我们感谢博士。马库斯·曼茨和欧文·韦斯曼提供试剂。我们也承认在医学和台大医院台大医学院提供的流式细胞仪分析，并第一次细胞分选核心设施和第二核实验室的服务，分别为。这项工作得到了科技部，台湾的支持部（MOST 102-2320-B-002-030-MY3）和国家卫生研究院，台湾地区（国家人权机构，EX102-10256SI和国家人权机构 - EX103-10256SI）。

C57BL / 6野生型小鼠购自国家实验动物中心（NLAC），台湾购买。所有小鼠饲养，并保持无特定病原体的条件下。协议和动物使用的程序进行审查和批准国家医学台湾大学的机构动物护理和使用委员会（许可证号：20120075）。另外，研究人员尽一切努力，以减少疼痛，痛苦，还是痛苦的潜力，而动物进行实验。描述的所有步骤均在室温戴手套。 1.制备AC-6饲养细胞的注意：所述的交流6.21（AC-6在短）基质细胞系18（由一威斯曼，斯坦福大学提供）应维持在RPMI补充有15％热灭活的小牛血清（FBS）。作为饲养细胞，AC-6细胞是γ-照射3000拉德（30GY）之一天共培养，以防止其扩散之前。注日在AC-6细胞往往会失去其分化配套能力强，如果在维护期间离开人满为患，或者如果他们通过人数超过20。 <ol><li>洗的AC-6细胞一次用1ml的Dulbecco磷酸盐缓冲盐水（DPBS）并且通过抽吸去除的DPBS。治疗的AC-6细胞用0.8mL胰蛋白酶溶液（0.05％胰蛋白酶和0.5mM EDTA的在DPBS）3-5分钟，在37℃，5％的CO 2，然后停止反应通过将其与10体积培养基稀释至使单细胞悬液。 </li><li>种子AC-6细胞以5.9x10 4个细胞 /孔的12孔板，37℃，5％的CO 2 O / N。 注意：AC-6细胞的密度是作为一个较低的密度将倾向于CDC代非常重要的。播种在5.9x10的密度4细胞/孔将允许AC-6的第二天，这是最适合于从体CLPs的pDC的推导到达汇合。 </li><li>照射AC-6细胞用γ-辐照3000 RAD（30戈瑞）。 注意：用于照射交流-6的剂量优化，以防止细胞增殖，但保持它们存活足够长的时间，以提供所需的CLPs从分化的DC的细胞因子和细胞 - 细胞接触。 </li><li>抽吸培养基并添加1毫升的完全RPMI（RPMI 10％热灭活FBS，50μg/ ml的庆大霉素和50μM的β巯基乙醇）。 </li></ol>从2小鼠分离骨髓细胞<ol><li>牺牲小鼠CO 2窒息和颈椎脱位。将小鼠解剖托盘和消毒用70％的乙醇。做一个切口在中间腹部和从鼠标包括皮肤覆盖下肢的末端部分去除皮肤。 </li><li>在半无菌罩解剖小鼠。为了释放股骨和胫骨，夹上面的股骨和胫骨下面膝关节。分离使用剪刀骨骼肌肉，然后将骨头含5毫升完全RPMI一个6cm培养皿中。 </li><li>移到一个无菌的培养罩。填写3毫升注射器连接到27G针完全RPMI。切断用剪刀骨头的两端，然后将27G针头从两端轻轻注射完全RPMI一次冲洗出骨髓的骨头。 </li><li>制备单细胞悬浮液通过温和移液细胞上下3-5倍于使用注射器没有针头培养皿。 </li><li>离心细胞5分钟，在500×g离心并弃去上清液。 </li><li>裂解红血细胞与1ml ACK缓冲液（150mM的氯化铵 ，10毫KHCO 3，0.1mM的EDTA）中培养1分钟，并通过用10体积的完全RPMI中稀释终止反应。允许细胞放置5分钟，以便通过重力沉淀下来死细胞团块和组织碎片。 注意：不要站在超过5分钟，因为这会导致细胞损失，由于沉降活细胞。 </li><li>慢慢倾析含细胞的上清液至新管离去碎片后面在原管。 </li><li>离心5分钟500 XG沉淀细胞，然后捞出弃去上清液。 </li><li>重悬总BM细胞（通常为4-6×10 7个骨髓细胞从一只小鼠获得）在100μlFACS缓冲液（1×PBS + 2％FBS + 1mM的EDTA）中。 </li></ol> 3.