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>
	<li>Ginhoux, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+analysis+reveals+that+adult+microglia+derive+from+primitive+macrophages.">Fate mapping analysis reveals that adult microglia derive from primitive macrophages.</a> Science. 330 (6005), 841-845 (2010).</li><li>Ginhoux, F., Lim, S., Hoeffel, G., Low, D., Huber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+differentiation+of+microglia.">Origin and differentiation of microglia.</a> Front Cell Neurosci. 7, 45 (2013).</li><li>Ginhoux, F., Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+and+macrophages:+developmental+pathways+and+tissue+homeostasis.">Monocytes and macrophages: developmental pathways and tissue homeostasis.</a> Nat Rev Immunol. 14 (6), 392-404 (2014).</li><li>Schulz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lineage+of+myeloid+cells+independent+of+Myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of Myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</li><li>London, A., Cohen, M., Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+monocyte-derived+macrophages:+functionally+distinct+populations+that+act+in+concert+in+CNS+plasticity+and+repair.">Microglia and monocyte-derived macrophages: functionally distinct populations that act in concert in CNS plasticity and repair.</a> Front Cell Neurosci. 7, 34 (2013).</li><li>Miron, V. E., Franklin, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+CNS+remyelination.">Macrophages and CNS remyelination.</a> J Neurochem. 130 (2), 165-171 (2014).</li><li>Mildner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+and+non-redundant+roles+of+microglia+and+myeloid+subsets+in+mouse+models+of+Alzheimer's+disease.">Distinct and non-redundant roles of microglia and myeloid subsets in mouse models of Alzheimer's disease.</a> J Neurosci. 31 (31), 11159-11171 (2011).</li><li>Boillee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onset+and+progression+in+inherited+ALS+determined+by+motor+neurons+and+microglia.">Onset and progression in inherited ALS determined by motor neurons and microglia.</a> Science. 312 (5778), 1389-1392 (2006).</li><li>Yamasaki, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+roles+of+microglia+and+monocytes+in+the+inflamed+central+nervous+system.">Differential roles of microglia and monocytes in the inflamed central nervous system.</a> J Exp Med. 211 (8), 1533-1549 (2014).</li><li>Wu, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+microglial+activation+is+neuroprotective+in+the+1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine+mouse+model+of+Parkinson+disease.">Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease.</a> J Neurosci. 22 (5), 1763-1771 (2002).</li><li>Cartier, N., Lewis, C. A., Zhang, R., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microglia+in+human+disease:+therapeutic+tool+or+target?.">The role of microglia in human disease: therapeutic tool or target?.</a> Acta Neuropathol. 128 (3), 363-380 (2014).</li><li>Ajami, B., Bennett, J. L., Krieger, C., McNagny, K. M., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infiltrating+monocytes+trigger+EAE+progression,+but+do+not+contribute+to+the+resident+microglia+pool.">Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool.</a> Nat Neurosci. 14 (9), 1142-1149 (2011).</li><li>Funk, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+peripheral+hematopoietic+stem+cells+and+monocytes+in+Parkinson's+disease.">Characterization of peripheral hematopoietic stem cells and monocytes in Parkinson's disease.</a> Mov Disord. 28 (3), 392-395 (2013).</li><li>Butovsky, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+inflammatory+monocytes+with+a+unique+microRNA+gene+signature+ameliorates+murine+ALS.">Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS.</a> J Clin Invest. 122 (9), 3063-3087 (2012).</li><li>Lewis, C. A., Solomon, J. N., Rossi, F. M., Krieger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+cells+in+the+central+nervous+system+of+a+mouse+model+of+amyotrophic+lateral+sclerosis+are+associated+with+blood+vessels+and+express+CX(3)CR1.">Bone marrow-derived cells in the central nervous system of a mouse model of amyotrophic lateral sclerosis are associated with blood vessels and express CX(3)CR1.</a> Glia. 57 (13), 1410-1419 (2009).</li><li>Sedgwick, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+direct+characterization+of+resident+microglial+cells+from+the+normal+and+inflamed+central+nervous+system.">Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.</a> Proc Natl Acad Sci U S A. 88 (16), 7438-7442 (1991).</li><li>Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+adult+ramified+microglia+separated+from+other+central+nervous+system+macrophages+by+flow+cytometric+sorting.