这项工作是由NIEHS的R01ES014470（KMZ）的支持。

再开始此协议，在出生后几天收集新生小鼠1-3（P1-3）在无菌容器中嵌套原笼床上用品的保护和温暖。它可以快速，高效地工作，通过该协议来优化小胶质收益率是很重要的。请参阅表1为完整的试剂列表。 1。仪器仪表，文化传媒，以及菜肴的制备<ol><li>制备出10毫升/小狗小胶质细胞完全培养基（MCM，1个最低必需培养基Earle的[存储]补充有:1 2mM L-谷氨酰胺，1mM丙酮酸钠，0.6％体积/体积的D-（+） - 葡萄糖，100微克/毫升青霉素/链霉素（P / S），4％V / V胎牛血清（FBS），6％v / v马血清），并添加到每个T-75瓶（1瓶/小狗）;帽和地方水平成培养箱（37℃，5％CO 2）中平衡1小时。 重要的是在开始剥离之前达到平衡这一媒体在烧瓶（次）。 </li><li>准备相再次，分装，并在温暖的37℃水浴MCM解剖（6毫升/小狗），并在一个单独的锥形管的MCM细胞悬浮（2毫升/小狗）。制备，分装，并冰上冷却介质切碎（MM; HBSS中补充有100微克/毫升的P / S，0.01M HEPES，3毫升/小狗）和夹层媒体（DM; MEM补充了100微克/毫升的P / S;为2ml /小狗）。 </li><li>消毒以下手术工具浸没于70％乙醇10分钟:剪刀＃1（1;杀头），钳子＃1（2，确保鼻子和去除皮质），剪刀＃2（1; nonangled，打开头皮），钳子＃2（1，去除头皮），钳子＃3（1，去除颅骨），钳子＃4（2，去除脑膜），剪刀＃3（1，剁碎的脑组织）。风干的无菌垫。 </li><li>擦拭用70％乙醇的水平气流工作站和解剖显微镜。 </li><li>免除糖尿病分为2无菌100毫米培养皿捕捉头（3毫升; 1碟/ 3的幼崽）和解剖（1毫升;1碟/小狗;前清扫冰上冷却）。分装成的MM无菌1 100毫米的培养皿用于切碎脑组织（1毫升; 1碟/小狗）。 </li></ol> 2。脑组织解剖<ol><li>在此之前安乐死，使用实验室组织轻轻消毒颈部和小狗的头部用70％乙醇。用剪刀＃1，很快就被除去头安乐死的小狗;捕获的头在先前准备的培养皿中。安乐死，并继续下一个动物之前完成一个小狗的解剖。 </li><li>头部转移到冷冻解剖盘，解剖显微镜下固定头使用镊子＃1（或者，头部可以通过眼窝刺入血管钳固定）捏鼻子，用剪刀＃2开头皮通过创建一个Y切:从头部的基地，并在朝着眼睛的角度开始。使用镊子＃3由横向剥落轻轻地取下头皮，暴露颅骨。 </li><li>使用镊子＃3，轻轻在底座上卸下结缔组织头。当颅底暴露出来，小心地打开颅骨通过螺纹钳子的大脑和颅骨（下半球间裂）之间的一个臂，抓地力;轻轻一拉头骨向上撕裂开来。 </li><li>根据打乱大脑的腹侧结缔组织使用镊子＃1，随后使用相同的工具取出大脑。通过使用大脑的腹侧下一个向上的力提起了脑颅骨。 </li><li>保持大脑在其解剖位置:背侧朝上，以可视化的解剖显微镜下的皮质，在中脑和皮质的交点插入镊子尖端固定大脑到位;彻底去除皮质脑膜与镊子＃4，然后进行删除腹侧脊膜。一旦脑膜已经完全去除，从用钳子＃4的脑的其余部分分离的皮层， 它是必不可少的，以完全除去脑膜组织最大化的小胶质细胞的纯度和yielð。 </li></ol> 3。脑组织切碎<ol><li>皮质转移到事先准备好的切碎的菜;继续使用无菌技术在II类生物安全柜切碎的一步。 </li><li>用剪刀＃3切末脑组织;转移组织糜到15毫升锥形管;冲洗剪刀和切碎的菜用1毫升的MM各，收集和分配冲洗进15毫升锥形管中组织糜此冲洗步骤将最大化小胶质细胞通过确保所有残留的组织转移至锥形管中的产率 。离开组织悬液静置1-2分钟，使组织糜，收于试管底部。 不要超过2分钟的MM不含有人体必需的营养素。 </li><li>小心地取出并丢弃2毫升上清液与通用1,000微升移液器与标准的枪头，使某些不破坏落户剁碎组织。 </li&gt; <li>加3毫升的MCM的碎组织，使用通用的1,000微升移液器使用标准的枪头全部力量磨碎的组织没有可辨别的实体组织应保持研磨时完成 。添加另一个3毫升MCM具有相同枪头，从移液管尖收集残留研磨样品。 </li></ol> 4。不断增长的皮层细胞<ol><li>离心机组织糜以沉淀细胞（215×g离心，5分钟;放疗;临床用离心机水平转头）。