这项工作是支持的美国国立卫生研究院神经疾病和中风（NIH / NINDS）授予R01-NS074387，R01-NS057711和R21-NS091555到的MGC /研究所; NIH / NINDS授予R01-NS061107，R01-NS076991，R01-NS082311和R21-NS084275到PRL;从利亚的快乐心助学金，密歇根大学综合癌症中心颁发给MGC和PRL大学;神经外科，医学密歇根大学的大学部;密歇根州的临床规范研究所与健康研究，由美国国立卫生研究院资助2UL1-TR000433支持;由NIH / NCI（国家癌症研究所）资助T32-CA009676支持的密歇根大学癌症生物学培训资助;密歇根培训的大学临床和基础神经科学由美国国立卫生研究院/ NINDS支持授予T32-NS007222;和密歇根大学医学院科学家培训项目的大学由美国国立卫生研究院/ NIGMS（通用医学科学研究所）的支持给予T32-GM007863。作者一再次感谢来自卡琳Muraszko博士和神经外科接收的学术领导和支持;到M.达尔格伦，D TomFord的，和S. Napolitan高超的行政支持;以M. Dzaman优秀的技术援助;和菲尔楼詹金斯向购买蔡司3D扫描电子显微镜的慷慨支持。我们也承认Kuchroo实验室在哈佛医学院从脑单个核细胞分离的密度离心媒体介导的战略的修改后的版本被吸引。

