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。 </li></ol>灌注和解剖<ol><li>用致死剂量的戊巴比妥（100μL，400mg / mL）安乐死小鼠。 </li><li>将动物置于仰卧位，用70％乙醇湿身体。 </li><li>用正确的剪刀剪下腹部皮肤，露出胸腔年。 </li><li>在隔膜中切开切口，用精细剪刀切割肋骨的侧面，以尾端朝向方向，以暴露心脏（并避免切割其他器官）。识别左心室（LV）和右心房（RA）。 </li><li>在LV内插入一个25 G针的蝴蝶导管。用精细剪刀在RA上切开，血液开始时，以20 mL PBS的流速以10mL / min的速度开始灌注，以从血管中除去细胞和血液;成功的灌注是随着血液流离失所而肺和肝脏的烫伤所引起的。 </li><li>用中等剪刀剪掉动物。用精细剪刀解剖脊柱，并通过切除脊柱腹侧的腹壁肌肉组织并通过切割尾部基部的脊柱将其与体部分离开。 </li><li>将含有PBS的10mL注射器和安装的200μL移液管吸头插入腰部脊柱冲洗颈部脊髓。 注意:200μL移液管尖端在最宽端（约1 cm）用剪刀切割，并紧紧插入预先填充PBS的10mL注射器上。 </li><li>去除头骨上方的皮肤，并使用镊子暴露大脑。在颅骨底部插入剪刀，并在矢状缝合后切开颅骨。将小镊子插入切口，轻轻抬起头骨。解剖大脑从鼠标头，并去除嗅球和小脑。 </li><li>在含有1.128mL PBS的含有消化混合物的1.5mL管中收集脑和脊髓（来自步骤1.1）。 </li></ol>细胞分离<ol><li>用小剪刀将1.5 mL管（步骤2.9）中的脑和脊髓细分为1-2 mm 3 （ 图1 ）。 </li><li>在孵育中孵育含有切碎的组织的1.5mL管30分钟在37℃下用5％CO 2 。 注意:在此步骤准备密度梯度。 </li><li>向1.5 mL管中加入20μL0.5 M EDTA（储备溶液），终浓度为10 mM，以停止酶反应。 </li><li>通过用5 mL移液管上下移动轻轻地制备细胞悬液。 </li><li>使用10mL注射器的柱塞，将细胞悬液通过50mL管中的尼龙网（70μm孔径）。 </li><li>用20 mL 5％胎牛血清（FBS）-PBS清洗尼龙网。 </li><li>在室温（18℃）下离心7分钟（300×g）。通过抽吸弃去上清液，并进行下一步骤。去除上清液要格外小心;不要打扰沉淀，这可以很容易地被抽吸吸管吸出。 </li></ol>密度梯度<ol><li>通过向密度梯度介质中加入适量的10x PBS来制备密度梯度介质的储备溶液。 注意:将一份10X PBS加入到9份密度梯度培养基中。 </li><li>如表1所述，用1x PBS稀释存储密度梯度培养基的不同百分比。 </li><li>用4mL 30％密度的梯度培养基重新悬浮沉淀（来自步骤3.7）并转移到15mL管中。 </li><li>将巴斯德吸管置于15 mL管的底部，并缓慢地铺上4 mL 37％密度梯度培养基。 </li><li>加入4mL 70％密度梯度培养基，如上所述。 </li><li>在室温（18℃）下将梯度离心40分钟（800xg）。确保离心机以最小或无制动器停止，以使相间不受干扰。 注意:管将显示分层。分层在70％ - 37％密度梯度界面的细胞是单核细胞免疫细胞（小胶质细胞和巨噬细胞亚群）;上层分别是细胞碎片和髓鞘（ 图2 ）。 </li><li&gt;使用转移移液管，轻轻取出髓鞘层。 </li><li>将含有巨噬细胞亚群细胞的3-4mL 70-37％密度梯度相间收集到干净的15mL管中。 </li><li>用PBS洗涤细胞3次，总体积为15mL。确保将含有细胞的密度梯度培养基用PBS稀释至少三次，并用制动器"开"离心7分钟（500×g，4℃）。 </li><li>用1 mL PBS重悬细胞沉淀，用于细胞标记或其他功能测定。 </li></ol>流式细胞术的细胞标记<ol><li>将细胞转移到流式细胞仪中（步骤4.10）。 </li><li>向步骤4.10中获得的每个细胞样品中加入1μL可固定死细胞染色剂。在冰上孵育30分钟，防光。 </li><li>用2mL PBS洗涤细胞一次并离心5分钟（300×g，4℃）。 </li><li>取出上清液，重悬细胞沉淀400μLPBS 1％BSA，0.1％叠氮化物。 </li><li>向100μL的细胞中加入1μgCD16 / CD32抗体以阻断Fc受体。在冰上孵育10-15分钟，防光。 </li><li>在PBS 1％BSA，0.1％叠氮化物或PBS 1％BSA，0.1％叠氮化物，0.25％皂苷的PBS中准备一级抗体混合物，用于细胞渗透用于细胞内染色。 </li><li>加入100μL的一级抗体混合物（2x），并在冰上孵育30分钟，避光保存。 注意:在进行实验前应对所有抗体进行滴定以确保最佳结果。 </li><li>用2mL PBS洗涤细胞并离心5分钟（300×g，4℃）。重复此步骤三次。 </li><li>如果第一抗体不直接与荧光团缀合，则添加100μL的二抗，并在冰上孵育30分钟，防止光照。 </li><li>用2mL PBS洗涤细胞，并在4℃以300xg离心5分钟）。重复此步骤三次。 </li><li>修复细胞用100μL1％PFA在室温下15分钟，防止光照。 注意:注意:PFA有毒。在通风橱中使用并佩戴个人防护装备。 </li><li>用2mL PBS洗涤细胞，并在4℃以300xg离心5分钟。重复此步骤三次。 </li><li>用200μLPBS重悬细胞沉淀，并进行流式细胞仪的获取和分析。按照图3中的门控策略。 </li></ol>

