这项工作是由卫生研究院（CIHR）（批准MOP-119503）和加拿大自然科学与工程理事会（NSERC）的加拿大学院资助。 NSERC还支持夏季助学金为YD和AA YD是由英属哥伦比亚大学（UBC）大学用4年的奖学金奖励支持，AA由CIHR与研究生硕士奖（CGS-M）的支持。我们感谢卡尔文Roskelley与用于生成图2中的图像在显微镜的帮助。我们也承认从UBC动物和流式细胞仪设备的支持。

小鼠按照加拿大理事会关于道德的动物研究动物护理指南由不列颠哥伦比亚省的动物护理委员会的批准大学的程序进行安乐死。 1.获得从小鼠股骨和胫骨单细胞骨髓暂停<ol><li>开关上的生物安全柜（BSC）之前15分钟开始的过程的吹扫柜内空气，并允许空气流稳定，用70％的乙醇，然后清洁/喷雾BSC表面。一切都保持无菌尽可能为协议的全部内容。不切非无菌条件下的骨头。 </li><li>通过在70％乙醇中浸泡制备清扫工具（1对解剖剪，2对镊子的），汉克平衡盐溶液，用5％胎牛血清（HBSS-FCS），无菌培养皿中英寸（100毫米×15毫米），1-毫升注射器，26½表针，圆锥形收集管（15毫升或50毫升）中，并纸巾在BSC。 </li><li>使用安乐死鼠标由该机构的动物研究伦理委员会批准的协议。带入BSC，放置在一个或两个纸巾顶部和喷雾用70％乙醇鼠标。 </li><li>在BSC中，打开一个无菌培养皿无菌，等分试样10毫升HBSS-FCS的到基和1 - 2 ml至盖子。 </li><li>放置在其朝上的背面胃鼠标和解除胸椎部分周围的皮肤与非优势手的手指。使用剪刀，使那里的皮肤被提起的切口。 </li><li>持到切口的两端，一端与每个手和小心从背面到胃拉开周围鼠标皮肤，继续拉动直到在胃切口相遇的两端。 </li><li>翻转朝上鼠标胃，并用一只手拉扯皮肤向下朝着腿的下半部分，保持拉回皮肤直至腿部被暴露。 </li><li>按住鼠标向上的脚，减少跟腱踝关节上方，连接的肌肉韧带在用剪刀胫骨的下端脚。这从松动的腿骨脚下。 </li><li>在保持腿由脚，切开肌肉和韧带用剪刀周围所有的膝关节在股骨的下端以从小腿断绝大腿肌肉。使用镊子轻轻一拉肌肉放松从腓骨和股骨的表面。注意不要割断股动脉，避免大量出血。 </li><li>用一只手抓着脚，按下开剪刀在髋关节股骨上端，旋转腿，轻轻拉，同时用剪刀晋级决赛自由股骨从髋关节分离从主体的腿。 </li><li>从这时开始，只持有与无菌镊子组织。紧握腿一端与拉断脚另一方。 注:这应该不会需要太多的力量，作为连接韧带步骤削减1.9。可替代地，切只是其中骨融合脚踝上方的胫骨。 </li><li>持通过用一种组镊子和胫骨和腓骨与另一个的近端股骨的远端的腿，小心弯曲膝关节将它们从股骨和髌骨分离的相反方向上的胫骨和腓骨。 </li><li>使用镊子拔下来剩下的韧带和肌肉还连着小腿骨头。这里，取出腓骨带钳子结缔组织沿着它太小，被刷新为骨髓细胞。放置在倒置培养皿盖的清洗胫骨。 </li><li>保持与一组镊子，并用另一个髌骨股骨下端，弯曲的关节除去的相反方向上的髌骨及周围组织。然后擦去剩余从股骨结缔组织;拉着他们走用镊子，并把它放在倒置的培养皿的盖子。丢弃髌骨。 </li><li>重复1.7如果需要的话1.13与另一条腿 - 。转移骨头含有1毫升无菌HBSS-FCS运输如果需要1.6毫升离心管。 </li><li>放置在倒置培养皿盖骨，用1 - 2毫升HBSS-FCS的，使用镊子清洁仍连接到胫骨和股骨的任何残留组织，然后骨骼转移至培​​养皿含有10ml HBSS-的基FCS。此外，清洁骨骼和用70％乙醇浸泡过的组织去除附着的肌肉组织。 </li><li>用剪刀剪下每个骨骼的一半。