Name: analysis of microglia and monocyte-derived macrophages from the central nervous system by flow cytometry
Uploaded: 2017-06-22T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry at JoVE.com

这项工作得到了国民革命联合会（ANR-12-MALZ-0003-02-P2X7RAD），法国阿尔茨海默病协会和Bpifrance协会的资助。我们的实验室也得到了Inserm，CNRS，Pierre et Marie-Curie大学以及“Investissements d&#39;avenir”ANR-10-IAIHU-06（IHU-A-ICM）的支持。我们要感谢CELIS细胞培养核心设施的协助。

本文所述的所有方法均已获得ICM研究所机构动物护理和使用委员会以及达尔文法国道德动物委员会的批准，并且符合协议01407.02。 准备<ol><li>通过对每只小鼠组合以下物质，在1.5 mL管中制备消化鸡尾酒：1 mL磷酸盐缓冲盐水（PBS）;将123μL消化酶（参见表格）以13wunsch / mL（储备溶液），终浓度1.6wunsch / mL;和5μLDNase I（见材料表），以100mg / mL（储备溶液），最终浓度为0.5mg / mL。 ...

<ol>
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已经证明，小胶质细胞和MDM在CNS中具有不同的功能和表型，因此这些巨噬细胞亚群的鉴定和分析对于更好地了解神经疾病9,18,25是必不可少的。使用两个标记（CD11b和CD45）的流式细胞术分析可以区分每个亚群（ 图3C ）。以前通过使用其他特异性标记（例如MDM的CCR2和小胶质细胞的CX3CR1）以及最近开发的具有Tmem119抗体（ 图3E - 3G ?...

小胶质细胞是中枢神经系统（CNS）的实质性组织沉积巨噬细胞。它们扮演两个关键的功能角色：免疫防御和维持CNS动态平衡。与从骨髓中的造血干细胞不断更新的MDM相反，小胶质细胞与原始造血祖细胞分化，起源于卵母细胞（YS），其在胚胎发育1,2,3期间定植于大脑。在啮齿动物中，转录因子Myb在所有骨髓来源的单核细胞和巨噬细胞的发育中起着至关重要的作用，但是对于YS衍生的小胶质细胞来说，这个因子是有限的，分化依赖于转录因子PU.1。 在健康的CNS中，小神经胶质细胞是动态细胞，不断地对其环境进行采样，入侵和测量入侵病原体或组织损伤5 。这种信号的检测启动了解决伤害的途径。小胶质细胞从分枝形态迅速转变为变形虫，随后是各种介质的吞噬和释放，例如促炎或抗炎细胞因子。因此，取决于它们的微环境，活化的小胶质细胞可获得不同引发状态的谱。 小胶质细胞对许多神经系统疾病的发展和进展产生深刻的影响。在阿尔茨海默病（AD） 7 ，肌萎缩性侧索硬化症（ALS） 8 ，多发性硬化症（MS） 9或帕金森病（PD） 10 ）的啮齿动物模型中，小胶质细胞被证明具有双重作用，诱导有害的神经毒性或作用以神经保护的方式，这是依赖于o特定疾病，疾病阶段，以及该疾病是否受到全身免疫隔室7,8,9,10,11的影响。在上述疾病中观察到的大多数CNS损伤包含异质骨髓细胞群体，不仅包括实质性小胶质细胞，还包括血管周围和脑膜巨噬细胞以及CNS浸润MDM。这些细胞类型可能差异有助于与损伤和修复相关的病理生理机制7,12,13,14,15。正在研究这些疾病模型的调查人员目前面临的挑战是确定外周单核细胞和巨噬细胞是否渗透到CNS，如果是这样，到dist从这些细胞诱导居民小神经胶质细胞。事实上，小胶质细胞是非常塑料的;当它们被激活时，小胶质细胞重新表达通常由外周单核细胞和巨噬细胞表达的标志物。因此，这个问题依赖于识别可以区分宿主小胶质细胞与浸润性单核细胞和巨噬细胞的标记。 由于缺乏特异性抗体，通过免疫组织学应用对这些人群对脑切片的鉴别是有限的。然而，流式细胞术分析是评估几种标记物的表达并区分细胞群体（例如，淋巴细胞，巨噬细胞/ MDM CD11b + CD45 高和小胶质细胞CD11b + CD45 med ）以及细胞亚群16的有效技术 ， 17，18 。该协议描述了隔离的过程通过使用优化的酶组织解离和密度梯度离心，在神经疾病模型中从小鼠CNS获得单核细胞;以及通过使用流式细胞术分离CNS中的小胶质细胞和MDM群体的方法。 另一种方法是通过使用与特异性抗体缀合的磁珠19,20,21来消除髓磷脂并纯化细胞。使用抗髓磷脂磁珠的髓磷脂去除更昂贵并影响分离细胞的生存力和产量22 。该步骤和以下免疫磁性分离的小胶质细胞，限制进一步研究特异性免疫细胞群体21,22 。 这些程序提供了一个简单的方法来研究巨噬细胞亚群在疾病发展中确定对巨噬细胞表型和激活状态的药物作用或基因修饰。

