这项研究是由西南德意志研究联合会（DFG WA 2804）和西南大学教育资助的实验和临床研究中心，最大的一个合作德尔布吕克分子医学中心在柏林的Charité医学院。资助者在研究设计，数据收集和分析，决定发表或准备手稿的作用。我们感谢罗伯特·韦斯特法尔先生在他的技术支持，在我们的实验室实习。

所有动物的程序，必须由当地机构执行前的动物福利委员会批准。于MR测量麻醉和生理监测（体温，呼吸频率）足够水平的必不可少的要求。 1。小鼠骨髓来源的树突状细胞生成<ol><li>提取C57BL / 6小鼠骨髓细胞如前所述，该协议的历史可以追溯到1992年13最初是由拉尔夫·斯坦曼（1943年至二○一一年），树突状细胞的发现者14组 12。 </li><li>简言之，提取后不久，胫骨和腓骨牺牲老鼠。下工作的层流罩（防止微生物污染），冲洗骨髓成一个小的陪替氏培养皿用洗涤培养基（5％FBS，1％青霉素/链霉素和1％HEPES的RPMI）12。 </li><li>通过细胞的细胞悬浮液转移斯特拉惯性（网目尺寸为100μm，尼龙）到无菌的50毫升离心管中，以去除骨和剩余的组织。经过多次洗涤步骤和裂解步骤12，活细胞使用台盼蓝和血球计数。 </li><li> 重悬在毫升-1（补充有10％FBS的RPMI 培养基中的浓度为400,000个细胞的骨髓细胞 ，1％青霉素/链霉素，1％HEPES和1％L-谷氨酰胺），含有75毫微克毫升-1鼠标粒巨噬细胞集落刺激因子（MGM-CSF）从293FT的小鼠GM-CSF的质粒转染的HEK细胞的上清液中获得。转移10ml的细胞悬浮液（4×10 6个细胞）在皮氏培养皿（100毫米×20毫米），在CO 2培养箱中培养（37℃，5％CO 2）。第3天，用10毫升新鲜培养基（MGM-CSF终浓度保持在75纳克毫升-1）补充旧的介质。第6天，取出10毫升的旧介质和补充用10毫升新鲜培养基（MGM-CSF终浓度= 75纳克毫升-1）。第8天，取出10毫升的旧介质，并补充10毫升新鲜培养基（MGM-CSF终浓度= 150毫微毫升-1）。 </li><li>第10天，1微克毫升-1脂多糖（LPS），24小时成熟分化的DC。 </li></ol> 2。标记树突状细胞与19架F-富<ol><li>在24小时熟化步骤（1.5），标签的DC与1毫米富氟全氟-15 - 冠醚颗粒（PFCE）（500-560纳米）也为24小时。准备大氟标记的粒子通过乳化PFCE，在Pluronic F-68（总体积2.5毫升）使用细胞破坏钛超声波发生器，采用连续脉冲程序（幅度100％，功率= 250 W），持续60秒。 </li><li>在上述培养步骤中，收获成熟和氟标记的DC使用组织培养细胞刮棒，转移到50毫升的离心管中，离心在500×g下在室温下5分钟。 </li><li>要洗去剩余的颗粒细胞和​​死细胞，留下收获细胞附着在聚-L-赖氨酸的菜肴。为了制备这一步，大衣的培养皿与聚-L-赖氨酸（1毫克毫升-1）在37℃下1小时，之后洗掉未结合的聚-L-赖氨酸用PBS。 </li><li>上述收集的细胞在无血清培养基中的聚-L-赖氨酸包被的板孵育，使细胞附着（1小时，在37℃下，5％CO 2）。 </li><li>洗掉培养基中，丢弃非贴壁细胞，其余的颗粒细胞和​​死细胞洗三次，用预温（37°C）的PBS。 </li><li>收获贴壁细胞治疗胰蛋白酶-EDTA（0.25％胰蛋白酶-EDTA，在37℃孵育3分钟）。 </li><li>另一个洗涤离心步骤后，重悬在无血清的无菌PBS将所需量的成熟的DC。 </li><li>为了判断细胞是否已成功标记，建议检查physicocheMICAL特性的细胞用流式细胞仪（FACS）。的侧向散射（SSC）揭示了一种增加的粒度在这些细胞中，如前面所述11。还应该记住的是，成纤维细胞可能污染的DC培养物，也可以采取19 F颗粒平行。因此，DC纯度应评估（使用流式细胞仪通过测量表面CD11c +细胞CD11b +细胞的百分比）前在体内应用。 </li></ol> 19 F-标记的树突状细胞在体内的应用3。 <ol><li>管理DC（10 6 - 10 7）在一个体积为50 - 100微升通过将鼠标定位在保持器中的C57BL / 6小鼠皮内注射到后肢并小心地插入到皮肤的上层的26½ģ针申请前（0.5毫升的结核菌素注射器针永久连接使用，以尽量减少死体积）。为了确保Ñ​​适当的皮内应用，重要的是，针的插入部分下方的皮肤是部分可见的。如果针是不可见的了，针已经插入过深进入皮肤层。对于未经训练的人，皮内的应用程序可以在麻醉下进行。 </li><li>图片四肢在体内的氟（第19楼）/质子（1H）磁共振成像（见下一节）注射后21小时。 </li></ol> 4， 在体内 19 F / 1ħ磁共振成像设立MR扫描的详 ​​细说明是指在Bruker MR扫描仪使用的的控制软件Paravison（版本5.1）。指的是供应商特定的功能和项目的名称已用斜体突出显示。对于其他MR扫描仪，这些步骤可能要进行调整，根据生产商的指引。 <ol><li>安排访问一个专门的小动物磁共振扫描仪。对于足够的图像质量，与磁场强度为9.4特斯拉以上且量身定制的射频线圈（ 例如 1 H / 19 F型双可调体积射频线圈，35毫米内径50毫米的长度;快速生物医学，维尔茨系统，德国）建议。 </li><li>此前MRI，准备麻醉和生理监测的核磁共振成像扫描仪的设置。确保的动物的呼吸用面罩床/保持器被连接到的异氟烷系统和温度探头和呼吸焊垫连接到远程动物监测系统（ 例如 SA仪器公司，型号1025，纽约，美国）。