局部用无菌保护盖培养皿同时削减，以确保骨头留在菜。可替代地，切割每个骨的端部，以允许冲洗在下一步骤。 </li><li>连接到1毫升注射器的26½摹针，并从培养皿制定1毫升HBSS-FCS。插入针的前端到骨片的端部和排出来的骨髓细胞（骨骼内软红组织）。针插入头部骨和开口端的，直到所有的红色骨髓被除去。观察骨骼白色的。 </li><li>通过注射器几次传骨髓任何可见团块以形成单细胞悬液。 </li><li>丢弃冲洗骨头。用10毫升吸管很好混合细胞悬浮液并转移至收集管。用5毫升HBSS-FCS的冲洗培养皿一次可收集残余细胞的菜。细胞置于冰上。 注意:如果存放2骨髓细胞悬浮液仍然是可行-在4℃下3小时。 </li></ol> 2.电镀和培养骨髓细胞（骨髓细胞） <ol><li>准备以下试剂和耗材。 <ol><li>通过在2毫摩尔Tris pH值7.2的终溶液制备0.84％氯化铵制备红细胞（RBC）溶胞缓冲液，然后通过0.2微米的过滤器过滤灭菌。 </li><li>通过添加10％FCS，20mM的HEPES，1×非必需氨基酸，55＃制备的BMC媒体181，M 2-巯基乙醇，50单位/ ml青霉素和链霉素，1mM丙酮酸钠和2mM L-谷氨酰胺向RPMI培养基1640。 </li><li>获得市售重组GM-CSF或使用的GM-CSF从与GM-CSF基因20转AG8.653骨髓瘤细胞含有上清液。确定GM-CSF的中通过ELISA上清液中的浓度为20纳克/毫升的等效添加到BMC介质。 </li></ol></li><li>降速骨髓细胞以314×g离心5分钟。粒料是红色的，由于红细胞​​。在BSC，使用连接到真空抽吸，通过敲击松开沉淀，然后加入10 mL RBC裂解缓冲液的无菌玻璃巴斯德吸管吸出上清液。涡旋悬浮细胞并在室温下在RBC裂解缓冲液孵育5分钟。 </li><li>加入10毫升的HBSS-FCS，然后在314 xg离心旋转细胞5分钟，吸出上清液。观察细胞沉淀，为白色的颜色。重悬BMC媒体所需的音量细胞。 </li><li>算上地区环境部门一道使用血球染色死细胞用0.4％台盼蓝后spended细胞。 注意:如果使用从一条腿（单胫骨和股骨）的骨髓，悬浮在5毫升BMC媒体，取10微升细胞悬液与70微升0.4％台盼的稀释蓝色，拌匀转移10微升1/8细胞稀释到血细胞计数器室。计数存活的细胞在四个象限:四个象限计数的平均×稀释因子×10 4 =每毫升的细胞数。股骨和从一条腿胫骨通常会产生约2 - RBC裂解后4×10 7个细胞。 </li><li>估计所需的基于在培养结束所需的实验的总细胞数下游实验骨髓培养10毫升餐具的数量。 注意:各盘需要在最初的接种2×10 6个骨髓细胞和产量5 - 10×10 6个细胞在第7天。 <ol><li>阿里QUOT细胞的总数为镀到一个新的管中，在314 xg离心降速5分钟，吸出上清液，并在BMC媒体4×10 6个细胞/ ml重悬细胞。 </li></ol></li><li>准备一个新的无菌100毫米×15毫米的培养皿9.5 BMC介质毫升含200毫微克的GM-CSF（重组或从含有上清液GM-CSF）的。加入500微升4×10 6个细胞/ ml的骨髓细胞的培养皿的中心为2×10 5个细胞/ ml在10ml的终浓度。 注意 :要使用无菌培养皿不是一个组织培养皿中是很重要的。此外，加入细胞时，细胞确保在中心仍然集中和播种之后，并在媒体的变化最小化干扰的菜肴。孵育细胞在37℃和5％的CO 2。这是在培养0天。 </li><li>在第3天，加入10毫升含200毫微克的GM-CSF的细胞的每道菜新鲜BMC介质。请注意，以尽量减少干扰Ø˚F浓缩悬浮液中的细胞。