<ol>
	<li>Bleeker, F. E., Molenaar, R. J., Leenstra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+molecular+understanding+of+glioblastoma.">Recent advances in the molecular understanding of glioblastoma.</a> J Neurooncol. 108, 11-27 (2012).</li><li>Stupp, 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=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> N Engl J Med. 352, 987-996 (2005).</li><li>Ostrom, Q. 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=CBTRUS+statistical+report:+primary+brain+and+central+nervous+system+tumors+diagnosed+in+the+United+States+in+2007-2011.">CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2007-2011.</a> Neuro Oncol. 16, iv1-63 (2014).</li><li>Louveau, 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=Structural+and+functional+features+of+central+nervous+system+lymphatic+vessels.">Structural and functional features of central nervous system lymphatic vessels.</a> Nature. 523, 337-341 (2015).</li><li>Kennedy, B. 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=Tumor-associated+macrophages+in+glioma:+friend+or+foe?.">Tumor-associated macrophages in glioma: friend or foe?.</a> J Oncol.. 2013, 486912 (2013).</li><li>Markovic, D. S., Glass, R., Synowitz, M., Rooijen, N., Kettenmann, 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+stimulate+the+invasiveness+of+glioma+cells+by+increasing+the+activity+of+metalloprotease-2.">Microglia stimulate the invasiveness of glioma cells by increasing the activity of metalloprotease-2.</a> J Neuropathol Exp Neurol. 64, 754-762 (2005).</li><li>Zhai, H., Heppner, F. L., Tsirka, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia/macrophages+promote+glioma+progression.">Microglia/macrophages promote glioma progression.</a> Glia. 59, 472-485 (2011).</li><li>Kmiecik, J., 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=Elevated+CD3++and+CD8++tumor-infiltrating+immune+cells+correlate+with+prolonged+survival+in+glioblastoma+patients+despite+integrated+immunosuppressive+mechanisms+in+the+tumor+microenvironment+and+at+the+systemic+level.">Elevated CD3+ and CD8+ tumor-infiltrating immune cells correlate with prolonged survival in glioblastoma patients despite integrated immunosuppressive mechanisms in the tumor microenvironment and at the systemic level.</a> J Neuroimmunol. 264, 71-83 (2013).</li><li>Yang, I., Han, S. J., Sughrue, M. E., Tihan, T., Parsa, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+infiltrate+differences+in+pilocytic+astrocytoma+and+glioblastoma:+evidence+of+distinct+immunological+microenvironments+that+reflect+tumor+biology.">Immune cell infiltrate differences in pilocytic astrocytoma and glioblastoma: evidence of distinct immunological microenvironments that reflect tumor biology.</a> J Neurosurg. 115, 505-511 (2011).</li><li>Wagner, 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=Microglial/macrophage+expression+of+interleukin+10+in+human+glioblastomas.">Microglial/macrophage expression of interleukin 10 in human glioblastomas.</a> Int J Cancer. 82, 12-16 (1999).</li><li>El Andaloussi, A., Lesniak, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+increase+in+CD4+CD25+FOXP3++regulatory+T+cells+in+tumor-infiltrating+lymphocytes+of+human+glioblastoma+multiforme.">An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme.</a> Neuro Oncol. 8, 234-243 (2006).</li><li>Kostianovsky, A. M., Maier, L. M., Anderson, R. C., Bruce, J. N., Anderson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+regulation+of+human+monocytic/microglial+activation.">Astrocytic regulation of human monocytic/microglial activation.</a> J Immunol. 181, 5425-5432 (2008).</li><li>Curtin, J. 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=HMGB1+mediates+endogenous+TLR2+activation+and+brain+tumor+regression.">HMGB1 mediates endogenous TLR2 activation and brain tumor regression.</a> PLoS Med. 6, e10 (2009).</li><li>Curtin, J. 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=Fms-like+tyrosine+kinase+3+ligand+recruits+plasmacytoid+dendritic+cells+to+the+brain.">Fms-like tyrosine kinase 3 ligand recruits plasmacytoid dendritic cells to the brain.</a> J Immunol. 176, 3566-3577 (2006).</li><li>Mineharu, Y., 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=Engineering+the+brain+tumor+microenvironment+enhances+the+efficacy+of+dendritic+cell+vaccination:+implications+for+clinical+trial+design.">Engineering the brain tumor microenvironment enhances the efficacy of dendritic cell vaccination: implications for clinical trial design.</a> Clin Cancer Res. 17, 4705-4718 (2011).</li><li>Larocque, 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=Exogenous+fms-like+tyrosine+kinase+3+ligand+overrides+brain+immune+privilege+and+facilitates+recognition+of+a+neo-antigen+without+causing+autoimmune+neuropathology.">Exogenous fms-like tyrosine kinase 3 ligand overrides brain immune privilege and facilitates recognition of a neo-antigen without causing autoimmune neuropathology.</a> Proc Natl Acad Sci U S A. 107, 14443-14448 (2010).</li><li>Curtin, J. 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=Treg+depletion+inhibits+efficacy+of+cancer+immunotherapy:+implications+for+clinical+trials.">Treg depletion inhibits efficacy of cancer immunotherapy: implications for clinical trials.