后者用来监视动物的重要的参数，例如体温和呼吸。 </li><li>麻醉小鼠用异氟醚的系统连接到鼠标室通过吸入麻醉状态。 0.2升分钟-1和0.1升分钟-1，分别调整空气流速和O 2和3％异氟醚（从蒸发器调整）约2分钟，直到所需的麻醉水平达到（以下脚趾捏没有响应）。最佳流速可能取决于正在使用的设置上。要知道，高流速可能导致脱水。上文所提到的，在任何时候都需要仔细监测动物的条件。 </li><li>将鼠标俯卧鼠标床/持有的小动物磁共振扫描仪（ 图1）。 </li><li>同时保持空气和O 2恒定的流速，调整异氟醚蒸发0.8 - 1.5％，直到达到最佳的呼吸模式。 50 - 70次每分钟的呼吸速率建议。 </li><li>在实验过程中保持身体的温度在36-37°C通过采用温水（或交替暖空气）循环系统。 </li><li>用无菌眼润滑软膏在测量过程中保持鼠标湿润的眼睛。 </li><li>在确保动物在成像保持器和连接到监测系统，移动夹持器的1 H / 19 F号RF线圈（即定位在动物扫描器磁体的等角点）的中心。 </li><li>鼠标等的设置位置，膝关节内的磁体的等角点，无论是一个自动化的方法（ 例如，使用一个马达驱动的动物保持器结合的定位激光），或通过手动计算需要移动的保持器的距离进磁体达到等角点。 </li><li>为了确认在扫描仪中的动物的正确定位和以后的规划形象片的几何形状（如大小，位置和旋转空间），收购侦察三种标准方向（轴向，冠状，矢状）使用快速和低分辨率图像的图像采集方法（ 如TriPilot协议，它采用了FLASH脉冲序列）。 </li><li>调谐射频线圈的1 H共振频率（ 例如400.1 MHz为9.4ŧ）和19 F的共振频率（ 例如 376.3 MHz为9.4ŧ）和匹配的线圈的特性阻抗50欧姆的动物MR扫描仪中使用的调谐监视器。调谐和匹配是必要的传输和信号接收达到最佳条件。 </li><li>执行必要的自动的系统设置，包括垫片来微调调谐射频鼠标（ 例如ADJ_SF）的拉莫尔频率和参考增益调整的RF振幅的均匀性的磁场（ 例如ADJ_SHIM），系统频率调整（ 如ADJ_REFG）。所有这些设置是尽可能地均匀，重要的是使磁性元件的静态和可变字段（B 0，B 1）的感兴趣的区域。 </li><li>沿三个正交方向采集的第二组的侦察员图像（如上述），之后，调整视场（FOV），使得两下肢芳E可见骨盆英尺（长×宽×高= 50x25x25毫米）。这些图像有助于规划的最终3D 1 H / 19 F扫描的方向。 </li><li>设置了TurboRARE的3D协议1 H-扫描：TR / TE = 1500/53毫秒，FOV = 50x25x25毫米，矩阵= 400x200x200，难得因数= 16（扫描时间约60分钟）。调整视场与侦察员图像的帮助下，开始扫描（ 交通灯 ）。 </li><li>与至少1,000毫秒的TR装入单脉冲FID序列。打开编辑扫描和设置核19楼“使用手动增益设置，取消选择“工具箱”的编辑方法自动参考收益。 </li><li>开始测量，而不记录数据实时显示MR信号通过GSP（去设置）。 19 F，如果频谱信号的采集窗口内太低，添加更多的平均值，在编辑的方法和/或调节的脉冲衰减器（TX0，否则SP0滑块），直到思此项目是清晰可见的。调整的基本频率，以中心19女性在0 Hz的谱峰在收购窗口。 应用基本频率 ，并按下停止 。请注意，在异氟醚麻醉作为可能会生成一个背景信号。在9.4特斯拉核磁共振成像之间的19 F-的含粒子和异氟醚的化学位移是1800赫兹左右。确保激发带宽小于3600赫兹，以避免激励异氟醚的19 F-标记的细胞或颗粒一起。上正确的19 F-标记的细胞所产生的信号的基本频率应设置。或者，另一种方法，可以用来作为麻醉实验者认为合适。 </li><li>克隆（副本）TurboRARE的3D协议，从以前的1 H扫描。转到编辑方法和取消选择自动的参考增益（如上）。将细胞核从编辑扫描 19女性 。改变矩阵128x64x64和稀土因数至少为40（最多64个）。对于TR / TE 1,000 / 6毫秒和64的平均值时，扫描时间是约70分钟。接收机增益设置为101（ 工具箱），并开始扫描，而不使用GOP（去管道）的任何进一步的调整。 </li><li>当扫描完成后收回的MR扫描仪鼠标座。小心拔下鼠标的持有人。如果鼠标不牺牲体外分析（ 如组织学或光谱，见下文）后直接MR测量，严密监控，直到它已完全从麻醉中恢复。体温调节是受麻醉，所以在恢复过程中，把鼠标被放置在一个温暖的温度调节垫在一个单独的笼子里。一旦鼠标已经完全从麻醉中恢复，它返回到其控股笼和动物室。 </li><li>要覆盖19 F 1 H图像的图像，使用“ 查看”菜单 Ø佩蒂翁在同一软件（Paravison）的。 底图功能下可以找到关联 。保存底衬，加载的新形象，并通过定义一个新的颜色查找表突出的19个F层。要区分的19楼和1 H扫描所选择的颜色，改变阈值（ 切查表 ）19 F信号将有选择的颜色和背景1 H形象将留在灰度。其他后处理程序（ 如 ImageJ的）可用来创建的1 H / 19 F图像的覆盖。 </li><li>如果在体内 19 F信号的量化是必要的，遵循一个简单的定量协议如前所述15,16。简单地说，可以通过将管内的视场角下一个鼠标的PFCE乳液，使用一种已知浓度的参考量化。收购后的MR图像，全TIFY使用的感兴趣区域（ROI）的分析，19 F信号的强度，并比较在体外校准曲线， 如图2所示。 </li></ol> 5。