细胞培养物的总体积现在是20毫升</li><li>观察放大100倍的光学显微镜下的细胞。观察大量非贴壁细胞（圆形）以及几个贴壁细胞（平）。在花的三个或四个类似瓣组细胞可以看出，指示增殖。 </li><li>在第6天进行一半媒体的变化。小心取出的10ml媒体到无菌50ml锥形管中，在314 XG降速5分钟，吸弃上清。 </li><li>重悬细胞沉淀在10ml含有20纳克/毫升GM-CSF，轻轻涡旋并添加到原始细胞培养皿为20ml的总体积的新鲜的BMC介质。观察在显微镜下的细胞。 注:第7天培养应在70％以上在汇合粘连扁平细胞和圆形非粘附细胞的稠密云。 </li></ol> 3.收集和准备BMDC和BMDMs的流式细胞仪<ol> &lt;李&gt;保持所有的缓冲区，在4℃。 </li><li>收获从第7天的细胞，第10天为收获细胞，将培养物上清液先将10毫升至50毫升的锥形收集管中，然后通过大约30度垂直地倾斜的菜，吹打其余10 1ml洗涤餐具从盘顶部的文化。重复此洗涤5次，每次由三分之一转动盘。 </li><li>传输剩余的10萃取池的到同一收集管并弃去板和贴壁细胞。 注:洗涤将除去非粘附和松散粘附的细胞;平贴壁细胞是对洗涤抗性。通常情况下，在5 - 10×10 6个非粘附细胞从一个板预期在第7天与2 -在第10天5×10 6个细胞，虽然Lutz 等人 。报告了10×10 6细胞在第10天的贴壁细胞通常不采取。然而，如果这些细胞需要进行比较，以非贴壁细胞，它们可以如下除去。添加5毫升0.7毫摩尔EDTA的在1×PBS中，pH为7.4至非贴壁细胞被除去后的盘，孵育在37℃下5分钟，吸液管上下以除去粘附的细胞，并收集在一个单独的试管中。 </li><li>降速收集的细胞在314×g离心5分钟，吸出上清液。悬浮在5毫升无菌BMC媒体在4°C，涡轻轻混合，并用台盼蓝和血球计数。 注意:每个培养皿将产生5 - 10×10 6个非粘附细胞从第7天培养。从现在起，执行4℃的所有步骤。 </li><li>对于流式细胞仪分析，转印每个样品2×10 5个细胞到5毫升聚苯乙烯圆底管，在4℃下（1×磷酸盐缓冲盐水，2毫摩尔EDTA和2％牛血清白蛋白）中添加300μl的FACS缓冲液中向每个样品。 </li><li>旋转细胞向下在314×g离心5分钟，吸出上清液。观察在可见白色的，不透明的粒料管的底部。 </li><li>在100μl未标记Fc受体Ab的悬浮细胞阻断Fc受体结合（从2.4G2产生骨髓瘤细胞系使用1/10上清液），并在4℃下孵育20分钟。然后加入300μl的FACS缓冲液中向每个样品，轻轻涡旋，然后旋细胞向下在314×g离心5分钟，吸出上清液。 </li><li>悬浮细胞在100μl的CD11c-PeCy7的0.2微克/毫升，Gr1的太平洋蓝0.25微克/毫升，MHCII-APC在0.05微克/毫升，和FL-HA在大约2微克/毫升，以确定巨噬细胞和树突状。 注:FL-HA用荧光素和鸡冠的透明质酸钠盐在室内制备如前所述21。 </li><li>孵育20分钟，在4℃下，以允许结合到细胞表面。添加300μl的FACS缓冲液中向每个样品，轻轻涡旋，然后旋细胞向下在314×g离心5分钟，在4℃，吸出上清液。注意:如果生物tinylated抗体在3.8时，重复3.8 - 9与荧光链霉。 </li><li>洗涤细胞与另一300μl的FACS缓冲液中，轻轻涡旋混合并在314 xg离心降速细胞5分钟。吸出上清液并悬浮细胞在200μl含有0.1 FACS缓冲液 - 每ml碘化丙锭或DAPI 0.2微克到标签不能存活的细胞。细胞现在准备流式细胞仪分析22。 注:参见结果与图3为群体的细节和细胞如何门控。 </li></ol>