返回沉淀的细胞的生物安全柜，取出，弃去上清液，轻轻重悬细胞沉淀（2毫升/管; MCM）;细胞悬浮添加到先前准备的T-75瓶中，盖上瓶和水平放置到湿润的标准组织培养箱（37℃，5％CO 2）。使细胞温育24小时。 </li><li> 24小时后，轻轻拍打瓶对手掌破坏组织碎片。之前和攻丝以确保完全去除杂物后，检查细胞。集烧瓶垂直;取出并丢弃媒体;添加新鲜的MCM到烧瓶的底部（12毫升; 37℃）;帽和放置放回培养箱中，水平。 </li><li>每天检查细胞的污染和监测的增长。 </li><li> 在体外 （DIV）约17-21天，小胶质细胞会准备好收成从混合胶质文化。如果要判断细胞准备，检查倒置相差显微镜下观察细胞。 小胶质细胞是准备收获时在〜40％汇合。它们显示为小，明亮，球形细胞，成长为对其他神经胶质细胞顶部的单层。 </li></ol> 5。从混合胶质细胞培养分离的小胶质细胞<ol><li>隔离的小胶质细胞，大力挖掘对烧瓶实验台。这有助于鼓动来自其他神经胶质细胞，由于小胶质细胞的低粘附性分开的小胶质细胞。 </li><li&gt;印章的T-75瓶瓶盖封口膜和旋转的混合胶质细胞培养（200转，37℃，5小时），使用温度控制，nonhumidified旋转摇床不要让细胞振摇5小时更长的目视检查。那小胶质细胞已经抬离烧瓶表面5小时后，用倒置相差显微镜， 小胶质细胞是光明的，球形的，自由浮动的细胞。会有星形胶质细胞和少突胶质细胞附着在烧瓶底部的残留层。 </li><li>收集含有胶质进入无菌的离心管中的上清液媒体，颗粒小胶质细胞（215×g离心，5分钟;水平转头）。 </li><li>悬浮在温水中的小神经胶质生长培养基（MGM的小胶质沉淀; MEM补充有:1mM丙酮酸钠，0.6％的v / vglucose，1mM的L-谷氨酰胺，100μg/ ml的P / S，5％体积/体积的FBS;〜2毫升/瓶），确定使用血球细胞浓度，并且板1×10 5个细胞/孔在24孔板格式上塑料或无菌玻璃盖玻片（500微升/孔; MGM）， 使用此程序的小胶质细胞平均产量为7.5×10 5细胞/小狗皮质。 </li><li> 24小时后镀，取出所有介质，并更换培养基细胞用新鲜的美高梅金殿（37℃）以去除漂浮细胞和碎片。 这一步是至关重要的细胞存活和保持静态的小胶质细胞。细胞投喂后立即可用于实验。 </li></ol> 6。例如治疗<ol><li>暴露的细胞在10纳克帕姆3 CSK 4（PAM）的/毫升，10纳克/毫升的脂多糖（LPS）或溶媒对照;孵育2或24小时后在加湿培养箱（37℃，5％CO 2）。 </li></ol> 7。对于功能性体外通过免疫荧光成像和免疫细胞小胶质细胞分析<ol><li>在微量离心管（1.5ml）和离心泵处理，收获细胞培养介质后ifuge使用桌面离心（2分钟; 900 XG），以清除介质。将上清转移到一个干净的离心管中，立即检测，或在-20℃储存待用。通过市售鼠TNF-αELISA试剂盒细胞培养上清分析TNF-α的浓度。 </li><li>用磷酸盐缓冲盐水（PBS）洗涤细胞，并固定细胞，用4％（重量/体积）低聚甲醛/ 4％（重量/体积）蔗糖/ PBS pH为7.4（4％PFA; 15分钟; RT）。下列固定，用PBS洗涤细胞5分钟轻微振荡轨道摇床。 <ol><li> 免疫/免疫细胞，经过固定，PBS洗涤，工艺小胶质细胞的免疫细胞化学。 开展轻轻摇动所有的步骤 。透化在PBS/0.1％Triton X-100细胞;块单元中一小时PBS/10％正常山羊血清。孵育细胞O / N在4℃下用兔抗NF-κB抗体（P65亚基，1/1，250）或兔抗伊巴-1抗体（1/750）在封闭缓冲液。视觉的IZE抗原:抗体复合物温育后用荧光标记的羊抗兔IgG二抗（1小时，1/1，000）。用PBS/0.1％Triton X-100（3×5分钟）洗涤除去未结合的第二抗体。 </li><li>计数器染色的细胞用4&#39;，6 -二脒基-2 -苯基吲哚/ PBS（DAPI; 0.1微克/毫升）。以可视化的细胞核从CX3CR1-GFP + /导出所有的小胶质细胞-小鼠中表达GFP。利用350nm的波长滤光器，以可视化的DAPI; 488nm的波长滤光器，使GFP可视化;和594 nm的波长滤光器，以可视化p65和IBA-1免疫复合物。 </li></ol></li><li>摩盖玻片使用水性安装的解决方案，并使用荧光显微镜可视化小胶质细胞。 </li></ol>