<table border="0" cellpadding="0" cellspacing="0" width="908">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">5-month-old Mice</td>
			<td style="width:223px;">Janvier</td>
			<td style="width:119px;">C57BL/6J</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Liberase TL Research Grade</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">5401020001</td>
			<td>Digestion enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Deoxyribonuclease I from bovine pancreas</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">DN25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Percoll</td>
			<td style="width:223px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0891-01&#160;</td>
			<td>Density gradient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Cell Strainer size 70 &#181;m Nylon</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">731751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Venofix 25G</td>
			<td style="width:223px;">BRAUN</td>
			<td style="width:119px;">4056370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Piston syringe 10 mL</td>
			<td style="width:223px;">Terumo</td>
			<td style="width:119px;">SS+10ES1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Pasteur pipette 230 mm</td>
			<td style="width:223px;">Dustcher</td>
			<td style="width:119px;">20420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">1,5 mL&#160; tube</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:119px;">0030 123.328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">15 mL&#160; tube</td>
			<td style="width:223px;">TPP</td>
			<td style="width:119px;">91015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">50 mL&#160; tube</td>
			<td style="width:223px;">TPP</td>
			<td style="width:119px;">91050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">5 mL polystyrene round bottom tube</td>
			<td style="width:223px;">BD Falcon</td>
			<td style="width:119px;">352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">D-PBS (1X) without Ca2+/Mg2+</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">D-PBS (10X) without Ca2+/Mg2+</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14200-067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fetal bovine serum</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Bovine Serum Albumin solution 30%</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Paraformaldehyde 32% Solution</td>
			<td style="width:223px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714-S</td>
			<td>Caution -Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Saponin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Sodium Azide</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">47036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">PerCPCy5.5 Rat anti-mouse CD11b (clone M1/70)</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">45-0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rat IgG2b K Isotype Control PerCP-Cyanine5.5</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">45-4031&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BV421 Rat anti-mouse CD45 (clone 30-F11)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">563890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BV421 Rat IgG2b, &#954; Isotype Control RUO</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">562603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rabbit anti-mouse TMEM119 (clone28-3)</td>
			<td style="width:223px;">Abcam</td>
			<td style="width:119px;">ab209064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">AlexaFluor 647 Donkey anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A31573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Anti-Mouse CD16/CD32 Purified</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">14-0161</td>
			<td>Mouse Fc Block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fixable Dead Cell Stain Kits</td>
			<td style="width:223px;">Invitrogen</td>
			<td style="width:119px;">L34969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mouse CCR2 APC-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">FAB5538A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rat IgG2B APC-conjugated Isotype Control</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">IC013A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mouse CX3CR1 PE-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">FAB5825P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Goat IgG PE-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">IC108P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Centrifuge</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:119px;">5804R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Cell analyzer</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">BD FACSVERSE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Data Analysis Software</td>
			<td style="width:223px;">FlowJo LLC</td>
			<td style="width:119px;">FlowJo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fine scissors</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">14090-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Standard Pattern Forceps</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">11000-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mayo Scissors</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">14010-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Dumont #5 Forceps</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of microglia and monocyte-derived macrophages from the central nervous system by flow cytometry

许多研究已经证明了免疫细胞，特别是巨噬细胞在中枢神经系统（CNS）病理学中的作用。 CNS中有两个主要的巨噬细胞群体：（i）小神经胶质细胞，其是CNS的驻极体巨噬细胞，并且在胚胎发生过程中衍生自卵黄囊祖细胞，以及（ii）可以渗透的单核细胞衍生的巨噬细胞（MDM）疾病期间的CNS来源于骨髓祖细胞。每个巨噬细胞亚群的作用根据正在研究的病理学而不同。此外，对于这些巨噬细胞亚群的组织学标记或区别标准尚未达成共识。然而，通过流式细胞术分析CD11b和CD45标记物的表达谱使我们能够区分小胶质细胞（CD11b + CD45 med ）与MDM（CD11b + CD45 高 ）。在本协议中，我们显示密度梯度离心并且流式细胞术分析可以用于表征这些CNS巨噬细胞亚群，并且如我们最近公布的那样研究由这些细胞表达的感兴趣的标记物。因此，这种技术可以进一步了解巨噬细胞在神经系统疾病小鼠模型中的作用，也可用于评估对这些细胞的药物作用。

密度梯度离心和抗体染色后，在流式细胞仪上获得细胞，并使用形态选择策略进行如下分析。点阵图前向散射面积（FSC-A） 与前向散射高度（FSC-H）之间定义了第一个门，以区分单个细胞与双峰（ 图3A ）。然后根据相对细胞大小和细胞粒度，将单细胞门控在FSC-A 与 Side-Scattered-Area（SSC-A）点图上以排除细胞碎片和固缩细胞（ <strong class="xf...

Watch this Scientific Journal Video about Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry at JoVE.com

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

该方案通过流式细胞术提供成年小鼠中枢神经系统中巨噬细胞亚群的分析，有助于研究由这些细胞表达的多种标志物。

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 ...

Research

JoVE Journal

Immunology and Infection

Culturing Microglia from the Neonatal and Adult Central Nervous System

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

科研

免疫与感染

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征：两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

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 ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

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

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. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

分离中枢神经系统组织和相关脑膜，用于免疫细胞的下游分析

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...