<ol>
	<li>Fleetwood, A. J., Lawrence, T., Hamilton, J. A., Cook, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+(CSF)+and+macrophage+CSF-dependent+macrophage+phenotypes+display+differences+in+cytokine+profiles+and+transcription+factor+activities:+implications+for+CSF+blockade+in+inflammation.">Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation.</a> J Immunol. 178 (8), 5245-5252 (2007).</li><li>Lari, 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=Macrophage+lineage+phenotypes+and+osteoclastogenesis--complexity+in+the+control+by+GM-CSF+and.">Macrophage lineage phenotypes and osteoclastogenesis--complexity in the control by GM-CSF and.</a> Bone. 40 (2), 323-336 (2007).</li><li>Brasel, K., De Smedt, T., Smith, J. L., Maliszewski, C. R. <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+murine+dendritic+cells+from+flt3-ligand-supplemented+bone+marrow+cultures.">Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures.</a> Blood. 96 (9), 3029-3039 (2000).</li><li>Angelov, G. S., Tomkowiak, M., Marcais, A., Leverrier, Y., Marvel, J. <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-generated+murine+plasmacytoid+and+conventional+dendritic+cells+differ+in+their+capacity+to+prime+naive+CD8+T+cells+and+to+generate+memory+cells+in+vivo.">Flt3 ligand-generated murine plasmacytoid and conventional dendritic cells differ in their capacity to prime naive CD8 T cells and to generate memory cells in vivo.</a> J Immunol. 175 (1), 189-195 (2005).</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 (6), 1693-1702 (1992).</li><li>Lutz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+advanced+culture+method+for+generating+large+quantities+of+highly+pure+dendritic+cells+from+mouse+bone+marrow.">An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.</a> J Immunol Methods. 223 (1), 77-92 (1999).</li><li>Labeur, M. 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=Generation+of+tumor+immunity+by+bone+marrow-derived+dendritic+cells+correlates+with+dendritic+cell+maturation+stage.">Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage.</a> J Immunol. 162 (1), 168-175 (1999).</li><li>Berthier, R., Martinon-Ego, C., Laharie, A. M., Marche, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-step+culture+method+starting+with+early+growth+factors+permits+enhanced+production+of+functional+dendritic+cells+from+murine+splenocytes.">A two-step culture method starting with early growth factors permits enhanced production of functional dendritic cells from murine splenocytes.</a> J Immunol Methods. 239 (1-2), 95-107 (2000).</li><li>Brasel, 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=Flt3+ligand+synergizes+with+granulocyte-macrophage+colony-stimulating+factor+or+granulocyte+colony-stimulating+factor+to+mobilize+hematopoietic+progenitor+cells+into+the+peripheral+blood+of+mice.">Flt3 ligand synergizes with granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor to mobilize hematopoietic progenitor cells into the peripheral blood of mice.</a> Blood. 90 (9), 3781-3788 (1997).</li><li>Segura, E., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+dendritic+cells+in+mice+and+humans.">Inflammatory dendritic cells in mice and humans.</a> Trends Immunol. 34 (9), 440-445 (2013).</li><li>West, M. 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=Enhanced+dendritic+cell+antigen+capture+via+toll-like+receptor-induced+actin+remodeling.">Enhanced dendritic cell antigen capture via toll-like receptor-induced actin remodeling.</a> Science. 305 (5687), 1153-1157 (2004).</li><li>Veldhoen, M., Hocking, R. J., Atkins, C. J., Locksley, R. M., Stockinger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+in+the+context+of+an+inflammatory+cytokine+milieu+supports+de+novo+differentiation+of+IL-17-producing+T+cells.">TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.</a> Immunity. 24 (2), 179-189 (2006).</li><li>Goodridge, H. 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=Activation+of+the+innate+immune+receptor+Dectin-1+upon+formation+of+a+'phagocytic+synapse.">Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse.</a> Nature. 472 (7344), 471-475 (2011).</li><li>Shalek, A. 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=Single-cell+RNA-seq+reveals+dynamic+paracrine+control+of+cellular+variation.">Single-cell RNA-seq reveals dynamic paracrine control of cellular variation.