</a> PLoS One. 3, e1983 (2008).</li><li>Ali, 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=Combined+immunostimulation+and+conditional+cytotoxic+gene+therapy+provide+long-term+survival+in+a+large+glioma+model.">Combined immunostimulation and conditional cytotoxic gene therapy provide long-term survival in a large glioma model.</a> Cancer Res. 65, 7194-7204 (2005).</li><li>Dewey, R. 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=Chronic+brain+inflammation+and+persistent+herpes+simplex+virus+1+thymidine+kinase+expression+in+survivors+of+syngeneic+glioma+treated+by+adenovirus-mediated+gene+therapy:+implications+for+clinical+trials.">Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials.</a> Nat Med. 5, 1256-1263 (1999).</li><li>Baker, G. J., 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=Natural+killer+cells+eradicate+galectin-1-deficient+glioma+in+the+absence+of+adaptive+immunity.">Natural killer cells eradicate galectin-1-deficient glioma in the absence of adaptive immunity.</a> Cancer Res. 74, 5079-5090 (2014).</li><li>Baker, G. J., 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=Mechanisms+of+glioma+formation:+iterative+perivascular+glioma+growth+and+invasion+leads+to+tumor+progression,+VEGF-independent+vascularization,+and+resistance+to+antiangiogenic+therapy.">Mechanisms of glioma formation: iterative perivascular glioma growth and invasion leads to tumor progression, VEGF-independent vascularization, and resistance to antiangiogenic therapy.</a> Neoplasia. 16, 543-561 (2014).</li><li>Ormerod, M. G., Novo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Flow cytometry : a basic introduction. , (2008).</li><li>Stirling, D. P., Yong, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+inflammatory+response+after+murine+spinal+cord+injury+revealed+by+flow+cytometry.">Dynamics of the inflammatory response after murine spinal cord injury revealed by flow cytometry.</a> J Neurosci Res. 86, 1944-1958 (2008).</li><li>Hirai, 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=The+prevalence+and+phenotype+of+activated+microglia/macrophages+within+the+spinal+cord+of+the+hyperostotic+mouse+(twy/twy)+changes+in+response+to+chronic+progressive+spinal+cord+compression:+implications+for+human+cervical+compressive+myelopathy.">The prevalence and phenotype of activated microglia/macrophages within the spinal cord of the hyperostotic mouse (twy/twy) changes in response to chronic progressive spinal cord compression: implications for human cervical compressive myelopathy.</a> PLoS One. 8, e64528 (2013).</li><li>Fleming, T. J., Fleming, M. L., Malek, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+expression+of+Ly-6G+on+myeloid+lineage+cells+in+mouse+bone+marrow.+RB6-8C5+mAb+to+granulocyte-differentiation+antigen+(Gr-1)+detects+members+of+the+Ly-6+family.">Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family.</a> J Immunol. 151, 2399-2408 (1993).</li><li>Wiesner, S. 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=De+novo+induction+of+genetically+engineered+brain+tumors+in+mice+using+plasmid+DNA.">De novo induction of genetically engineered brain tumors in mice using plasmid DNA.</a> Cancer Res. 69, 431-439 (2009).</li><li>Holland, E. 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=Combined+activation+of+Ras+and+Akt+in+neural+progenitors+induces+glioblastoma+formation+in+mice.">Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice.</a> Nat Genet. 25, 55-57 (2000).</li><li>Lynes, J., 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=Lentiviral-induced+high-grade+gliomas+in+rats:+the+effects+of+PDGFB,+HRAS-G12V,+AKT,+and+IDH1-R132H.">Lentiviral-induced high-grade gliomas in rats: the effects of PDGFB, HRAS-G12V, AKT, and IDH1-R132H.</a> Neurotherapeutics. 11, 623-635 (2014).</li><li>de Vries, N. 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=Rapid+and+robust+transgenic+high-grade+glioma+mouse+models+for+therapy+intervention+studies.">Rapid and robust transgenic high-grade glioma mouse models for therapy intervention studies.</a> Clin Cancer Res. 16, 3431-3441 (2010).</li><li>Uhrbom, 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=Ink4a-Arf+loss+cooperates+with+KRas+activation+in+astrocytes+and+neural+progenitors+to+generate+glioblastomas+of+various+morphologies+depending+on+activated+Akt.">Ink4a-Arf loss cooperates with KRas activation in astrocytes and neural progenitors to generate glioblastomas of various morphologies depending on activated Akt.</a> Cancer Res. 62, 5551-5558 (2002).</li><li>Marumoto, 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=Development+of+a+novel+mouse+glioma+model+using+lentiviral+vectors.">Development of a novel mouse glioma model using lentiviral vectors.</a> Nat Med. 15, 110-116 (2009).</li><li>Assanah, 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=Glial+progenitors+in+adult+white+matter+are+driven+to+form+malignant+gliomas+by+platelet-derived+growth+factor-expressing+retroviruses.">Glial progenitors in adult white matter are driven to form malignant gliomas by platelet-derived growth factor-expressing retroviruses.</a> J Neurosci. 26, 6781-6790 (2006).</li></ol>