+Phenotypic+differences+defined+and+direct+ex+vivo+antigen+presentation+to+myelin+basic+protein-reactive+CD4++T+cells+compared.">Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.</a> J Immunol. 154 (9), 4309-4321 (1995).</li><li>Martin, E., Boucher, C., Fontaine, B., Delarasse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+inflammatory+phenotypes+of+microglia+and+monocyte-derived+macrophages+in+Alzheimer's+disease+models:+effects+of+aging+and+amyloid+pathology.">Distinct inflammatory phenotypes of microglia and monocyte-derived macrophages in Alzheimer's disease models: effects of aging and amyloid pathology.</a> Aging Cell. , (2016).</li><li>Bennett, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+studying+microglia+in+the+mouse+and+human+CNS.">New tools for studying microglia in the mouse and human CNS.</a> Proc Natl Acad Sci U S A. 113 (12), E1738-E1746 (2016).</li><li>Jin, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+glutamine+transporter+SNAT1+in+Rett+syndrome+microglia:+a+mechanism+for+mitochondrial+dysfunction+and+neurotoxicity.">Dysregulation of glutamine transporter SNAT1 in Rett syndrome microglia: a mechanism for mitochondrial dysfunction and neurotoxicity.</a> J Neurosci. 35 (6), 2516-2529 (2015).</li><li>Bedi, S. S., Smith, P., Hetz, R. A., Xue, H., Cox, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomagnetic+enrichment+and+flow+cytometric+characterization+of+mouse+microglia.">Immunomagnetic enrichment and flow cytometric characterization of mouse microglia.</a> J Neurosci Methods. 219 (1), 176-182 (2013).</li><li>Nikodemova, M., Watters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+live+microglia+with+preserved+phenotypes+from+adult+mouse+brain.">Efficient isolation of live microglia with preserved phenotypes from adult mouse brain.</a> J Neuroinflammation. 9, 147 (2012).</li><li>Mizutani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fractalkine+receptor+but+not+CCR2+is+present+on+microglia+from+embryonic+development+throughout+adulthood.">The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.</a> J Immunol. 188 (1), 29-36 (2012).</li><li>Lecoeur, H., Ledru, E., Gougeon, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytofluorometric+method+for+the+simultaneous+detection+of+both+intracellular+and+surface+antigens+of+apoptotic+peripheral+lymphocytes.">A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes.</a> J Immunol Methods. 217 (1-2), 11-26 (1998).</li><li>Richter, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioma-associated+microglia+and+macrophages/monocytes+display+distinct+electrophysiological+properties+and+do+not+communicate+via+gap+junctions.">Glioma-associated microglia and macrophages/monocytes display distinct electrophysiological properties and do not communicate via gap junctions.</a> Neurosci Lett. 583, 130-135 (2014).</li><li>Mahad, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+CCR2+and+CCL2+at+the+blood-brain+barrier:+relevance+for+multiple+sclerosis+pathogenesis.">Modulating CCR2 and CCL2 at the blood-brain barrier: relevance for multiple sclerosis pathogenesis.</a> Brain. 129 (Pt 1), 212-223 (2006).</li></ol>

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 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（ 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 ...

analysis-microglia-monocyte-derived-macrophages-from-central-nervous

Nanyang Technological University

Research

JoVE Journal

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

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

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

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

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

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

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

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

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 ...

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

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

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

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

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

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

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

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

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

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 between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

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 ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

小鼠肾-居民CD8+T 细胞的分离，用于流细胞测量分析

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...