</a> Nature. 510 (7505), 363-369 (2014).</li><li>Vander Lugt, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+programming+of+dendritic+cells+for+enhanced+MHC+class+II+antigen+presentation.">Transcriptional programming of dendritic cells for enhanced MHC class II antigen presentation.</a> Nat Immunol. 15 (2), 161-167 (2014).</li><li>Shibata, 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=GM-CSF+regulates+alveolar+macrophage+differentiation+and+innate+immunity+in+the+lung+through+PU.1.">GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1.</a> Immunity. 15 (4), 557-567 (2001).</li><li>Lacey, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+GM-CSF-+and+Macrophage-CSF+Dependent+Macrophage+Responses+by+In+Vitro+Models.">Defining GM-CSF- and Macrophage-CSF Dependent Macrophage Responses by In Vitro Models.</a> J Immunol. 188 (11), 5752-5765 (2012).</li><li>Poon, G. 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=Hyaluronan+Binding+Identifies+a+Functionally+Distinct+Alveolar+Macrophage-like+Population+in+Bone+Marrow-Derived+Dendritic+Cell+Cultures.">Hyaluronan Binding Identifies a Functionally Distinct Alveolar Macrophage-like Population in Bone Marrow-Derived Dendritic Cell Cultures.</a> J Immunol. 195 (2), 632-642 (2015).</li><li>Helft, 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=GM-CSF+Mouse+Bone+Marrow+Cultures+Comprise+a+Heterogeneous+Population+of+CD11c(+)MHCII(+)+Macrophages+and+Dendritic+Cells.">GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells.</a> Immunity. 42 (6), 1197-1211 (2015).</li><li>Stockinger, B., Zal, T., Zal, A., Gray, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cells+solicit+their+own+help+from+T+cells.">cells solicit their own help from T cells.</a> J. Exp. Med. 183 (3), 891-899 (1996).</li><li>de Belder, A. N., Wik, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+properties+of+fluorescein-labelled+hyaluronate.">Preparation and properties of fluorescein-labelled hyaluronate.</a> Carbohydr Res. 44 (2), 251-257 (1975).</li><li>Stewart, C. C., Stewart, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotyping.">Immunophenotyping.</a> Current Protocols in Cytometry. , (2001).</li><li>Dearman, R. J., Cumberbatch, M., Maxwell, G., Basketter, D. A., Kimber, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+ligand+activation+of+murine+bone+marrow-derived+dendritic+cells.">Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells.</a> Immunology. 126 (4), 475-484 (2009).</li><li>Abdi, K., Singh, N. J., Matzinger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-Activated+Dendritic+Cells:+'Exhausted'+or+Alert+and+Waiting.">Lipopolysaccharide-Activated Dendritic Cells: 'Exhausted' or Alert and Waiting.</a> J Immunol. 188 (12), 5981-5989 (2012).</li><li>Contreras, I., 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=Impact+of+Leishmania+mexicana+Infection+on+Dendritic+Cell+Signaling+and+Functions.">Impact of Leishmania mexicana Infection on Dendritic Cell Signaling and Functions.</a> PLoS Negl Trop Dis. 8 (9), (2014).</li><li>Feng, T., Cong, Y. Z., Qin, H. W., Benveniste, E. N., Elson, C. O. <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+Mucosal+Dendritic+Cells+from+Bone+Marrow+Reveals+a+Critical+Role+of+Retinoic+Acid.">Generation of Mucosal Dendritic Cells from Bone Marrow Reveals a Critical Role of Retinoic Acid.</a> J Immunol. 185 (10), 5915-5925 (2010).</li><li>Grauer, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+maturation+states+of+rat+bone+marrow-derived+dendritic+cells+using+an+improved+culture+technique.">Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique.</a> Histochem Cell Biol. 117 (4), 351-362 (2002).</li><li>Suzuki, 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=Pulmonary+macrophage+transplantation+therapy.">Pulmonary macrophage transplantation therapy.</a> Nature. 514 (7523), 450-454 (2014).</li><li>Happle, 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=Pulmonary+transplantation+of+macrophage+progenitors+as+effective+and+long-lasting+therapy+for+hereditary+pulmonary+alveolar+proteinosis.">Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis.</a> Sci Transl Med. 6 (250), (2014).</li></ol>