<ol>
	<li>Ranshoff, R. M. P. V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Physiology:+Unique+Stimuli,+Specialized+Responses.">Microglia Physiology: Unique Stimuli, Specialized Responses.</a> Annu. Rev. Immunol. 27, 119-145 (2009).</li><li>Lawson, L. J., Perry, V. H., Dri, P., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+the+distribution+and+morphology+of+microglia+in+the+normal+adult+mouse+brain.">Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain.</a> Neuroscience. 39, 151-170 (1990).</li><li>Gomez Perdiguero, E., Schulz, C., Geissmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+homeostasis+of+&amp;#34;resident&amp;#34;+myeloid+cells:+The+case+of+the+microglia.">Development and homeostasis of &amp;#34;resident&amp;#34; myeloid cells: The case of the microglia.</a> Glia. 61, 112-120 (2013).</li><li>Kierdorf, K., 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=Microglia+emerge+from+erythromyeloid+precursors+via+Pu.1-+and+Irf8-dependent+pathways.">Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways.</a> Nat. Neurosci. 16, 273-280 (2013).</li><li>Saijo, K., Glass, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+origin+and+phenotypes+in+health+and+disease.+Nature+reviews.">Microglial cell origin and phenotypes in health and disease. Nature reviews.</a> Immunology. , 11-775 (2011).</li><li>Davalos, 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=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nat. Neurosci. 8, 752-758 (2005).</li><li>Nimmerjahn, A., Kirchhoff, F., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+are+highly+dynamic+surveillants+of+brain+parenchyma+in+vivo.">Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.</a> Science. 308, 1314-1318 (2005).</li><li>Hu, 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=Cytokine+and+free+radical+production+by+porcine+microglia.">Cytokine and free radical production by porcine microglia.</a> Clin. Immunol. Immunopathol. 78, 93-96 (1996).</li><li>Muzio, M., Polentarutti, N., Bosisio, D., Prahladan, M. K., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors:+a+growing+family+of+immune+receptors+that+are+differentially+expressed+and+regulated+by+different+leukocytes.">Toll-like receptors: a growing family of immune receptors that are differentially expressed and regulated by different leukocytes.</a> J. Leukocyte Biol. 67, 450-456 (2000).</li><li>Lee, S. J., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+and+inflammation+in+the+CNS.">Toll-like receptors and inflammation in the CNS.</a> Curr. Drug Targets. Inflamm. Allergy. 1, 181-191 (2002).</li><li>Block, M. L., Zecca, L., Hong, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia-mediated+neurotoxicity:+uncovering+the+molecular+mechanisms.">Microglia-mediated neurotoxicity: uncovering the molecular mechanisms.</a> Nat. Rev. Neurosci. 8, 57-69 (2007).</li><li>Halle, 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=The+NALP3+inflammasome+is+involved+in+the+innate+immune+response+to+amyloid-beta.">The NALP3 inflammasome is involved in the innate immune response to amyloid-beta.</a> Nat. Immunol. 9, 857-865 (2008).</li><li>Chen, G. Y., Nunez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+inflammation:+sensing+and+reacting+to+damage.">Sterile inflammation: sensing and reacting to damage.</a> Nat. Rev. Immunol. 10, 826-837 (2010).</li><li>Duewell, P., 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=NLRP3+inflammasomes+are+required+for+atherogenesis+and+activated+by+cholesterol+crystals.">NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.</a> Nature. 464, 1357-1361 (2010).</li><li>Stewart, C. 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=CD36+ligands+promote+sterile+inflammation+through+assembly+of+a+Toll-like+receptor+4.">CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4.</a> 11, 155-161 (2010).</li><li>Beraud, 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=alpha-Synuclein+Alters+Toll-Like+Receptor+Expression.">alpha-Synuclein Alters Toll-Like Receptor Expression.</a> Front. Neurosci. 5, 80 (2011).</li><li>Banati, R. B., Gehrmann, J., Schubert, P., Kreutzberg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+of+microglia..">Cytotoxicity of microglia..</a> Glia. 7, 111-118 (1993).</li><li>Combs, C. K., Karlo, J. C., Kao, S. C., Landreth, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Amyloid+stimulation+of+microglia+and+monocytes+results+in+TNF+alpha-dependent+expression+of+induciblenitric+oxide+synthase+and+neuronal+apoptosis.">beta-Amyloid stimulation of microglia and monocytes results in TNF alpha-dependent expression of induciblenitric oxide synthase and neuronal apoptosis.</a> J. Neurosci. 21, 1179-1188 (2001).</li><li>Kim, Y. S., Joh, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia,+major+player+in+the+brain+inflammation:+their+roles+in+the+pathogenesis+of+Parkinson's+disease.">Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease.</a> Exp. Mol. Med. 38, 333-347 (2006).</li><li>Colton, C., Wilcock, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+activation+states+in+microglia.">Assessing activation states in microglia.</a> CNS Neurol. Disord. Drug Targets. 9, 174-191 (2010).</li><li>Nakajima, K., Tohyama, Y., Kohsaka, S., Kurihara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ceramide+activates+microglia+to+enhance+the+production/secretion+of+brain-derived+neurotrophic+factor+(BDNF)+without+induction+of+deleterious+factors+in+vitro.">Ceramide activates microglia to enhance the production/secretion of brain-derived neurotrophic factor (BDNF) without induction of deleterious factors in vitro.</a> J. Neurochem. 80, 697-705 (2002).