作者什么都没有透露。

这个协议描述一种可再生的方法的PBMC的分离和流式细胞仪分析已渗入早期鼠脑肿瘤微环境。在由被立体定位植入到小鼠脑的纹状体的实验者指定的浓度产生神经胶质瘤细胞悬浮液。小鼠然后安乐死在由实验设计指定的预定的时间点和他们的大脑收获和加工以分离这些免疫标记与荧光标记的初级抗体的组合胶质瘤浸润的PBMC;免疫标记细胞，然后通过流式细胞术进行定量。虽然这里给出的示范着重于早期时间点，神经胶质瘤浸润的PBMC可在任何时间点后的肿瘤细胞植入直至鼠标濒死进行评估。该方法是高度模块化作为任意数量的不同抗体的组合可以被用于检查感兴趣我免疫细胞ncluding T细胞，B细胞，NK细胞，单核细胞和巨噬细胞。请注意，协议进行了优化与雌性C57BL / 6J小鼠8-10周龄使用。此年龄范围之外的小鼠可能改变循环的PBMC，这可能会导致细胞计数在等效时间点不属于代表此处呈现的数据的百分比。也可能需要表征实验如果替代肿瘤模型使用或如果小鼠比对B6背景其它用于由于这样的事实，菌株可在不同的PBMC型材（Jaxpheno6;小鼠Phenome数据库; Jackson实验室）。性别与环境条件下也能PBMC的频率和百分比之间的差距的来源。 此方法已被使用四种细胞表面的抗体，使检测的组合表现出的Gr-1 + / CD11b的+髓样细胞和NK1.1 + NK细胞，细胞类型牵连ガ的排斥升-1缺失神经胶质瘤。列入细胞内GZMB抗体的进一步使细胞毒性潜力的NK细胞亚群的检测。提供代表结果的免疫细胞浸润到初GL26-CIT胶质瘤在同系的C57BL / 6J小鼠，要么表达加仑-1的正常水平或已减少了因的shRNA介导的基因敲低的水平。示范通过表明胶质瘤浸润的PBMC可再现地分离，并尽快后24小时肿瘤植入定量表现出技术的灵敏度和重现性。除了 ​​揭示肿瘤浸润GR-1 的高 /的CD11b +髓样细胞群，演示也揭示了GR-1 INT ​​人口/的CD11b +细胞 ，其身份目前是不确定的。另外的实验是必要的，以进一步破译该细胞群的特征。我们还注意到，我们没有发现CD45的容易区分人口<suP&gt; INT / GR-1 - / CD11b的高 /NK1.1 -先前由他人23,24描述小胶质细胞相一致。造成这种结果的简单解释是，这里使用的特定隔离协议排除在七十〇分之三十七密度离心媒体界面的积累。同样重要的是要指出，从70分之37密度离心接口收集细胞似乎不包括神经胶质瘤细胞本身。这是显而易见的事实，GL26-CIT胶质瘤细胞，100-200K之间在SSC-A和100​​K中出现FSC-A采用相当于PMT电压为那些在这个演示中使用（数据未显示）均来自FSC-缺席A对SSC-A地块（参见图5a步骤1）。 抗Gr-1抗体识别两者Ly6G和的Ly6C细胞表面蛋白所特有的多形和单核骨髓细胞， 分别为25。使用抗GR-1 antibodIES从而排除了这两种细胞群之间的区别。在我们的实验室的初步结果现在开始揭示早期GR-1 + / CD11b的+细胞浸润GAL-1缺失的神经胶质瘤微环境中更准确的描述光。 （:1A8克隆）和抗的Ly6C（克隆:AL-21）结合的抗Ly6G实验用抗CCR2抗体抗体一起现在表明的Gr-1 高 / CD11b的+细胞浸润初期加仑-1缺失肿瘤微（数据未显示）与高的Ly6C / CCR2 高炎性单核细胞相一致，尽管进一步的实验都必须确认此结果。 由于细胞失去期间参与分离胶质瘤浸润的PBMC重复离心和再悬浮的步骤可能发生，很可能观察到细胞计数实际上比什么都实际存在于完整的脑。然而，电子之间的差异xperimental组应如果七十○分之三十七密度离心媒体接口的等效体积的提取和如果每个样品的整个体积由流式细胞仪进行分析。如果不遵守这些步骤将导致样品之间的不公平比较，将妨碍公正的分析。量化进入大脑肿瘤微环境的PBMC的确切数目不能不是限制特定于此协议。替代方法，如免疫组织细胞计数策略也受到错误，由于基于体视计数和等效图像阈值对组织切片的显微照片的要求总细胞数的推断，其可以不同的背景染色的水平，从而可能导致假阴性或假阳性信号。然而，不同的分析方法之间的时程分析的比较应该显示类似的整体造型与时间相关的免疫涌入曲线。一个另外的缺点，以免疫组化方法是有效的流式细胞术结合在福尔马林固定的脑组织切片的上下文等同抗原性表位的某些抗体的无力。 还有在这个协议可以作为那些不熟悉其方法的局限性的几个关键步骤。一个这样的步骤是从颅骨大脑的收获而不损坏它。我们建议练的技术多次运行适当的实验之前。然而，由于大脑将最终被研磨，到大脑的浅层轻微损坏可能无关紧要胶质瘤浸润的PBMC的准确定量。没有经验的研究者可以最初发现难以第二步骤是密度离心媒体梯度的倾倒。该实验家"，形成一个简洁的界面，而覆盖在上面2毫升37％密度离心媒体的能力在70％的层是至关重要的PBMC可重放隔离。虽然这一步可能最初证明具有挑战性的，它极大地丰富的PBMC和大大降低的时间段另有要求试样分析如果全脑组织其中待cytometrically分析流动。 PBMC富集也减少了由流式细胞仪捕获的，因而减少.fcs文件大小和帮助解决稀有脑浸润免疫细胞群的事件的总数。为了建立一个干净的密度梯度离心接口，建议使用具有平滑柱塞作用的P-1000微量浇37％密度离心媒体的最佳效果。角度的15ml离心管中约有20-30度高于台面。休息微量的尖端上的管3-5毫升标记的70％密度离心介质层的表面上方的一侧。倒入平稳，慢慢等，以最大限度地减少37％，密度离心媒体，因为它extru的公开向外势头从宫为了微量移液器尖端，以避免干扰底层70％密度离心媒体。最后，一​​个事实，即脑组织结构在分离的过程中必然破坏神经胶质瘤浸润的PBMC意味着额外的小鼠将需要获得时间点匹配的组织学数据。 通过注入的致癌基因的质粒DNA进入正常啮齿动物大脑中产生较新的神经胶质瘤模型已开发近年来26-32。这些"内生"肿瘤模型规避造成立体定向移植体外癌细胞，更加紧密地模仿的临床神经胶质瘤的组织学特点的潜在假象。本文所描述的方法是非常适合于表征这些更临床相关的模型系统免疫浸润事件的研究。研究在新生小鼠免疫涌入也可以使用这种方法虽然新生儿备忘录的密度进行Ë脑小于成年人，这可能需要的酶消化步骤的优化，以防止过度的脑组织消化。该技术的其它应用包括调查关于替代类的炎性脑疾病除了恶性肿瘤如实验性变应性脑脊髓炎（EAE）和免疫​​反应的病毒感染。