The authors have no financial conflicts of interest.

在这个手稿，我们提供了从正在从Lutz 等人的6适于单个小鼠骨髓培养产生的GM-CSF衍生的巨噬细胞和DC的方法。 MHCII表达和FL-HA结合未成熟DC和巨噬细胞区分在这种文化中（ 见图3C），而此前已经很难。这一点，与Helft 等 19另一份报告，都表明以前被认为只的DC GM-CSF诱导BMDC文化中的异质性。 Helft 等 19用完全不同的培养条件下产生的BMDCs但也发?...

几个体外培养的方法已被描述，以产生骨髓来源的巨噬细胞（BMDMs），并使用一种或生长因子的组合骨髓衍生的DC（DC培养上清）。 BMDMs可以通过使用细胞巨噬细胞集落刺激因子（M-CSF）或GM-CSF 1,2-培养骨髓细胞生成。为DC培养上清，加入FLT3配体与骨髓培养产生了非粘附经典和浆的DC（CD11c的高 / MHCII 高和CD11c的LO，B220分别+）后的培养3,4- 9天。与此相反，后7至10天在培养与单独的GM-CSF 5,6-产生的非贴壁细胞，GM-CSF和IL-4 7，或GM-CSF和FLT3配体8,9-生成的BMDCs更密切类似的炎症的DC （CD11c的高 ， 高 MHCII +细胞CD11b）10。而这些在体外培养物被用于产生巨噬细胞或区议会，目前还不清楚，如果每一种文化产生了纯洁的人群。例如，虽然在GM-CSF培养贴壁细胞被描述为巨噬细胞5中 ，从相同的培养非粘附细胞用作树突6,11-13，与推定它们是均匀和任何观察到的变异性是由于发展14,15的不同阶段。此外，研究发现GM-CSF是用于体内 16,17肺泡巨噬细胞发展的一个重要的生长因子，并可以在体外用于生成肺泡状巨噬细胞16,17,18。 除了坚持，从GM-CSF治疗骨髓的文化产生巨噬细胞和DC的过程非常相似的异质性暗示可能GM-CSF骨髓文化中存在。这确实似乎是的情况下为两个论文报告BMDMs在BMDC培养物的非粘附部分的存在。在一个论文中，他们identifi编细胞的CD11c +，CD11b的+ MHCII 中旬 ，MERTK +和CD115 +，它表示最密切相似肺泡巨噬细胞，不得不激活T细胞19的能力下降的基因表达特征的群体。使用MHCII和FL-HA的第二个文件，以便找出一个肺泡巨噬细胞样的人口（的CD11c +，MHCII 中/低 ，FL-HA 高点 ），这是由不成熟的不同（的CD11c +，MHCII 中旬 ，FL-HA 低点 ）和成熟的DC（CD11c的+，MHCII 高 ），这两个表型和功能18。这些论文都表明，GM-CSF BMDC文化是异质的，含有巨噬细胞和DC群表示从BMDC文化解读数据时，应采取照顾。 这个协议描述如何分离骨髓，培养骨髓细胞在GM-CSF，以及识别肺泡巨噬细胞样人口由在骨髓培养未成熟和成熟DC通过流式细胞术采用FL-HA结合和MHCII表达。这个过程是基于Lutz 等人 6的既定的程序，并能产生5 -在一个10毫升培养的第7天10×10 6个非贴壁细胞。培养是可用的从数天7到10和在7这提供了一种简单的方法来生长，并在大量隔离在体外肺泡状巨噬天产生巨噬细胞，未成熟和成熟DC，以及一些前体细胞的异质群体。