</li><li>Uesugi, M., Nakajima, K., Tohyama, Y., Kohsaka, S., Kurihara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonparticipation+of+nuclear+factor+kappa+B+(NFkappaB)+in+the+signaling+cascade+of+c-JunN-terminal+kinase+(JNK)-+and+p38+mitogen-activated+protein+kinase+(p38MAPK)-dependent+tumor+necrosis+factor+alpha+(TNFalpha)+induction+in+lipopolysaccharide+(LPS)-stimulated+microglia.">Nonparticipation of nuclear factor kappa B (NFkappaB) in the signaling cascade of c-JunN-terminal kinase (JNK)- and p38 mitogen-activated protein kinase (p38MAPK)-dependent tumor necrosis factor alpha (TNFalpha) induction in lipopolysaccharide (LPS)-stimulated microglia.</a> Brain Res. , 1073-1074 (2006).</li><li>Beraud, 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=Microglial+Activation+and+Antioxidant+Responses+Induced+by+the+Parkinson's+Disease+Protein+alpha-Synuclein.">Microglial Activation and Antioxidant Responses Induced by the Parkinson's Disease Protein alpha-Synuclein.</a> J. Neuroimmun. Pharmacol. 8, 94-117 (2013).</li><li>Su, X., 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=Synuclein+activates+microglia+in+a+model+of+Parkinson's+disease.">Synuclein activates microglia in a model of Parkinson's disease.</a> Neurobiol. Aging. 29, 1690-1701 (2008).</li><li>Minghetti, L., Levi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+as+effector+cells+in+brain+damage+and+repair:+focus+on+prostanoids+and+nitric+oxide.">Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide.</a> Prog. Neurobiol. 54, 99-125 (1998).</li><li>Hirsch, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+and+Parkinson's+disease.">Glial cells and Parkinson's disease.</a> J. Neurol.. 247 (2), 58-62 (2000).</li><li>Liu, B., 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=Role+of+nitric+oxide+in+inflammation-mediated+neurodegeneration.">Role of nitric oxide in inflammation-mediated neurodegeneration.</a> Ann. NY Acad. Sci. 962, 318-331 (2002).</li><li>Shie, F. S., Nivison, M., Hsu, P. C., Montine, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+microglialinnate+immunity+in+Alzheimer's+disease+by+activation+of+peroxisome+proliferator-activated+receptor+gamma.">Modulation of microglialinnate immunity in Alzheimer's disease by activation of peroxisome proliferator-activated receptor gamma.</a> Curr. Med. Chem. 16, 643-651 (2009).</li><li>Rogers, J., Mastroeni, D., Leonard, B., Joyce, J., Grover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation+in+Alzheimer's+disease+and+Parkinson's+disease:+are+microglia+pathogenic+in+either+disorder.">Neuroinflammation in Alzheimer's disease and Parkinson's disease: are microglia pathogenic in either disorder.</a> Int. Rev. Neurobiol. 82, 235-246 (2007).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murinemicroglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murinemicroglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</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+antigenpresentation+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 antigenpresentation to myelin basic protein-reactive CD4+ T cells compared.</a> J. Immunol. 154, 4309-4321 (1995).</li><li>Ford, A. L., Foulcher, E., Goodsall, A. L., 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=Tissue+digestion+with+dispase+substantially+reduces+lymphocyte+and+macrophage+cell-surface+antigen+expression.">Tissue digestion with dispase substantially reduces lymphocyte and macrophage cell-surface antigen expression.</a> J. Immunol. Methods. 194, 71-75 (1996).</li><li>Pino, P. A., Cardona, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+brain+and+spinal+cord+mononuclear+cells+using+percoll+gradients.">Isolation of brain and spinal cord mononuclear cells using percoll gradients.</a> J. Vis. Exp. , (2011).</li><li>Veremeyko, T., Starossom, S. C., Weiner, H. L., Ponomarev, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+microRNAs+in+microglia+by+real-time+PCR+in+normal+CNS+and+during+neuroinflammation.">Detection of microRNAs in microglia by real-time PCR in normal CNS and during neuroinflammation.</a> J. Vis. Exp. , (2012).</li><li>Su, X., Federoff, H. J., Maguire-Zeiss, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+alpha-synuclein+overexpression+mediates+early+proinflammatory+activity.">Mutant alpha-synuclein overexpression mediates early proinflammatory activity.</a> Neurotox. Res. 16, 238-254 (2009).</li><li>Jung, 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=Analysis+of+fractalkine+receptorCX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptorCX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol. Cell. Biol. 20, 4106-4114 (2000).</li><li>Imai, T., 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=Identification+and+molecular+characterization+of+fractalkine+receptor+CX3CR1,+which+mediates+both+leukocyte+migration+and+adhesion.">Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.</a> Cell. 91, 521-530 (1997).</li><li>Cardona, A. E., 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=Control+of+microglial+neurotoxicity+by+the+fractalkine+receptor.">Control of microglial neurotoxicity by the fractalkine receptor.</a> Nat. Neurosci. 9, 917-924 (2006).</li><li>Streit, W. J., Walter, S. A., Pennell, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+microgliosis.">Reactive microgliosis.</a> Prog. Neurobiol. 57, 563-581 (1999).</li><li>Frank, M. G., Wieseler-Frank, J. L., Watkins, L. R., Maier, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+highly+enriched+and+quiescent+microglia+from+adult+rat+hippocampus:immunophenotypic+and+functional+characteristics.">Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus:immunophenotypic and functional characteristics.</a> J. Neurosci. Methods. 151, 121-130 (2006).</li></ol>