胶质瘤是一类从转化胶质中枢神经系统（CNS）内所产生的神经上皮脑癌。在所有的胶质瘤，世界卫生组织（WHO）Ⅳ级神经胶质瘤，或胶质母细胞瘤（GBM），是最常见和致命的1。 GBM是高度耐火到当前标准的转交它由肿瘤切除术，以随后辐射加伴随和辅助化疗与替莫唑胺2尽可能。这些致命的癌症随身携带的只有15〜18个月的生存率从最初诊断时，只有5％的患者生存的疾病预后极差5 年 1之后。 的血脑屏障（BBB）的存在，缺乏专业的抗原呈递细胞的（的APC），和脑4内的以前未知的存在真正的淋巴结构导致基底膜免疫特权的概念。然而，大量的研究现在SHO瓦特这些脑癌确实产生外周免疫细胞主要是粒细胞中的起源，其中包括单核细胞，巨噬细胞和髓源抑制细胞（肌源性干细胞）5的募集。 GBM还影响脑驻留小胶质细胞的活性而成为亲致瘤6,7。淋巴样细胞例如CD8 + T细胞8和CD56 +自然杀伤细胞9也是在肿瘤微环境中存在的，但在少得多的数量，想到了一个事实是由于策动胶质瘤衍生的因子对肿瘤相关巨噬细胞的免疫抑制功能（噬细胞）10。 CD4 + T细胞也存在于GBM，但许多这类人群也表达CD25和FoxP3的，免疫调节性T（ 调节性T细胞）11机。 GBM的整体免疫抑制状态的高潮在促进免疫逃逸和肿瘤进展12。 13-19。这项工作的高潮已经导致了旨在评估组合的细胞毒性和免疫刺激治疗的患者初诊GBM（ClinicalTrials.gov标识符:NCT01811992）临床试验。 我们最近的工作表明，小鼠GL26和大鼠CNS-1 GBM细胞通过产生大量的β半乳糖苷结合凝集素半乳糖凝集素1（GAL-1）20块抗肿瘤NK细胞的免疫监视。这证明通过抑制加仑-1在神经胶质瘤细胞使用shRNA介导的基因敲除的表达。 体外 experimenTS表明加仑-1缺失神经胶质瘤细胞中培养正常增殖，但颅内植入进同源的C57BL / 6J或RAG1之后不久进行快速排斥- / -小鼠 ，从而建立T-或B细胞的独立性对这种形式的肿瘤排斥。 NK细胞免疫耗竭具有抗唾液酸GM 1抗血清或导致颅内GAL-1缺失的脑胶质瘤生长完全恢复单克隆抗体NK1.1，建立NK细胞GAL-1缺失的神经胶质瘤抑制的作用。我们现在表明的Gr-1 + / CD11b的+髓样细胞的免疫耗竭足以防止加仑-1缺失胶质瘤排斥尽管NK细胞的存在，从而揭示在NK介导的伽援用于骨髓细胞一个不可或缺的辅助作用的-1缺陷型肿瘤溶解（未公布的数据）。这一出人意料的结果使我们制定一个全面的协议，用于外周血单核细胞的分离和分析（外周血单个核细胞），其渗透到脑肿瘤微环境很快颅内植入后，让我们可以更好地表征，上游GAL-1缺失的胶质瘤抑制免疫渗透活动。 该方法是这里演示用鼠标GL26胶质瘤细胞组成性表达mCitrine荧光蛋白，叫GL26-CIT，它通过荧光显微镜21个州允许直接肿瘤细胞的可视化。这些细胞被立体定位植入进同源C57BL / 6J小鼠的脑和被允许之前小鼠安乐死生长24，48，或72小时。胶质瘤浸润的PBMC，然后分离和免疫标记使用抗-CD45，-GR-1，​​-CD11b和-NK1.1细胞表面的抗体与细胞内的免疫标记为颗粒酶B（GZMB）。这种特异性抗体的组合允许肿瘤浸润的Gr-1 + / CD11b的+髓样细胞的鉴定和NK1.1 + NK细胞，细胞类型，我们有bEEN牵连GAL-1缺失的肿瘤排斥。的GAL-1缺失GL26-CIT胶质瘤免疫浸润轮廓，这里称为GL26-CIT-gal1i，然后比较该胶质瘤表达加仑-1称为GL26-CIT-NT的正常水平，包含一非易失的针对对照shRNA的发夹。该协议开始于上GL26-CIT胶质瘤如何来培养细胞的说明在体外 ，随后是关于如何原位嫁接这些细胞进同源C57BL / 6J小鼠的纹状体的解释。然后，它前进到枚举涉及分离和胶质瘤浸润的PBMC用于流式细胞术分析的免疫标记的步骤。协议结尾标准数据分析和图形表示的解释。 该演示可以发现，GR-1 + / CD11b的+骨髓细胞和NK1.1 + NK细胞优先积累在48中的GAL-1缺失的脑肿瘤微环境中小时肿瘤植入，因此这有助于解释为什么这些肿瘤迅速进行完整的肿瘤溶解大约一周后的肿瘤植入20。该方法是很容易适应一些，其中对免疫渗入脑颞数据，需要在体内不同的实验设计。单个实验者可以从大脑收获执行协议流胶质瘤浸润的PBMC的流式细胞术分析在约4-6小时取决于待分析的样品的数目。该方法还可以用实验的目的是表征循环的PBMC在荷瘤小鼠对与那些渗入脑如此识别由肿瘤微环境特异性诱导的免疫抑制的表型比较的信息相结合。这和类似的方法应用应有助于更好地理解所涉及的外周免疫细胞贩运到大脑肿瘤微环境的因素。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modification of Eagles Medium</td>
			<td>Gibco</td>
			<td>12430-054</td>
			<td>with 4.5g/L D-glucose, L-glutamine, 25mM HEPES and phenol red&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-buffered Saline&#160;</td>
			<td>Gibco</td>
			<td>14190-144&#160;</td>
			<td>without calcium chloride or Magnesium chloride</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Omega Scientific Inc.</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>10,000U/ml Penicillin; 10,000&#956;g/ml Streptomycin&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>200mM</td>
		</tr>
		<tr>
			<td>G418 sulfate&#160;</td>
			<td>Omega Scientific Inc.</td>
			<td>GN-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td>from Streptomyces alboniger</td>
		</tr>
		<tr>
			<td>HyClone HyQTase non-mammalian trypsin alternative&#160;</td>
			<td>Thermo Scientific</td>
			<td>SV30030.01</td>
			<td>in DPBS with EDTA&#160;</td>
		</tr>
		<tr>
			<td>Phase contrast hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>0.1mm deep</td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 0.9% NaCl injection, USP</td>
			<td>Hospira</td>
			<td>NDC: 0409-4888</td>
			<td>10ml vials</td>
		</tr>
		<tr>
			<td>0.6ml conical polypropylenemi microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezole hydrochloride injection</td>
			<td>Orion Pharma</td>
			<td>NDC: 52483-6298</td>
			<td>5mg/ml solution</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection</td>
			<td>Fort Dodge</td>
			<td>NDC: 0409-2051</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Dexmedetomidine hydrochloride injection</td>
			<td>Zoetis</td>
			<td>NDC: 54771-2805</td>
			<td>0.5mg/ml solution</td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride injection</td>
			<td>Lloyd</td>
			<td>NDC: 61311-481</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Carprofen injection</td>
			<td>Pfizer</td>
			<td>NDC: 61106-8501</td>
			<td>50mg/ml solution</td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride injection</td>
			<td>Reckitt Benckiser</td>
			<td>NDC: 12496-0757</td>
			<td>0.3mg/ml ampuls</td>
		</tr>
		<tr>
			<td>1ml tuberculin syringes&#160;</td>
			<td>Covidien</td>
			<td>8881501400</td>
			<td></td>
		</tr>
		<tr>
			<td>26G x &#189; (0.45mm x 13mm) syringe needles&#160;</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical clippers</td>
			<td>3M</td>
			<td>9661</td>
			<td></td>
		</tr>
		<tr>
			<td>Providone-iodine solution</td>
			<td>Aplicare</td>
			<td>82-217</td>
			<td>NDC: 52380-1905</td>
		</tr>
		<tr>
			<td>Sterile petrolatum ophthalmic ointment&#160;</td>
			<td>Dechra</td>
			<td>NDC: 17033-211</td>
			<td></td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol prep pads</td>
			<td>Kendall</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Covidien</td>
			<td>8044</td>
			<td>non-woven, 4&#34; x 4&#34;, 4-ply&#160;</td>
		</tr>
		<tr>
			<td>1.7ml conical polypropylene microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-281</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse stereotactic frame</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical lamp</td>
			<td>Philips Burton</td>
			<td>CS316W</td>
			<td>Coolspot II Variable Spotlight</td>
		</tr>
		<tr>
			<td>Curved dissecting forceps</td>
			<td>Ted Pella Inc.</td>
			<td>5431</td>
			<td></td>