<table><tbody><tr><td>Flow Cytometer</td><td>BD&amp;nbsp;</td><td>LSR-II</td><td></td></tr><tr><td>Automated Inverted Microscope&amp;nbsp;</td><td>Leica&amp;nbsp;</td><td>DMI4000 B</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Thermo Fisher</td><td>ST-40R</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>Nuaire</td><td>NU-425-600</td><td></td></tr><tr><td>Syringe 1 ml</td><td>BD</td><td>309659</td><td></td></tr><tr><td>26 1/2 Gauge Needle</td><td>BD</td><td>305111</td><td></td></tr><tr><td>50 ml Conical Tube&amp;nbsp;</td><td>Corning</td><td>357070</td><td>*Falcon brand</td></tr><tr><td>Eppendorf tubes (1.5 ml)</td><td>Corning</td><td>MCT-150-C</td><td></td></tr><tr><td>5 ml polystyrene round bottomed tubes</td><td>Corning</td><td>352052</td><td></td></tr><tr><td>Dissection Tools</td><td>Fine Science Tools&amp;nbsp;</td><td>*Various&amp;nbsp;</td><td>*Dissection scissors, dumont forcep and standard forcep&amp;nbsp;</td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>Reichert</td><td>1490</td><td></td></tr><tr><td>Sterile 100 mm x 15 mm Petri Dish</td><td>Corning</td><td>351029</td><td>*Falcon brand</td></tr><tr><td>2-Mercaptoethanol</td><td>Thermo Fisher</td><td>21985-023</td><td></td></tr><tr><td>Ammonium Chloride</td><td>BDH</td><td>BDH0208-500G</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Fisher Bioreagents</td><td>BP1600-1</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma</td><td>B7651-5MG</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134-1KG</td><td>Ethylenediaminetetraacetic acid</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher</td><td>16000-044</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Thermo Fisher</td><td>14175-095&amp;nbsp;</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher</td><td>15630-080</td><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G8540-100G</td><td></td></tr><tr><td>LPS Ultrapure</td><td>Invivogen</td><td>tlrl-3pelps</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution&amp;nbsp;</td><td>Thermo Fisher</td><td>11140-050</td><td></td></tr><tr><td>Penicillin/Streptomycin 100x</td><td>Thermo Fisher</td><td>15140-122</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>BDH</td><td>BDH0268-500G</td><td></td></tr><tr><td>Potassium Chloride</td><td>BDH</td><td>BDH9258-500G</td><td></td></tr><tr><td>Recombinant GM-CSF</td><td>Peprotech</td><td>315-03-A</td><td></td></tr><tr><td>Rooster Comb Sodium Hyaluronate&amp;nbsp;</td><td>Sigma</td><td>H5388-1G</td><td>*Used to make fluoresceinated hyaluronan</td></tr><tr><td>RPMI-1640&amp;nbsp;</td><td>Thermo Fisher</td><td>21870-076</td><td>No sodium pyruvate no glutamine. Warm media to 37&amp;nbsp;&amp;#176;C before using.</td></tr><tr><td>Sodium Chloride</td><td>Fisher&amp;nbsp;</td><td>5271-10&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma</td><td>50876-1Kg</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>P5290-100G</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Fisher Bioreagents</td><td>BP152-5</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Anti-Fc Receptor (unlabeled), Tissue Culture Supernatant</td><td>N/A</td><td>N/A</td><td>Clone: 2.4G2</td></tr><tr><td>Anti-CD11c PeCy7</td><td>eBioscience</td><td>25-0114-82</td><td>Clone: N418</td></tr><tr><td>Anti-Gr-1 efluor450</td><td>eBioscience</td><td>48-5931-82</td><td>Clone: RB6-8C5</td></tr><tr><td>Anti-MHCII APC</td><td>eBioscience</td><td>17-5321-82</td><td>Clone: M5/114.15.2</td></tr><tr><td>Biotinylated Anti-MerTK</td><td>Abcam</td><td>BAF591</td><td>Goat polyclonal IgG</td></tr><tr><td>Streptavidin PE</td><td>eBioscience</td><td>12-4317-87</td><td></td></tr><tr><td>Propidium Iodide</td><td>Sigma</td><td>P4170-25MG</td><td></td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole)</td><td>Sigma</td><td>D9542-5MG</td><td></td></tr></tbody></table>