淋巴结磁共振波谱（MRS） <ol><li>牺牲了鼠标后，取出腘窝淋巴结和小5毫米核磁共振管，并加入100微升2％的煤灰。 </li><li>安装内标的19 F光谱线圈（ 如环形线圈），它具有至少为5毫米的开口用于保持的NMR管中，淋巴结。 </li><li>线圈内的NMR管中放置和移动线圈的磁体的等角点。 </li><li>调谐射频线圈19 F共振频率（ 例如 376.3 MHz为9.4ŧ）和相匹配的线圈的特性阻抗50欧姆的动物MR扫描仪中使用的调谐监视器。调谐和匹配是必要的，以达到最佳的传输和信号条件接待处。 </li><li> 垫补工具箱 0内所有垫片参数设置和按申请。一般是没有必要用匀场的方法，由于样品的体积是相对小的。如果有足够的19 F信号是可用的，自动化的方法可施加垫片，系统频率和上面所描述的接收器的增益。 </li><li>与至少1,000毫秒的TR装入单脉冲FID序列。打开编辑扫描的细胞核，并设置19楼使用手动增益设置，取消选择“工具箱”的编辑方法自动参考收益。设置数的平均值为1。 </li><li>启动顺序使用GSP。 19 F，如果频谱信号的采集窗口内太低，在编辑的方法和/或添加更多的平均值调节的脉冲衰减器（TX0，否则SP0滑块），直到一个信号是清晰可见的。调整的基本频率，使19 F SPEctral峰是在0 Hz采集窗口的中心，并适用于基本频率。再次调整脉冲衰减器，使信号最大化，达到90°激发。当信号是最大的新闻停止 。 </li><li>设置在“编辑”，64和256之间的方法 （或更多的，如果必要，这取决于信号）的平均值和使用的GOP开始采集。检测到的信号线性相关与平均值的数目。在量化过程中牢记，检测到的信号需要进行归一平均。此外，已知浓度的校准标准或校准曲线是必要的信号（ 图2），归一化的19 F的细胞数来计算。 </li><li>一旦扫描完成后，取出样品，将样品和进行步骤5.3。 </li></ol>

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Med. 58 (4), 725-734 (2007).</li><li>Waiczies, H., Lepore, S., Janitzek, N., Hagen, U., Seifert, 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=Perfluorocarbon+particle+size+influences+magnetic+resonance+signal+and+immunological+properties+of+dendritic+cells.">Perfluorocarbon particle size influences magnetic resonance signal and immunological properties of dendritic cells.</a> PLoS ONE. 6 (7), e21981 (2011).</li><li>Matheu, M. P., Sen, D., Cahalan, M. D., Parker, I. <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+Bone+Marrow+Derived+Murine+Dendritic+Cells+for+Use+in+2-photon+Imaging.">Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging.</a> J. Vis. Exp. (17), e773 (2008).</li><li>Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, 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=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> Journal of Experimental Medicine. 176 (6), 1693-1702 (1992).</li><li>Lanzavecchia, A., Sallusto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ralph+M.+Steinman.+1943-2011.">Ralph M. Steinman. 1943-2011.</a> Cell. 147 (6), 1216-1217 (1943).</li><li>Srinivas, M., Turner, M. S., Janjic, J. M., Morel, P. A., Laidlaw, D. 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=In+vivo+cytometry+of+antigen-specific+t+cells+using+19F.">In vivo cytometry of antigen-specific t cells using 19F.</a> MRI. Magn. Reson. Med. 62 (3), 747-753 (2009).