<ol>
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作者没有什么可以披露的

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 ）的方法验证了该策略。我们以前发表的18例进行了CCR2和CX3CR1抗体的实验（ 图3F ，3G ）。 CD11b + CD45与CD11b + CD45 高群体相反， med群体高度表达Tmem119和CX3CR1，而不是CCR2。 CD11b + CD45 高群体由具有或不具有CCR2表达的各种亚群组成。这些巨噬细胞亚群（血管周围巨噬细胞，浸润巨噬细胞等 ）的鉴定和表征是广泛的研究领域。此外，这些群体的标记物可以根据病理过程而改变。实际上，当单核细胞穿过血脑屏障26时，CCR2被下调。 CD45和CD11b抗体识别细胞表面抗原，因此不需要透化治疗。因此，该技术也可用于通过细胞分选来纯化每个巨噬细胞亚群以进行mRNA分析或其他功能测定。值得注意的是使用酶和密度梯度可以在一定程度上诱导小胶质细胞的活化。我们以前观察到，分离后，小胶质细胞和巨噬细胞表达诱导型一氧化氮合酶（iNOS）。然而，使用该方法可以突出显示在病理学与正常状态下观察到的增加的iNOS表达水平。此外，该技术实现了具有高比例活细胞的分离良好的细胞群（单体）。 为了成功复制此协议，有几个关键参数。梯度的设置是最关键的。我们的经验表明，将15 mL管中的梯度与50 mL管相比，可以实现更好的分离。如果在70-37％的界面中没有细胞可见，则应考虑用于细胞分离的CNS组织的量。我们通常从一只小鼠脑中收集〜40万个细胞。虽然这种产量从各种实验条件波动s，一个更好的可视化的70-37％界面是使用PBS和酚红的37％密度梯度层。 借助流式细胞仪中的不同激光和滤光片，该方案可以量化由每个巨噬细胞亚群18表达的几种标记物。该技术还可用于评估其他免疫细胞如同一样品中的T细胞和B细胞的参与。实际上，使用免疫磁性富集的技术只允许研究以前选择的群体21 。 总的来说，这种技术构成了一种用于表征神经系统疾病小鼠模型中不同巨噬细胞亚群的有用工具。这种独特的选择可以评估治疗对炎症过程在病理状况中的影响。