generation and identification of gm-csf derived alveolar-like macrophages and dendritic cells from mouse bone marrow

巨噬细胞和树突状细胞（DC）是在组织和淋巴器官发挥抵御病原体是重要角色发现先天免疫细胞。然而，它们难以在足够数量的分离，以研究它们的详细，因此， 在体外模型已经制定出来。 体外骨髓来源的巨噬细胞和树突状细胞的培养物用于免疫学研究成熟的和有价值的方法。这里，描述了用于培养和使用该细胞因子粒细胞巨噬细胞集落刺激因子（GM-CSF）的原代小鼠骨髓细胞的单培养识别两者的DC和巨噬细胞的方法。此协议是基于首先由Lutz 等人开发的既定程序。在1999年的骨髓来源的DC。培养是异质的，并且MHCII和荧光标记的透明质酸（FL-HA）是用来区分未成熟和成熟DC的巨噬细胞。这些GM-CSF巨噬细胞亲韦迪体外衍生的巨噬细胞可以非常类似于在两个表型和功能肺泡巨噬细胞的一个方便的来源。

总结了该方法的主要步骤的流程图如图1所示。在培养的不同时间的骨髓培养物密度和形态在图2中示出。在第1天，将细胞小和稀疏，用3天有更多的细胞，一些较大和一些已开始附着。第6天有一定的粘附和非粘附部分（ 图2A）。培养可以从第7天收获- 10与存在于第10天。图2B的CD11c +细胞的比例更高示收获前一天7培?...

Watch this Scientific Journal Video about Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow at JoVE.com

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

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. ...

generation-identification-gm-csf-derived-alveolar-like-macrophages

Research

JoVE Journal

Immunology and Infection

17.9K 次观看.  University of British Columbia. 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. 

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

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

科研

生物学

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

免疫与感染

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

从小鼠中分离和培养骨髓来源的巨噬细胞

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

新生儿骨髓来源巨噬细胞的吞噬试验评价 

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

作者聚焦：开发一种从新生小鼠中分离骨髓来源的巨噬细胞的可重复方法，以促进早期免疫反应

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...

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

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此视频中的章节

文化髓系树突状细胞从骨髓前体

使用骨髓源性巨噬细胞巨噬细胞极化的调查

骨髓衍生的巨噬细胞产

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

从小鼠中分离和培养骨髓来源的巨噬细胞

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备