作者宣称，该研究是在没有可能被理解为潜在的利益冲突的任何商业或财务关系进行的。

本程序提供了一种有效的方法皮层小胶质细胞从新生小鼠的隔离。这个过程有:1）保持小胶质细胞免疫表型和功能性两方面的益处，如通过荧光成像，免疫细胞化学和ELISA测定，以及，2）使小胶质细胞在其他神经胶质细胞（星形胶质细胞和oligodentrocytes）的存在之前，成熟隔离，这可能会在培养的神经胶质成熟期间促进重要细胞 - 细胞相互作用。重要的是，分离的细胞具有较高的生存能力和均匀性与保存功能和免疫体外 。 有迹象表明，必须特别注意​​和勤勉尽责地为小胶质细胞具有最高的纯度和收率的成功分离执行几个步骤。这些步骤是:1）在整​​个实验过程保持无菌，健康的文化; 2）从肺心病删除所有脑膜蒂斯斯显微切割过程中，皮质切碎后切碎板和剪刀3）适当的清洗; 4）平衡MCM中的T-75瓶培养箱中显微切割开始前; 5）再投喂24小时后隔离。 由于小胶质细胞是中枢神经系统的驻留免疫细胞，它们是高度敏感环境的侮辱。因此，避免文化污染病原体是保持静止的小胶质细胞的免疫表型和功能属性的关键。日常检查细胞监测污染和增长是不可或缺的避免破坏细胞培养和更好地了解小胶质细胞的生长率。此外，有必要刷新MGM的24小时后的镀敷的小胶质细胞，以维持细胞活力和功能。 一个主要障碍在小胶质细胞中分离，以克服是确保碎CNS组织是自由内皮细胞，其中如果存在的话，可以通过形成密集的族群中的胶质文化阻碍小胶质增生。出于这个原因，重要的是向大脑的显微切割过程中彻底去除CNS脑膜。根据我们的经验，P3新生儿的脑脊膜都更容易比P1新生儿除去，由于组织越发达，更容易在解剖显微镜下可视化。此外，为了避免从头骨移除脑部后润湿脑的背侧与DM是很重要的。这可以通过将其转移到DM时，保持大脑的解剖位置（背侧）来完成。保持大脑干背侧允许更好的可视化，更容易去除脑膜。 我们已经发现，最有效的方式，除去脑膜是插入在纵裂的最后部分上的力＃4和剥离脑膜关闭，横向。一旦背脑膜已被清除，然后我们雷莫通过反转脑（腹侧的一面朝上），并轻轻抓嗅球和剥离背面朝向脑的后部已经腹侧脑膜。然后，我们轻轻地剥离，并在如何运用眼线改变的横向运动消除任何剩余腹脑膜组织。皮层从大脑的其余部分随后的分离是用于分离仅皮质小神经胶质细胞是至关重要的。本文详述的方法也是灵活的，因为它可以被用来从皮层下区域隔离的小胶质细胞，以及。不过，需要注意的是细胞产量可能降低由于小胶质细胞密度淡漠细胞结构的大脑区域40的变异性是很重要的。 我们已发现，以最大限度地提高小胶质细胞的最终产率，以从在切碎过程中使用的材料冲洗并收集冲洗是很重要的。这些冲洗确保所有残留组织的完整集合，可能已经不切碎剪刀，培养皿和吸管尖用于分装的组织糜入锥形管中。重要的是，这些冲洗液转移剁碎CNS组织的锥形管后立即发生，以优化细胞活力。这也是细胞活力的细胞不留的MM，时间超过2分钟时间，因为MM是缺乏必要的营养是至关重要的。 最后，关键的是要准备的T-75烧瓶，MCM，并将其放置在潮湿的培养箱中（37℃，5％CO2）开始之前的显微解剖协议。泰德 - 75瓶已根据其上限，允许气体交换，促进媒体平衡加法混合胶质细胞培养的前一个多孔衬里。我们发现，如果不这样做会导致一个次优的小胶质细胞的收获。同样重要的是，以密封在T-75烧瓶中，盖用Parafilm将烧瓶放置在旋转摇床上以进出烧瓶在旋转的同时最大限度地减少气体交换之前。 <p类="jove_content"&gt;本程序详细说明隔离皮层小胶质细胞从新生小鼠产生胶质较高的平均收益率（7.5×10 5细胞/小狗皮层）比利用消化酶和/或珀先前报道的方法的有效方法梯度（3-5×10 5细胞/整个CNS）30,33,34。就证明了荧光成像，免疫细胞化学，酶联免疫吸附和通过该方法分离的小胶质细胞能够进行形态学和生化反应，当暴露于免疫原性刺激如帕姆和LPS的。因此，本文描述的过程提供了调查在体外条件下的小胶质细胞生物学的实验方法。