流式细胞仪分析的CLP的排序<ol><li> 1-2分钟，以阻断Fc受体添加的抗CD16 / 32（杂交瘤上清克隆2.4G2，50微升/反应或1-2微克/反应）进细胞在FACS缓冲液（步骤2.9）。 注意：抗CD16 / 32也被称为Fc的块，这是添加到防止抗体的非特异性结合到表达的Fc受体，包括粒细胞，单核细胞，淋巴细胞和B淋巴细胞。 </li><li>同时染色细胞用下面的荧光染料标记的抗体（为4×10 7个细胞）在冰上15分钟，以避免环境光线。抗体：PE-缀合谱系标记物（各0.2微克），包括抗CD3（17A2），抗CD8（53-6.7），抗B220（RA3-6B2），抗CD19（eBio1D3），抗CD11b（M1 / 70），抗Gr-1（RB6-8C5），抗Thy1.1的（HIS51）的抗NK1.1（PK136），抗TER119（TER-119）和抗MHC-II（NIMR-4），0.2微克的抗c-Kit的-PerCP-经Cy5.5 （2B8），1微克的抗Sca-1-FITC（D7），0.4微克抗M-CSFR-APC（AFS98）和0.2微克的抗IL-7Rα-PE-Cy7的（A7R34）。 </li><li> 5分钟，在500×g下洗涤细胞用3ml FACS缓冲液，并离心。 </li><li>悬浮细胞在300微升FACS通过一个40微米的细胞过滤缓冲和过滤单元。 </li><li>与另外的100微升FACS缓冲液洗管以回收任何剩余的细胞，并过滤到无菌的FACS使用相同的细胞过滤排序管。 </li><li>立即用适当的过滤器和电压信号检测染色后进行流式细胞仪分析。使用488纳米的激光来检测FITC-，PerCP-Cy5.5标记抗体，561纳米的激光来检测PE-和PE-Cy7的-labelded抗体和633纳米拉斯呃检测APC标记的抗体。 </li><li>理出根据下列标志物的CLPs林-使用细胞分选仪的IL-7Rα+（如在染色步骤3.2） -的c-kit INT的Sca-1 INT ​​的M-CSFR。收集分选的细胞在一个15毫升管装有8毫升完全RPMI作为缓冲。 </li><li>记录分选的细胞的绝对数量为在运行的端部所示的细胞分选仪。典型地，5×10 4的CLPs可以从一个鼠标对BM而获得。 </li></ol> 4.共培养的CLPs和AC-6 <ol><li>离心分选的细胞从步骤3.7 10分钟，在500×g离心，除去上清液和重悬细胞沉淀用足够的完全RPMI来获得周围5×10 4细胞/ ml的细胞密度。种子500个细胞/孔进12孔平板含有在步骤1.4制备的饲养细胞。 </li><li>添加100毫微克/毫升FL 19（HU-FLT3L-Ig的内部产生的使用由M.曼茨，Universi提供了一种表达系统TY医院苏黎世，瑞士），37℃，5％CO 2显微镜下定期可视化监控DC的发展。 注：市售的重组人FL或鼠标FL可以用作替代HU-FLT3L-Ig的支持直流发展。 </li><li>收集细胞上清液中的第12天，并用0.5一次洗井ml完全RPMI培养基中组合所得到的上清液。添加0.5 ml的新鲜培养基和报废贴壁细胞用细胞刮刀。 </li><li>组合来自两个部件的含细胞的培养基，离心5分钟，在500×g。 </li><li>倒出上清，重悬细胞于50μlFACS缓冲液中。加入50微升的抗CD16 / 32杂交瘤上清液孵育1-2分钟，以阻断Fc受体。 </li><li>枚举和染色的所有细胞与下列抗体（每0.05微克）抗CD11c-APC（N418），抗CD11b-FITC，并且抗B220-PE。 </li><li>洗净离心机的细胞描述d。在步3.3。 </li><li>悬浮细胞在100微升FACS缓冲液，门上的CD11c +和分析cDC上（表面CD11c +细胞CD11b + B220 - ）和的pDCs（表面CD11c +细胞CD11b - B220 +）17。 </li></ol>