		</tr>
		<tr>
			<td>Colibri retractors&#160;</td>
			<td>FST</td>
			<td>17000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless precision power drill</td>
			<td>Dremel&#160;</td>
			<td>1100-01</td>
			<td>Stylus model; 7.2-Volt Lithium-Ion with Docking Station</td>
		</tr>
		<tr>
			<td>Engraving Cutter</td>
			<td>Dremel</td>
			<td>105</td>
			<td>1/32&#34; or 0.8 mm bit diameter</td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>75</td>
			<td>Hamilton Microliter&#174; Syringes 700 series; 5&#956;l volume</td>
		</tr>
		<tr>
			<td>Microliter syringe needles</td>
			<td>Hamilton</td>
			<td>7762-06</td>
			<td>33G, small hub RN NDL, 1.5 in, point style 3, 6/PK</td>
		</tr>
		<tr>
			<td>Ethilon 3-0 black monofilament nylon sutures&#160;</td>
			<td>Ethicon</td>
			<td>1663</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir bar</td>
			<td>VWR</td>
			<td>74950-296</td>
			<td>7.9mm diameter x 50mm length (5/16&#34; diameter x 2&#34; length)</td>
		</tr>
		<tr>
			<td>1L glass screw-cap storage bottle</td>
			<td>Corning</td>
			<td>1395</td>
			<td>Type 1, Class A borosilicate glass</td>
		</tr>
		<tr>
			<td>Heparin sodium&#160;</td>
			<td>Sagent</td>
			<td>NDC: 25021-400</td>
			<td>1,000U/ml solution</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Cole Parmer Instruments Group</td>
			<td>77200-60</td>
			<td>Master Flex Easy Load II console drive</td>
		</tr>
		<tr>
			<td>compressed carbogen (95% O2 / 5% CO2)</td>
			<td>available from local vendor</td>
			<td>N/A</td>
			<td>A-type, large cylinder&#160;</td>
		</tr>
		<tr>
			<td>20G x 1 &#189;&#34; aluminum hub blunt needles</td>
			<td>Kendall</td>
			<td>8881202363</td>
			<td>0.9mm x 38.1mm</td>
		</tr>
		<tr>
			<td>Polyurethane ice bucket</td>
			<td>Fisherbrand</td>
			<td>02-591-45</td>
			<td>Capacity: 0.152 oz. (4.5L)</td>
		</tr>
		<tr>
			<td>Collagenase (Type I-S)</td>
			<td>Sigma Aldrich</td>
			<td>C1639</td>
			<td>from Clostridium histolyticum&#160;</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS002007</td>
			<td>&#62;2,000 Kunitz units per mg dry weight</td>
		</tr>
		<tr>
			<td>Antistatic polystyrene hexagonal weighing dishes</td>
			<td>Ted Pella Inc.</td>
			<td>20157-3</td>
			<td>top I.D. 115mm, base I.D. 85mm, 203ml volume&#160;</td>
		</tr>
		<tr>
			<td>Stainless steel single edged razor blades&#160;</td>
			<td>Garvey</td>
			<td>40475</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone rongeurs</td>
			<td>FST</td>
			<td>16001-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>FST</td>
			<td>13014-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Large dissection scissors</td>
			<td>Ted Pella Inc.</td>
			<td>1316</td>
			<td></td>
		</tr>
		<tr>
			<td>Small dissection scissors</td>
			<td>FST</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt end forceps</td>
			<td>Ted Pella Inc.</td>
			<td>13250</td>
			<td></td>
		</tr>
		<tr>
			<td>7ml glass Dounce tissue grinder</td>
			<td>Kontes</td>
			<td>KT885300-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll Density Centrifugation Media&#160;</td>
			<td>GE Healthcare</td>
			<td>GE17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipettes</td>
			<td>Genesee Scientific</td>
			<td>12-104</td>
			<td>single use; sterile</td>
		</tr>
		<tr>
			<td>70&#956;m sterile nylon mesh cell strainers&#160;</td>
			<td>Fisherbrand</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11)</td>
			<td>Biolegend</td>
			<td>103128</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti-mouse NK1.1 (clone: PK136)</td>
			<td>eBiosciences&#160;</td>
			<td>17-5941-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5)</td>
			<td>BD Pharmigen</td>
			<td>553128</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70)&#160;</td>
			<td>Biolegend</td>
			<td>101228</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400628</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG2a, &#954; (clone: eBM2a) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>17-4724</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat IgG2b, &#954; (clone: eB149/10H5) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>12-4031-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control</td>
			<td>Biolegend</td>
			<td>400632</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse anti-mouse granzyme B (Clone: GB11)&#160;</td>
			<td>Biolegend</td>
			<td>515403</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse IgG1, &#954; (Clone: MOPC-21) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400151</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm</td>
			<td>BD</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-103</td>
			<td>conical bottom&#160;</td>
		</tr>
		<tr>
			<td>50ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-108</td>
			<td>conical bottom</td>
		</tr>
		<tr>
			<td>12x75mm round-bottom polypropylene FACS tubes&#160;</td>
			<td>Fisherbrand</td>
			<td>14-956-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria Special Order Research Product flow cytometer/cell sorter</td>
			<td>BD&#160;</td>
			<td>650033</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II Biological Safety Cabinet</td>
			<td>The Baker Company</td>
			<td>SG603</td>
			<td>model: SterilGARD III Advance</td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells

我们的实验室已最近表明，天然杀伤（NK）细胞能够颅内植入后CNS-1恶性胶质瘤很快消除原位移植小鼠GL26和大鼠如果癌细胞在它们的β半乳糖苷结合凝集素的galectin表达呈现缺陷的-1（加仑-1）。更近期的工作现在显示的Gr-1 + / CD11b的+髓样细胞群是这种效果的关键。为了更好地了解由NK细胞和骨髓细胞协作以赋予加仑-1缺失的肿瘤排斥我们已经开发了全面的协议用于分离和胶质瘤浸润外周血单核细胞（PBMC）的分析的机制。该方法是在这里证明了外周血单个核细胞浸润比较成的肿瘤微环境GAL-1表达GL26胶质瘤与那些通过shRNA的敲除呈现GAL-1缺失。该协议开始的描述如何培养并准备GL26 CELLS接种入同系的C57BL / 6J小鼠的大脑。然后，它解释了涉及从早期脑肿瘤微环境胶质瘤浸润的PBMC的分离和流式细胞术分析的步骤。该方法是适应若干体内 ，其中免疫渗入脑颞数据是必需的实验设计。该方法灵敏度高，重现性好，为神经胶质瘤浸润外周血单个核细胞可以从颅内肿瘤一旦被隔离24小时后移植瘤与时间点匹配的肿瘤遍布独立的实验中观察到类似的细胞计数。单个实验者可以从大脑收获执行该方法流胶质瘤浸润的PBMC的流式细胞术分析中大约4-6小时取决于待分析的样品的数目。替代胶质瘤模型和/或细胞特异性的检测抗体，也可以使用在实验者​​自行决定，以评估其它几个与免疫的渗透Ë细胞类型，而不需要改​​变对整体过程的兴趣。