小胶质细胞，CNS实质的监视巨噬细胞，包括成年哺乳动物大脑的总细胞群的约12％。小胶质细胞来源于卵黄囊骨髓前体细胞，改变细胞密度和形态在不同的细胞结构区域的成人中枢神经系统1-5范围内。在一个健康的成年人的大脑，小胶质细胞小，分枝或极性细胞，精，动态过程。相较于周边的巨噬细胞的形态，小胶质展示，可能会出现蜂窝闲置健康的大脑静止，低调的表型，但在体内成像研究表明，小胶质细胞的过程中动态地伸出和缩回，以监测他们的微环境的方式让人想起"采样和测量"6,7。 小胶质细胞是高度和差分响应于环境和病理生理改变在大脑中，切换从监视者与效应国家通常被视为他们的休息和激活状态，分别为。这在激活开关可以通过膜结合的模式识别受体（的PRRs），如Toll-样受体（TLRs），其中病原体相关分子模式响应（的PAMP），包括细菌和病毒衍生的脂蛋白的啮合来介导，核酸，碳水化合物和8-11。除的PAMP，模式识别受体也已显示出诱导小胶质细胞活化对被称为危险/损伤相关分子模式（D-AMPS），无菌的，非致病性的分子，其代表在中枢神经系统内环境稳定的扰动，如细 ​​胞损伤12-16。一旦接合，模式识别受体引发的细胞内信号级联放大，结果在变化中的小胶质细胞形态和基因表达，具体而言，活化的小胶质细胞适应的变形虫状的表型，转运的p65的NF-κB亚基（p65蛋白）的细胞的细胞核，并且上调了机生产线CTION和促炎细胞因子，如肿瘤坏死因子-α（TNF-α），白介素-1β（IL-1β），随着活性氧（ROS）的16-24的分泌。虽然积分在CNS中的先天免疫应答，这些分泌的分子也已发现增加神经细胞的氧化应激，从而诱发并加重神经变性疾病状态，如帕金森氏症和阿尔茨海默氏病25-29。 然而，在病理状态的小胶质细胞活化的机制尚未完全了解。因此，小胶质细胞的分离是一个强大的调查工具为这些生物过程，因为许多在小胶质细胞激活体内功能可以在培养中概括。有几种方法可用于通过Percoll梯度以下CNS组织30,31的酶消化分离的小胶质细胞，包括隔离。然而，酶消化可以改变细胞通过减少细胞表面抗原的表达32，结果在每个动物的低细胞产量比本文所描述的方法中的免疫表型。具体来说，我们报告每小狗皮质的平均收益率小胶质细胞7.5×10 5细胞，而以前报道的整个中枢神经系统隔离的方法通过Percoll梯度产量3-5×10 5细胞30,33,34。本程序规避通过隔离基于其低粘附性小胶质细胞，从而保留了小胶质细胞免疫表型和功能的使用消化酶。 在本研究中，我们描述了从新生儿杂CX3CR1-GFP（CX3CR1-GFP + / - ）衍生的混合胶质细胞培养的小胶质细胞的分离通过在旋转摇床上，先前的延长机械搅拌和C57BL / 6小鼠皮层出版法24,35。我们利用前小鼠品系的小胶质细胞易于可视化，这些小鼠表达内源性CX3CR1轨迹的控制下的GFP -单核细胞特异性启动子36-38。这种方法可以产生高纯度的小胶质细胞培养物具有保藏免疫体外 ，当与细菌脂多糖（LPS）或帕姆3 CSK 4挑战就证明了形态变化，p65蛋白的核转位，和TNF-α的分泌，TLR4和TLR1 / 2激动剂元。