<ol>
	<li>Hartwig, 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=Fcgamma+receptor-mediated+antigen+uptake+by+lung+DC+contributes+to+allergic+airway+hyper-responsiveness+and+inflammation.">Fcgamma receptor-mediated antigen uptake by lung DC contributes to allergic airway hyper-responsiveness and inflammation.</a> Eur. J. Immunol. 40, 1284-1295 (2010).</li><li>Merad, M., Sathe, P., Helft, J., Miller, J., Mortha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dendritic+cell+lineage:+ontogeny+and+function+of+dendritic+cells+and+their+subsets+in+the+steady+state+and+the+inflamed+setting.">The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting.</a> Annu. Rev. Immunol. 31, 563-604 (2013).</li><li>Mildner, A., Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Function+of+Dendritic+Cell+Subsets.">Development and Function of Dendritic Cell Subsets.</a> Immunity. 40, 642-656 (2014).</li><li>Swiecki, M., Colonna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+functions+of+plasmacytoid+dendritic+cells+during+viral+infections,+autoimmunity,+and+tolerance.">Unraveling the functions of plasmacytoid dendritic cells during viral infections, autoimmunity, and tolerance.</a> Immunol. Rev. 234, 142-162 (2010).</li><li>Liu, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPC:+professional+type+1+interferon-producing+cells+and+plasmacytoid+dendritic+cell+precursors.">IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors.</a> Annu. Rev. Immunol. 23, 275-306 (2005).</li><li>Merad, M., Manz, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+homeostasis.">Dendritic cell homeostasis.</a> Blood. 113, 3418-3427 (2009).</li><li>Ghosh, H. S., Cisse, B., Bunin, A., Lewis, K. L., Reizis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+expression+of+the+transcription+factor+e2-2+maintains+the+cell+fate+of+mature+plasmacytoid+dendritic+cells.">Continuous expression of the transcription factor e2-2 maintains the cell fate of mature plasmacytoid dendritic cells.</a> Immunity. 33, 905-916 (2010).</li><li>Karsunky, H., Merad, M., Cozzio, A., Weissman, I. L., Manz, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flt3+ligand+regulates+dendritic+cell+development+from+Flt3++lymphoid+and+myeloid-committed+progenitors+to+Flt3++dendritic+cells+in+vivo.">Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo.</a> J. Exp. Med. 198, 305-313 (2003).</li><li>Manz, M. G., Traver, D., Miyamoto, T., Weissman, I. L., Akashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+potentials+of+early+lymphoid+and+myeloid+progenitors.">Dendritic cell potentials of early lymphoid and myeloid progenitors.</a> Blood. 97, 3333-3341 (2001).</li><li>D'Amico, A., Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+progenitors+of+mouse+dendritic+cells+and+plasmacytoid+predendritic+cells+are+within+the+bone+marrow+hemopoietic+precursors+expressing+Flt3.">The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3.</a> J. Exp. Med. 198, 293-303 (2003).</li><li>Sathe, P., Vremec, D., Wu, L., Corcoran, L., Shortman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+differentiation:+myeloid+and+lymphoid+pathways+to+murine+plasmacytoid+dendritic+cells.">Convergent differentiation: myeloid and lymphoid pathways to murine plasmacytoid dendritic cells.</a> Blood. 121, 11-19 (2013).</li><li>Shigematsu, H., 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=Plasmacytoid+dendritic+cells+activate+lymphoid-specific+genetic+programs+irrespective+of+their+cellular+origin.">Plasmacytoid dendritic cells activate lymphoid-specific genetic programs irrespective of their cellular origin.</a> Immunity. 21, 43-53 (2004).</li><li>Reizis, B., Bunin, A., Ghosh, H. S., Lewis, K. L., Sisirak, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmacytoid+dendritic+cells:+recent+progress+and+open+questions.">Plasmacytoid dendritic cells: recent progress and open questions.</a> Annu. Rev. Immunol. 29, 163-183 (2011).</li><li>Inaba, 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=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> J. Exp. Med. 176, 1693-1702 (1992).</li><li>Naik, S. H., 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+plasmacytoid+and+conventional+dendritic+cell+subtypes+from+single+precursor+cells+derived+in+vitro+and+in+vivo.">Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo.</a> Nat. Immunol. 8, 1217-1226 (2007).</li><li>Onai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+clonogenic+common+Flt3+M-CSFR++plasmacytoid+and+conventional+dendritic+cell+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow.</a> Nat. Immunol. 8, 1207-1216 (2007).</li><li>Chen, Y. 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=A+type+I+IFN-Flt3+ligand+axis+augments+plasmacytoid+dendritic+cell+development+from+common+lymphoid+progenitors.">A type I IFN-Flt3 ligand axis augments plasmacytoid dendritic cell development from common lymphoid progenitors.</a> J. Exp. Med. 210, 2515-2522 (2013).</li><li>Whitlock, C. A., Tidmarsh, G. F., Muller-Sieburg, C., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cell+lines+with+lymphopoietic+activity+express+high+levels+of+a+pre-B+neoplasia-associated+molecule.">Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule.</a> Cell. 48, 1009-1021 (1987).</li><li>Onai, N., Obata-Onai, A., Tussiwand, R., Lanzavecchia, A., Manz, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+Flt3+signal+transduction+cascade+rescues+and+enhances+type+I+interferon-producing+and+dendritic+cell+development.">Activation of the Flt3 signal transduction cascade rescues and enhances type I interferon-producing and dendritic cell development.</a> J. Exp. Med. 203, 227-238 (2006).</li><li>Szilvassy, S. 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=Leukemia+inhibitory+factor+upregulates+cytokine+expression+by+a+murine+stromal+cell+line+enabling+the+maintenance+of+highly+enriched+competitive+repopulating+stem+cells.">Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells.</a> Blood. 87, 4618-4628 (1996).</li><li>Arcanjo, 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=Biochemical+characterization+of+heparan+sulfate+derived+from+murine+hemopoietic+stromal+cell+lines:+a+bone+marrow-derived+cell+line+S17+and+a+fetal+liver-derived+cell+line+AFT024.">Biochemical characterization of heparan sulfate derived from murine hemopoietic stromal cell lines: a bone marrow-derived cell line S17 and a fetal liver-derived cell line AFT024.</a> J. Cell. Biochem. 87, 160-172 (2002).</li><li>Onai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonogenic+progenitor+with+prominent+plasmacytoid+dendritic+cell+developmental+potential.">A clonogenic progenitor with prominent plasmacytoid dendritic cell developmental potential.</a> Immunity. 38, 943-957 (2013).</li><li>Whitlock, C. A., Muller-Sieburg, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+B-lymphoid+cultures+from+murine+bone+marrow+establishment+and+cloning+by+using+stromal+cell+line+AC+6.21.">Long-term B-lymphoid cultures from murine bone marrow establishment and cloning by using stromal cell line AC 6.21.</a> Methods Mol. Biol. 75, 231-248 (1997).</li><li>Chen, Y. -. L., Chang, S., Chen, T. -. T., Lee, 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=Efficient+Generation+of+Plasmacytoid+Dendritic+Cell+from+Common+Lymphoid+Progenitors+by+Flt3+Ligand.">Efficient Generation of Plasmacytoid Dendritic Cell from Common Lymphoid Progenitors by Flt3 Ligand.</a> PLoS ONE. 10 (8), e0135217 (2015).</li></ol>