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><tbody><tr><td>5 month-old Mice</td><td>Janvier</td><td>C57BL/6J</td><td></td></tr><tr><td>Liberase TL Research Grade</td><td>Sigma-Aldrich</td><td>5401020001</td><td>Digestion enzyme</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare Life Sciences</td><td>17-0891-01</td><td>Density gradient medium</td></tr><tr><td>Cell Strainer size 70 &amp;micro;m Nylon</td><td>Corning</td><td>731751</td><td></td></tr><tr><td>Venofix 25 G</td><td>BRAUN</td><td>4056370</td><td></td></tr><tr><td>Piston syringe 10 mL</td><td>Terumo</td><td>SS+10ES1</td><td></td></tr><tr><td>Pasteur pipette 230 mm</td><td>Dustcher</td><td>20420</td><td></td></tr><tr><td>1.5 mL tube</td><td>Eppendorf</td><td>0030 123.328</td><td></td></tr><tr><td>15 mL tube</td><td>TPP</td><td>91015</td><td></td></tr><tr><td>50 mL tube</td><td>TPP</td><td>91050</td><td></td></tr><tr><td>5 mL polystyrene round bottom tube</td><td>BD Falcon</td><td>352054</td><td></td></tr><tr><td>D-PBS (1x) without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>D-PBS (10x) without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Thermo Fisher Scientific</td><td>14200-067</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E4884</td><td></td></tr><tr><td>Bovine Serum Albumin solution 30%</td><td>Sigma-Aldrich</td><td>A7284</td><td></td></tr><tr><td>Paraformaldehyde 32% Solution</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>Caution -Toxic</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>PerCPCy5.5 Rat anti-mouse CD11b (clone M1/70)</td><td>eBioscience</td><td>45-0112</td><td></td></tr><tr><td>Rat IgG2b K Isotype Control PerCP-Cyanine5.5</td><td>eBioscience</td><td>45-4031</td><td></td></tr><tr><td>BV421 Rat anti-mouse CD45 (clone 30-F11)</td><td>BD Biosciences</td><td>563890</td><td></td></tr><tr><td>BV421 Rat IgG2b, &amp;kappa; Isotype Control RUO</td><td>BD Biosciences</td><td>562603</td><td></td></tr><tr><td>Rabbit anti-mouse TMEM119 (clone28 - 3)</td><td>Abcam</td><td>ab209064</td><td></td></tr><tr><td>AlexaFluor 647 Donkey anti-rabbit IgG</td><td>Life Technologies</td><td>A31573</td><td></td></tr><tr><td>Anti-Mouse CD16/CD32 Purified</td><td>eBioscience</td><td>14-0161</td><td>Mouse Fc Block</td></tr><tr><td>Fixable Dead Cell Stain Kits</td><td>Invitrogen</td><td>L34969</td><td></td></tr><tr><td>Mouse CCR2 APC-conjugated Antibody</td><td>R&amp;amp;D</td><td>FAB5538A</td><td></td></tr><tr><td>Rat IgG2B APC-conjugated Isotype Control</td><td>R&amp;amp;D</td><td>IC013A</td><td></td></tr><tr><td>Mouse CX3CR1 PE-conjugated Antibody</td><td>R&amp;amp;D</td><td>FAB5825P</td><td></td></tr><tr><td>Goat IgG PE-conjugated Antibody</td><td>R&amp;amp;D</td><td>IC108P</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804R</td><td></td></tr><tr><td>Cell analyzer</td><td>BD Biosciences</td><td>BD FACSVERSE</td><td></td></tr><tr><td>Data Analysis Software</td><td>FlowJo LLC</td><td>FlowJo</td><td></td></tr><tr><td>Fine scissors</td><td>F.S.T</td><td>14090-11</td><td></td></tr><tr><td>Standard Pattern Forceps</td><td>F.S.T</td><td>11000-13</td><td></td></tr><tr><td>Mayo Scissors</td><td>F.S.T</td><td>14010-15</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>F.S.T</td><td>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（ 图3B ）。然后，小胶质细胞和基于其CD11b和CD45标记的表达水平鉴定MDM（ 图3C ，3D ）。最后，分析每个巨噬细胞亚群的其他表达标志物（ 图3E- 3G ）。微胶囊据报道，与MDM 23相反，ia据报道表达CX3CR1，但不表达CCR2标记。