<table border="0" cellpadding="0" cellspacing="0" width="680">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="160">
			<td height="160" style="height:160px;width:233px;">Glucose</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:155px;">G8270</td>
			<td style="width:143px;">Make 20% Stock Solution with MilliQ water; filter sterilize; store at 4 &#176;C; shelf life: 3-6 months. Used to make MCM and MGM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Sodium pyruvate 100 mM (100x)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30239.01</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Penicillin/Streptomycin 10,000 units/ml (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15140-122</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM, MGM, MM, and DM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">L-glutamine 200 mM (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">25030-081</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Fetal Bovine Serum (Defined)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30070.03</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:233px;">Minimum Essential Medium Earle&#39;s (MEM)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">15-010-CV</td>
			<td style="width:143px;">Without L-glutamine. Contains Earle&#39;s salts. Used to make MCM, MGM, and DM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Horse Serum</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">16050</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">21-021-CV</td>
			<td style="width:143px;">Without calcium and magnesium. Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">HEPES 1 M</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15630-031</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">T-75 Flask</td>
			<td style="width:149px;">Corning</td>
			<td style="width:155px;">430641</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">4&#39;,6-Diamidino-2-Phenylindole, dilactate (DAPI)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">D3571</td>
			<td style="width:143px;">Used to stain cell nucleus.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-Iba1</td>
			<td style="width:149px;">Wako</td>
			<td style="width:155px;">019-19741</td>
			<td style="width:143px;">Used at 1/750 dilution for ICC staining of Iba1.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-NF&#954;B (p65)</td>
			<td style="width:149px;">Abcam</td>
			<td style="width:155px;">7970</td>
			<td style="width:143px;">Used at 1/1250 dilution for ICC staining for p65.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:233px;">Alexafluor 594 Goat anti-Rabbit IgG (H+L)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">A11012</td>
			<td style="width:143px;">Used at 1/1000 dilution for visualization of antigen:antibody complex in ICC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">10 ml Disposable Serological Pipet</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">13-678-11E</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">50 ml Disposable Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-8</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">15 ml Disposible Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-12</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Sterile Polystyrene Petri Dish</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">875713</td>
			<td style="width:143px;">100 mm x 15 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Straight Metzembaum (scissor #1)</td>
			<td style="width:149px;">Roboz Surgical</td>
			<td style="width:155px;">RS-6010</td>
			<td style="width:143px;">1; 5 inch; used for removing head</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Scissor: Vannas 
			(scissor #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">15000-08</td>
			<td style="width:143px;">1; non-angled; 2.5mm cutting edge; used to open scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Student Vannas (scissor #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91501-09</td>
			<td style="width:143px;">1; curved; used to mince brain tissue</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Forcep: Dumont #7 
			(forcep #1)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91197-00</td>
			<td style="width:143px;">2; used to secure nose and remove cortices&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #2 
			(forcep #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11223-20</td>
			<td style="width:143px;">1; used to remove scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #3 
			(forcep #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11231-30</td>
			<td style="width:143px;">1; used to remove skull</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #5a 
			(forcep #4)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11253-21</td>
			<td style="width:143px;">1; used to remove meninges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table of specific equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Name of Equipment</td>
			<td style="width:149px;">Name of Company</td>
			<td style="width:155px;">Catalogue Number</td>
			<td style="width:143px;">Comments</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Zoom Stereo Dissection Microscope&#160;</td>
			<td style="width:149px;">Olympus</td>
			<td style="width:155px;">SZ4060</td>
			<td style="width:143px;">Microscope is placed inside Laminar-Horizontal Flow Cabinet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Laminar-Horizontal Flow Cabinet</td>
			<td style="width:149px;">Nuaire</td>
			<td style="width:155px;">NU-201-330</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Biological Safety Cabinet</td>
			<td style="width:149px;">Labconco</td>
			<td style="width:155px;">3440001</td>
			<td style="width:143px;">Class II</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:233px;">Water-Jacketed CO2 Incubator</td>
			<td style="width:149px;">VWR</td>
			<td>97025-836</td>
			<td style="width:143px;">Set to 37 &#176;C, 5% CO2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out buckets</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006441</td>
			<td style="width:143px;">To be used with Swing-out rotor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out Rotor</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006445</td>
			<td style="width:143px;">Max Radius: 19.2 (cm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Sorvall Legend RT+ Centrifuge 
			(clinical centrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75-004-377</td>
			<td style="width:143px;">With swing-out rotor</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">AccuSpin Micro 17 microcentrifuge 
			(tabletop microcentrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>S98645</td>
			<td style="width:143px;">With microliter rotor (24 x 1.5/2.0 ml; 
			Cat #: 75003524)&#160;&#160;</td>
		</tr>
	</tbody>
</table>

isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates

从中枢神经系统组织的小胶质细胞的隔离是用于研究小胶质细胞生物学体外强大的调查工具。本发明方法详述的程序用于通过机械搅拌，用旋转摇动器隔离的小胶质细胞的新生鼠皮层。这个小胶质细胞的分离方法产率，表现出在体内正常的，非病理性状况指示静态小胶质细胞的形态和功能特征的高纯度皮质小神经胶质细胞。此过程还保留了小胶质细胞的免疫表型和生化功能就证明了形态变化，NF-κB（p65蛋白）的P65亚基的核转运，和标志促炎性细胞因子的分泌，肿瘤坏死因子-α（TNF-α的诱导），经脂多糖（LPS）和Pam 3 CSK 4（PAM）的挑战。因此，本分离方法保留了两个静态和激活的免疫vated小胶质细胞，提供调查在体外条件下小胶质细胞生物学的实验方法。

