a medium point tip forceps to grasp the abdominal skin near the urethra opening and use sharp dissecting scissors to create a small incision in the raised skin.Insert the lower blade of the scissors into the incision between the skin and the peritoneum and make a lateral incision from the groin to the chin. Gently pull back the skin to expose the intact peritoneal cavity and thoracic wall and make a lateral incision through the peritoneum. Lift the sternum and carefully incise the diaphragm.Then move the gastrointestinal organs to the side and locate the subhepatic inferior vena cava or IVC. Using hemostatic forceps, clamp the suprahepatic IVC to maintain a localized perfusion and attach a syringe filled with pre-warmed perfusion buffer to a 23 gauge blood collection and infusion set. Gently depress the plunger until the tubing and needle are filled with perfusion buffer and insert the needle parallel to the subhepatic IVC.Proper cannulation of the IVC ensures that the dissociation buffer is locally distributed throughout the liver and peripheral tissues optimizing the tissue digestion. Use a four centimeter hemostatic clamp to secure the needle in place and gently depress the plunger to begin the perfusion. If the needle is appropriately positioned, the color will rapidly flush from the liver and the lobes will become enlarged.Promptly cut the portal vein to allow the perfusion buffer to flow freely through the liver, occasionally gently massaging the lobes between the thumb and forefinger to facilitate the removal of the blood. When only five milliliters of perfusion buffer are left, replace the perfusion buffer syringe with a syringe filled with pre-warmed dissociation buffer, continuing to gently massage the lobes and perfuse the liver until it is fully digested. Confirm a successful digestion by gently pressing the blunt edge of a pair of forceps into the left lateral lobe.An indentation should appear on the surface that slowly fills once the forceps is removed. Then remove the needle and use a short straight blade from a pair of dissecting scissors to carefully cut through the connecting ligaments to excise the whole liver and the gallbladder. Transfer the liver to a 10 centimeter dish containing 10 milliliters of ice cold DMEM and use toothed forceps to grip the severed suprahepatic IVC.Using medium point forceps, apply a rapid stroking motion to each lobe to release the cells from the Glisson's Capsule and pass the saturated DMEM through a 70 micrometer cell strainer in a 50 milliliter conical tube on ice. High yields of viable cells can only be achieved by properly dissociating the liver cells from the Glisson's Capsule. When all of the cells have been collected, invert the tube to gently mix the cell suspension and pellet the cells by centrifugation.Transfer the supernatant to a new 50 milliliter conical tube for three more low-spin centrifugations. After the last centrifugation, transfer the supernatant to a new 50 milliliter tube and centrifuge the cells one more time at a higher speed. Then resuspend the non-parenchymal cell pellet in FACS buffer for counting and dilute the liver cells to the appropriate concentration according to their downstream application in labeled five milliliter round bottom polystyrene tubes.Mice fed a high fat diet or a high fat high cholesterol diet exhibit an increased infiltration of classically activated M1 macrophages in affected tissues such as the aorta. RON receptor expressing aortic CD45 positive F4/80 positive macrophages which demonstrate an anti-inflammatory phenotype are decreased in high fat high cholesterol diet fed mice while pro-inflammatory CD45 positive F4/80 positive CD11c positive macrophages are found in increased numbers in aortas isolated from these animals. Further, sorted RON receptor expressing macrophages derived from digested aortas display an increased expression of Arginase 1 gene, a well-established M2 macrophage marker.Indeed, the characterization of liver-resident macrophages reveals the prevailing phenotype of RON receptor expressing subpopulations with CD11c positive pro-inflammatory macrophage populations demonstrating a decreased expression of genes that are strongly associated with the ant-inflammatory M2 phenotype. Similar trends are observed in the macrophage populations isolated from digested white adipose tissue with pro-inflammatory phenotype macrophage populations exhibiting a decreased surface expression of the RON receptor. Once mastered, this technique can be completed in 20 to 30 minutes per animal if it is performed properly.Following this procedure, other methods such as flow cytometry, fluorescent-activated cell sorting, and/or quantitative PCR can be performed to further characterize the phenotype of the isolated liver-derived tissue-resident macrophages. After watching this video, you should have a good understanding of how to properly isolate tissue-resident macrophages from diseased livers. Thanks for watching and good luck with your experiments.

Research

JoVE Journal

Immunology and Infection

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

科研

免疫与感染

Proteomic Profiling of Macrophages by 2D Electrophoresis

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

抑制接枝反应性免疫的调节性巨噬细胞的功能表征

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

成年日本血吸虫细胞外囊泡的分离与鉴定

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...