Authors have nothing to disclose.

这里，我们描述的体外培养体系为鲁棒生成的DC，和pDC细胞特别是从少数的CLPs的。该培养体系的独特性是由于使用AC-6细胞，基质细胞系，如送料器。这种方法已被证明提供的不仅是细胞因子，如IL-7，SCF，M-CSF和FL 20，而且在细胞-细胞接触21必要支持直流发展。 AC-6细胞已被先前用于促进体外直流发展的研究从多个祖细胞8,16,22。我们发现，这是至关重要的种子?...

树突细胞（DC）是专业抗原呈递细胞，在控制免疫反应1起着重要的作用。而DC是异质的，它们大致可分为两个群体，常规的DC（cDC上）和浆树突（pDC细胞）2,3-。除了 ​​淋巴器官，和cDC上的pDCs还发现在组织中，包括肺，肠，皮肤和2。 cDC上和的pDC的形态不同，与cDC上呈现枝蔓状凸起和的pDCs更加浆细胞样的形状。此外，常见的小鼠DC标记，CD11c的，更高度表达于cDC上比上的pDC。此外，cDC上可进一步分成的CD11b + CD24 - cDC上和CD11b - CD24 + cDC上，这两者都表达MHC II类的更高水平和共刺激分子比的pDC 2。成熟的pDCs，另一方面，已经显示出选择性表达的Siglec-H和BST2 4。 Functionally，cDC上有更好的抗原呈递细胞比是pDC的;然而，pDC细胞可以产生类型的大量的I型干扰素后，病毒感染或炎症刺激5。 既cDC上和的pDCs是短暂的，因此，必须不断从骨髓（BM）的6,7-内祖细胞补充。继转移的常见髓系祖细胞（中医）和公共淋巴祖细胞（的CLPs）到致死照射的小鼠表明cDC上和pDC细胞可以从两个谱系8-10产生。然而，有表达RAG1 / 2和具有重新排列的DJ片段在IgH基因基因座11,12的pDC的子集。这些细胞也与B淋巴细胞，包括B220标志物的表达，核酸感应受体（TLR7 / TLR9），和转录因子（SPIB和BCL11A）13共享分子的相似性，提供所有认为是淋巴谱系的签名。因此，电S可以是用于体外代因为谱系相似的pDC的很好的选择。 同时兼具cDC上和的pDC在人类和小鼠的频率很低6，DCS，尤其cDC上， 可以在细胞因子，如GM-CSF或11,14 Flt3配体的存在下，从BM或祖细胞体外生成（佛罗里达州）采用无饲养系统11,15,16。然而不幸的是，它是不可能产生大量使用FL 11,15,16 体外的pDC的。先前我们表明pDC细胞可以有效地在体外从使用AC-6馈线系统17的CLPs生成。使用AC-6基质细胞系在培养系统的优点是，它提供了细胞 - 细胞接触和支持大量的pDC的产生从体CLPs细胞因子的分泌。虽然生产该系统是相当强劲，手续如下所述小心复制需要我n阶确保有效产生的pDC的。