保留了小胶质细胞的体外分离过程中的免疫表型和功能是至关重要的，以便能够利用这些细胞作为调查模型胶质生物学。为了说明小胶质immunofunctionality的使用本发明方法的成功保存，我们分离皮质小神经胶质细胞从新生儿P3（CX3CR1-GFP + / -小鼠和C57BL / 6）和处理的培养物与任何LPS或帕姆。 如图1A所示 ，通过搅动小胶质细胞的分离通过旋转振动保留了静态表型归因于在正常体内条件下的小胶质细胞。评估胶质纯度，CX3CR1-GFP + / -细胞培养物进行染色的巨噬细胞的抗原伊巴-1和计数器以可视化的细胞核用DAPI染色。如通过预先选择的窗口显示在图1A和图1B，根据低倍率，小胶质隔离在高纯度的细胞培养ntly所述方法的结果，如大于95％的细胞显示出GFP，伊巴-1，和DAPI的共定位。 接下来，我们试图通过与LPS挑战的细胞，以确认小胶质细胞体外的反应。相比于载体处理，LPS处理CX3CR1-GFP + / -小胶质适于从一个小的，双极型形成了鲜明的形态变化到一个变形虫状形状， 如图1B所示该形态变化也概括在皮质小神经胶质细胞分离从C57BL / 6新生儿帕姆处理（ 图2）。遗传上不同的小胶质细胞之间的这种保守的形态反应，不同的刺激激活，强调可重复性和本议定书的适用性方面的各种实验模型。 尽管这种变化形态是指示激活39的，有必要至determine如小胶质细胞中分离的旋转振动保持其在活化后易位p65的能力，表明功能性的TLR介导的细胞内信号传导的保存。为了证实这一信号级联，我们对待小胶质细胞的C57BL / 6新生儿帕姆隔离。如示于图2中，免疫细胞化学染色对p65的证明的是，在正常情况下，p65蛋白被漫本地化的整个细胞质中，而一个2小时刺激帕姆后，p65蛋白的免疫反应性显着地错开定位于细胞核。 经证实，小胶质细胞通过提出的方法分离本文中保留的分子机制到p65的转位到细胞核中，我们接下来想确认孤立的小胶质细胞保持它们的生化immunofunctionality通过测试其分泌的标志促炎性细胞因子TNF-α，在激活能力。这表现在图3中，在相比于载体处理的小胶质细胞，将细胞暴露于帕姆或LPS增加TNF-α的分泌到培养基中（P &lt;0.0001;单向ANOVA），指示性官能TLR1 / 2 andTLR4的信号通路，分别。 因此结合在一起，这些数据建立的小胶质细胞是通过隔离旋转振动造成的高纯度的细胞培养物保藏与免疫表型和功能性， 在体外 。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51005/51005fig1highres.jpg" src="/files/ftp_upload/51005/51005fig1.jpg" /> 。图1的旋转振动产生高纯度的小胶质细胞培养物保藏与免疫小胶质细胞的分离从CX3CR1-GFP + / -新生小鼠在P3和用载体（A），或细菌脂多糖（B）中处理24小时。固定细胞，用4％PFA，免疫细胞化学染色，伊巴-1和计数器用DAPI染色。 （ 一 ）治疗小胶质细胞用PBS表现出双极性，静态表型。 （二）经LPS刺激，小胶质细胞适应的变形虫样表型，表明激活。在面板下面10X放大率合并的通道（A）和（B）表明&gt; 95％的细胞是GFP/Iba-1阳性细胞。 10X比例尺:100微米; 40X比例尺:20微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51005/51005fig1highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51005/51005fig2highres.jpg" src="/files/ftp_upload/51005/51005fig2.jpg" /> 图2:通过小胶质细胞中分离旋转振荡易位核因子-κB在帕姆茶到细胞核llenge。小胶质细胞，从C57BL / 6新生儿在P3中分离，并与车辆或帕姆处理2小时。固定细胞，用4％PFA和染色通过免疫组织化学的p65。细胞计数用DAPI染色以可视化的细胞核。在车辆处理的小胶质细胞，P65是定位在整个细胞质中，而刺激帕姆诱导的p65核易位。 40X比例尺:20微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51005/51005fig2highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51005/51005fig3highres.jpg" src="/files/ftp_upload/51005/51005fig3.jpg" /> 图3。小胶质细胞分离与旋转振动释放的TNF-α在暴露于帕姆或LPS。小胶质细胞的分离从C57BL / 6新生小鼠在P3和与车辆处理，帕姆，或LPS 2小时。细胞培养物上清液收集并分析了通过ELISA检测TNF-α的释放。 *** p &lt;0.0001（N = 2;单因素方差分析）。数据以均数±标准差。

Watch this Scientific Journal Video about Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates at JoVE.com

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

一键小胶质细胞生物学的研究成功是小胶质细胞免疫功能的体外从中枢神经系统组织分离过程中的保存。通过旋转振动造成的高纯度和immunofunctional细胞培养物中分离的小胶质细胞所评估的荧光成像，免疫细胞化学，ELISA和以下的小胶质细胞活化的促炎性刺激脂多糖（LPS）和Pam 3 CSK 4（PAM）。

皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

isolation-cortical-microglia-with-preserved-immunophenotype

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

15.7K 次观看.  Georgetown University Medical Center. 一键小胶质细胞生物学的研究成功是小胶质细胞免疫功能的体外从中枢神经系统组织分离过程中的保存。通过旋转振动造成的高纯度和immunofunctional细胞培养物中分离的小胶质细胞所评估的荧光成像，免疫细胞化学，ELISA和以下的小胶质细胞活化的促炎性刺激脂多糖（LPS）和Pam 3 CSK 4（PAM）。 

视频: Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

科研

免疫与感染

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

小鼠牙龈细胞的分离，处理和分析

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

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 ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

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

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

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

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

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

隔离和不同的老年小鼠胸腺移植的移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

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

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

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

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.