下面浇注策略用于一个典型的实验:FSC-A对SSC-A→SSC-H与SSC-W→FSC-H与FSC-W→CD45与数→GR-1与CD11b的→ NK1.1与计数。盖茨放置的Gr-1 + / CD11b的+髓样细胞和NK1.1 + NK细胞，然后分层基于GZMB表达（图5A）。 Backgating那些鉴定为NK1.1 + NK细胞和Gr-1 + / CD11b的+髓样细胞在我们的实验上的FSC-A对SSC-A确认NK细胞的小尺寸淋巴和骨髓细胞的相对大尺寸（细胞图5B）。原始数据文件（ 即 ，FCS文件）市售流式细胞仪数据分析软件进行分析，如的FlowJo。 总共18只C57BL / 6J小鼠用于证实这种方法。九（9）被移植了GL26-CIT-NT和9被移植了GL26-CIT-gal1i上的同一个D唉使用细胞（3×10 4）的当量数。三只小鼠每组安乐死在24，48和72小时后的肿瘤植入。数据表明，植入GL26-CIT-gal1i胶质瘤细胞进同源C57BL / 6J小鼠的大脑迅速诱导CD45 + PBMC中（图6A）的募集。基于在演示中使用特异性抗体鸡尾酒它可以进一步表明的Gr-1 + / CD11b的+髓样细胞和NK1.1 + NK细胞特异性内48小时肿瘤植入的（图输入 ​​伽-1缺失肿瘤微图6B和6C）;胶质瘤浸润骨髓细胞远胜NK细胞的然而总数。 <img alt="图1" src="/files/ftp_upload/53676/53676fig1.jpg" /> 图1:GL26-CIT细胞颅内准备定植（A。）代表10X明场和GL26-CIT-NT（左图像）和GL26-CIT-gal1i（右图像）在培养细胞表面荧光显微照片。 （B）GL26-CIT-NT（左线）和GL26-CIT-gal1i（右线）全细胞裂解液的Western印迹。伽马微管蛋白（γ-浴盆。）显示为上样对照。 （C）的GL26-CIT小区从约50％汇合的T75组织培养瓶离心在550×g离心（最大RCF）中5分钟，在4℃下后沉淀。 （D）的含GL26-CIT细胞（白点）血细胞计数器的明视场视图1:1稀释用台盼蓝评估细胞数目和生存力。细胞应&gt; 90％存活，以确保重现性肿瘤的生长。应取自标记为1到5的平方的细胞计数的平均值，以增加细胞数估计的准确度。在未补充的DMEM重悬在3×10 4个/微升颅内impla（E）GL26-CIT细胞ntation。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53676/53676fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53676/53676fig2.jpg" /> 图2:立体定向灌输胶质瘤细胞的成C57BL / 6J小鼠的纹状体（A）外科手术室布局，可以显示所需要的手术器械和试剂。 （ 二）特写图一C57BL / 6J小鼠利用，在立体定位框架与合适的针安置在0.5毫米AP，2.5毫米ML和-3.0毫米DV相对于前囟门。 （ 三）背鼠标颅骨呈现前囟门的位置和靶位点的肿瘤细胞植入的看法。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53676/53676fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3:鼠标穿心灌注（A）进行解剖，研磨和小鼠脑组织的酶消化所需的试剂。 （二）在层流罩，致力于终端的鼠标外科手术穿心灌注氧合和肝素台氏的解决方案表示需要鼠标的仪器和试剂的布局。 （C）的心脏显示出正确的仪器及所需的穿心灌注展示位置示意图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53676/53676fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53676/53676fig4.jpg" /> 图4:脑胶质瘤浸润的隔离外周血单个核细胞（一）战略包含早期原位胶质瘤植入小鼠脑组织的解剖。顶部面板演示同侧半球矢状平分远离对侧大脑半球。底部面板演示需要分离含有肿瘤植入物（紫色突出显示）目标组织的两个附加冠状削减。 （B）的单细胞脑组织沉淀（白色箭头）酶消化后，通过70微米的尼龙网和离心过滤。 （三）妥善堆积密度离心媒体之前离心梯度。白色箭头指示两个密度离心介质层之间形成的干净的界面。 （D）的离心表明形成在37％密度离心介质层白色箭头顶部的脂质层）和PBMC频带后密度离心分离介质梯度（由第概述Ë黑色虚线），在这两个层之间的接口。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53676/53676fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53676/53676fig5.jpg" /> 图5:流式细胞门控策略用于识别胶质瘤浸润的Gr-1 + / CD11b的+髓系细胞及NK细胞（A）的步骤1:总免疫标记的细胞从一个密度离心媒体梯度的PBMC频带分离被选通到排除细胞碎片（FSC-A小于〜50 K）。步骤2和3:双峰歧视选通过滤出细胞聚集。步骤4:CD45的门，以确定胶质瘤浸润免疫细胞。同种型对照显示为灰色的剪影。第5步:CD45 +细胞分层基于GR-1和CD11b。同种型对照为GR-1和CD<html>部11b重叠在情节和由金圆包围。红色圆圈表示GR-1 + / CD11b的+骨髓细胞。第5步":GR-1 + / CD11b的+分层的基础上GZMB表达髓样细胞。作为前提，这些细胞不标注有防GZMB抗体在同型对照（灰色剪影）。第6步:GR-1 的低电池由黑色矩形在步骤5中所列的分层基于NK1.1表达。同种型对照显示为灰色的剪影。第6步":基于GZMB表达NK1.1 高细胞分层。这些细胞确实标注有防GZMB抗体高于同型对照（灰色剪影）。的Gr-1 + / CD11b的+髓样细胞（B）的Backgating到FSC-A对SSC-甲证明，认为NK细胞具有较小的淋巴尺寸相比，较大尺寸的Gr-1 + / CD11b的+髓样细胞。"_blank"&gt;点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/53676/53676fig6.jpg" /> 图6:外周血单个核细胞浸润比较入GL26-CIT-NT与GL26-CIT-gal1i肿瘤微环境在颅内肿瘤生长的前三天（A）总 CD45 +免疫细胞。 （B）CD45 + / GR-1 + / CD11b的+骨髓细胞。 （C），CD45 + /NK1.1 + NK细胞。从PBMC中从GL26-CIT-gal1i胶质瘤分离数据点由线条流畅连接，田地那些从GL26-CIT-NT胶质瘤分离由虚线连接。从三只小鼠胶质瘤浸润的PBMC每肿瘤类型在每个时间点进行分析。与上GL26-CIT-gal1i曲线​​的每一个数据点相关联的数字代表的褶皱感应特定的外周血单个核细胞类型的过度GL26慈叔NT在指定小时后肿瘤植入（HPI）。数据代表计数±平均值（SEM）的标准误差的免疫细胞的平均数。由2路方差分析进行统计分析。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53676/53676fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

这里介绍的是胶质瘤浸润外周血单核细胞的分离和流式细胞术分析能够产生在进入早期脑肿瘤微环境的免疫细胞的数目和激活状态依赖于时间的定量数据一个直接的方法。

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

isolation-flow-cytometric-analysis-glioma-infiltrating-peripheral

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

15.7K 次观看.  University of Michigan. 这里介绍的是胶质瘤浸润外周血单核细胞的分离和流式细胞术分析能够产生在进入早期脑肿瘤微环境的免疫细胞的数目和激活状态依赖于时间的定量数据一个直接的方法。 

视频: Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

科研

免疫与感染

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

协同刺激信号封锁的同种抗原刺激诱导人外周血单个核细胞同种异体抗原特异性无能

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...