</li><li>Srinivas, M., Heerschap, A., Ahrens, E. T., Figdor, C. G., de Vries, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(19)F+MRI+for+quantitative+in+vivo+cell+tracking.">(19)F MRI for quantitative in vivo cell tracking.</a> Trends Biotechnol. 28 (7), 363-370 (2010).</li><li>Lammermann, T., Bader, B. L., Monkley, S. J., Worbs, T., Wedlich-Soldner, 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=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Liu, M. S., Long, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluoroctylbromide+as+a+diagnostic+contrast+medium+in+gastroenterography.">Perfluoroctylbromide as a diagnostic contrast medium in gastroenterography.</a> Radiology. 122 (1), 71-76 (1977).</li><li>Kwiatkowska, K., Sobota, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+in+phagocytosis.">Signaling pathways in phagocytosis.</a> Bioessays. 21 (5), 422-431 (1999).</li><li>Mitragotri, S., Lahann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+approaches+to+biomaterial+design.">Physical approaches to biomaterial design.</a> Nat. Mater. 8 (1), 15-23 (2009).</li><li>Hoult, D. I., Richards, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signal-to-noise+ratio+of+the+nuclear+magnetic+resonance+experiment.">The signal-to-noise ratio of the nuclear magnetic resonance experiment.</a> J. Magn. Reson. 24 (1), 71-85 (1976).</li><li>Kovacs, H., Moskau, D., Spraul, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryogenically+cooled+probes+-+a+leap+in+NMR+technology.">Cryogenically cooled probes - a leap in NMR technology.</a> Prog. Nucl. Magn. Reson. Spectrosc. 46 (2-3), 131-155 (2005).</li><li>Waiczies, H., Millward, J. M., Lepore, S., Infante-Duarte, C., Pohlmann, 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=Identification+of+Cellular+Infiltrates+during+Early+Stages+of+Brain+Inflammation+with+Magnetic+Resonance+Microscopy.">Identification of Cellular Infiltrates during Early Stages of Brain Inflammation with Magnetic Resonance Microscopy.</a> PLoS ONE. 7 (3), e32796 (2012).</li><li>Haacke, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Magnetic resonance imaging physical principles and sequence design. , (1999).</li></ol>

作者宣称，他们有没有竞争经济利益。

这种方法采用19 F / 1ħMRI跟随移动到淋巴结的DC提供了学习的机会， 体内的免疫细胞的迁移模式。树突状细胞是快速迁移的免疫细胞，是能够通过三维结构不紧密粘附的特异性底物17的情况下的机动的很好的例子。虽然所描述的技术的低空间分辨率（μm的范围内）是不与高的分辨率（nm范围内），使用这种技术，可以实现与多光子显微镜，它仍然是可能的，研究细胞?...