<table>
	<tbody>
		<tr>
			<td>Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5</td>
			<td>Biolegend</td>
			<td>108408</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse NK1.1 (PE), clone PK136</td>
			<td>Biolegend</td>
			<td>108708</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse&#160; CD11b (PE), cloneM1/70</td>
			<td>Biolegend</td>
			<td>101208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD19 (PE), clone eBio1D3</td>
			<td>Biolegend</td>
			<td>115508</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse B220 (PE), clone RA3-6B2</td>
			<td>Biolegend</td>
			<td>103208</td>
			<td>linage marker/FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3 (PE), clone 17A2</td>
			<td>Biolegend</td>
			<td>100308</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8a (PE), clone 53-6.7</td>
			<td>Biolegend</td>
			<td>100707</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse MHC-II (PE), clone NIMR-4</td>
			<td>Biolegend</td>
			<td>107608</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Ter119 (PE), clone TER-119</td>
			<td>Biolegend</td>
			<td>116208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Thy1.1 (PE), clone HIS51</td>
			<td>eBioscience</td>
			<td>12-0900-83</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse M-CSFR (APC), clone AFS98</td>
			<td>Biolegend</td>
			<td>135510</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8</td>
			<td>Biolegend</td>
			<td>105824</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse Sca-1 (FITC), clone D7</td>
			<td>Biolegend</td>
			<td>108106</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse IL-7Ra (PE/Cy7), clone A7R34</td>
			<td>Biolegend</td>
			<td>135014</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c (PerCP/Cy5.5), clone N418</td>
			<td>Biolegend</td>
			<td>117328</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (FITC), clone M1/70</td>
			<td>Biolegend</td>
			<td>101206</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>FACS sorting tube&#160;</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo LLC</td>
			<td></td>
			<td>Flow analysis sofrware</td>
		</tr>
	</tbody>
</table>

in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system

浆细胞样树突细胞（pDC细胞）是强有力的I型干扰素（IFN-I）的生产被响应于感染或在炎症反应的活化细胞。不幸的是，的pDC功能的研究是由它们的低频在淋巴器官受阻，并在体外直流代现有的方法主要有利于生产cDC上的过度的pDC。在这里，我们提出了一个独特的方法来有效地生成常见淋巴祖（的CLP） 在体外 pDC的。具体地，该协议描述细节如何从骨髓纯化的CLPs和通过用γ照射的AC-6饲养细胞在Flt3配体的存在下共培养产生的pDC。该培养系统的一个独特的特征是所述的CLPs迁移对AC-6细胞的下面，并成为鹅卵石区形成细胞，用于扩展的pDC的关键步骤。形态不同的DC，即的pDC和cDC上，产生后约2周为7的组合物最佳条件下0-90％的pDC。通常情况下，用这种方法产生的pDC的数目大约是100倍的CLPs播种的数目。因此，这是一种新的系统，用以有力产生大量以促进进一步的研究进入这些细胞的发育和功能所需的pDC的。

共有4-6x10 7骨髓细胞通常是孤立于股骨和一个6-8周龄，野生型C57BL / 6小鼠胫骨。到的CLPs梳理，总骨髓细胞用针对谱系标记物（CD3，CD8，B220，CD19，CD11b的，GR-1，​​Thy1.1的，NK1.1，TER119，和MHC-II）中，抗PE结合抗体-c-Kit的-PerCP /经Cy5.5，的抗Sca-1-FITC，抗M-CSFR-APC和抗IL-7Rα-PE / Cy7的，分析和分类用细胞分选仪。排序策略CLP 示于图1，典型地，5×10 4的CLPs从一个C57...

Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

in-vitro-generation-murine-plasmacytoid-dendritic-cells-from-common

Research

JoVE Journal

Immunology and Infection

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

8.3K 次观看.  National Taiwan University College of Medicine. In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand. 

视频: In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

科研

免疫与感染

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

使用黑色素瘤和鳞状癌干细胞生成新型支气干细胞疫苗

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

不成熟，成熟和耐受性树突状细胞的生成与代谢不同表型

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...