最近证实，Tmem119是小胶质细胞19的特异性标志物。使用对每个标记物特异的检测器抗体，通过流式细胞术评估以下标记（Tmem119，CX3CR1和CCR2）对于Tmem119和CX3CR1标记而言，所有CD11b + CD45mRNA细胞均显示为阳性，而不是CCR2标记，这验证了该门控策略（ 图3E- 3G ）。 在没有密度梯度分离的情况下，由于存在许多细胞和髓磷脂碎片，巨噬细胞亚群不能在斑点图FSC-A 与 SSC-A中定义（ 图3H ）。值得注意的是，透化治疗诱导细胞收缩，因此巨噬细胞亚群出现sm如前所述，FSC 对 SSC点图（ 图3I ，J ） 24 。使用可固定死细胞染色试剂盒测定细胞的活力。密度梯度后，85-95％的细胞存活（ 图3K ）。在单个样品（ 图3L ）中分离脑和脊髓细胞之后，以及单独的脑或脊髓（ 图3M ，3N ）可以在点图上由CD45和CD11b标记物定义小胶质细胞和MDM群体）。 对于流式细胞术标记，重要的是要知道抗体识别的蛋白质和/或表位是否是细胞外或细胞外的。在胞内抗原的情况下，需要透化缓冲液。 Tmem119是跨膜蛋白，但表位被识别通过抗体染色为细胞​​内19 。在没有细胞透化的情况下，没有标记（ 图30 ）。没有这种渗透步骤可能会导致假阴性结果。由于Tmem119抗体不直接与荧光团缀合，所以用二抗标记细胞;阴性对照是单次二抗（黑线）（ 图3E ，30-3R ）。当使用直接缀合的抗体时，有两种评估特异性标记的主要策略:同种型抗体作为阴性对照或荧光减少一（FMO）对照。 FMO对照包含除了该样品的对照抗体外的混合物中的每个标记抗体。因此，FMO对照对于每个标记是必需的，而每个样品只需要一种标记混合物用于同种型抗体对照。为了标记脑免疫细胞，我们选择了等位基因ype抗体控制，因为在来自一只小鼠脑的密度梯度之后获得有限数量的细胞（40万个细胞）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55781/55781fig1.jpg" /> 图1:细胞解离过程的步骤。 （ A ）将组织置于具有消化混合物酶的管中。 （ B ）用剪刀将组织切成小块。 （ C ）将小片组织孵育30分钟（37℃）以进行有效的组织消化。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55781/55781fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55781/55781fig2.jpg" /> 图2:Cen后密度梯度图微调步骤离心后，髓鞘存在于密度梯度的顶部，含有巨噬细胞群体的免疫细胞在37％和70％密度梯度层之间的间期发现。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55781/55781fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55781/55781fig3.jpg" /> 图3:巨噬细胞的代表性流式细胞术分析。应用逻辑门控策略:（ A ）双标识别是指点图FSC-A 与 FSC-H。 （ B ）形态门控策略是指点图FSC-A 与 SSC-A。 （ C ）巨噬细胞亚群是指CD45 与 CD11b的点图。 （ D ）细胞的点图以同种型抗体为CD45和CD11b染色对照。这两个巨噬细胞亚群表达的标记:Tmem119（ E ），CX3CR1（ F ）和CCR2（ G ）（门P2中的10,000个事件）的分析。在密度梯度（ H ）之前或密度梯度分离和透化处理（ I ）或不含（ J ）之后，显示脑和脊髓细胞的细胞大小（FSC-A）和粒度（SSC-A） 。 （ K ）流式细胞术直方图说明密度梯度后死细胞的百分比。点阵图中显示的门分别说明脑和脊髓（ L ）或脑（ M ）或脊髓（ N ）中的CD11b + CD45 细小胶质细胞群体和CD11b + CD45 高 MDM群体。分析Tmem119表达通过渗透（ O ）或非透化（ P ）的脑和脊髓细胞中的巨噬细胞亚群，以及脑（ Q ）或脊髓（ R ）透化细胞中的巨噬细胞亚群。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55781/55781fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>体积为1管（mL） </td><td>密度梯度介质1x </td><td> PBS 10x </td><td> PBS 1x </td></tr><tr><td>密度梯度介质30％ </td><td> 1.5 </td><td> 0.15 </td><td> 3.35 </td></tr><tr><td>密度梯度介质37％ </td><td> 1.85 </td><td> 0.185 </td><td> 2.965 </td></tr><tr><td>密度梯度介质70％ </td><td> 3.5 </td><td> 0.35 </td><td> 1.15 </td></tr></tbody></table>表1:密度梯度介质的不同百分比的组成。

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 ...

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Nanyang Technological University

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Immunology and Infection

23.6K 次观看.  Inserm U 1227, CNRS UMR 7225. 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。 

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

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

大脑和脊髓的单核细胞的分离，使用Percoll梯度

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

科研

免疫与感染

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

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 ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

神经科学

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

使用流式细胞术进行小鼠脑组蛋白修饰定量

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

作者聚焦:基因表达调控研究的增强

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

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

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此视频中的章节

大脑和脊髓的单核细胞的分离，使用Percoll梯度

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

评估视网膜小胶质细胞吞噬功能

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