细胞在体内的跟踪是在一些生物医药领域的一个重要方面。对于这一点，非侵入性的成像技术，可以选择性地本地化细胞过一段时间是极​​其宝贵的。三维磁共振成像（MRI）的发展之前，不限于显微分析或组织切片的免疫细胞迁移的跟踪。细胞跟踪的帮助下，MRI在过去的几年中取得了巨大的关注，不仅为免疫学研究的免疫细胞在体内的行为，同时也为临床和干细胞研究。在90年代中期，最初的研究氧化铁纳米粒子发起级联发展与MRI追踪细胞。氧化铁颗粒缩短MR弛豫时间（T2 *）标记的细胞，从而导致信号损耗MR图像。氧化铁颗粒已雇用标签的巨噬细胞，少突胶质细胞progenitors 3和许多其它类型的细胞。这些颗粒也被临床被FDA批准用于标记细胞疫苗在黑色素瘤患者4。由于在体内或体外与氧化铁颗粒标记的细胞依赖于缩短的T2 *信号，后者还可以带来体内的易感性相关T2 *的影响，如微出血，铁沉淀物或气泡，因此可能很难识别标记的细胞在体内的来自其他背景T2 *信号灭绝5。 在这篇文章中，我们描述了一种技术用于跟踪在体内的树突状细胞（DC）采用19 F / 1ħ磁共振成像（MRI）。这种细胞追踪技术才被引入几年后，于2005年6 19 F在MRI首次确认申请报告已7。其中一个重要的ADVA氧化铁颗粒细胞标记的19 F超过ntage是低的生物组织中发生的19 F，这使得它可以非常有选择性与基本上无背景图像跟踪细胞。此外，有可能获得从传统的1H核磁共振成像的解剖图像，叠加的19 F标记的细胞从移植的MR信号。因此，19 F / 1ħ核磁共振成像是相当有关的研究在体内细胞迁移。用这种方法研究的细胞都标有19架F-富。综合来自主要由碳原子和氟原子组成的全氟碳（PFC），通常用于制备颗粒。这些化合物是不溶于水和乳化之前， 在体外或体内应用。通常的PFC颗粒大小已受聘于其他群体在体内 19 F-MRI跟踪实验介于100纳米和245纳米6,8-10。然而，我们已经表明，标签与全氟-15 -冠-5 -醚（PFCE）随着粒径（&gt; 560nm的颗粒增加的树突状细胞中的效率）11

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>C57BL/6 mice</td>
 <td>Charles River, Berlin</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td>Gibco</td>
 <td>21875-091</td>
 <td></td>
 </tr>
 <tr>
 <td>FBS Superior</td>
 <td>Biochrom AG</td>
 <td>S 0615</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Gibco-Invitrogen</td>
 <td>15630-056</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15140-122</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030-024</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's PBS</td>
 <td>Sigma Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>PFA</td>
 <td>Santa Cruz</td>
 <td>sc-281692</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfluoro-15-crown-5-ether </td>
 <td>ChemPur</td>
 <td>391-1996</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F-68 </td>
 <td>Sigma Aldrich</td>
 <td>P5556</td>
 <td></td>
 </tr>
 <tr>
 <td>Petri dishes (35 x 10 mm)</td>
 <td>VWR, Germany</td>
 <td>391-1996</td>
 <td></td>
 </tr>
 <tr>
 <td>27 &frac12; G syringes</td>
 <td>VWR, Germany</td>
 <td>612-0151</td>
 <td></td>
 </tr>
 <tr>
 <td>Nylon cell strainers (100 &mu;m mesh) </td>
 <td>VWR, Germany</td>
 <td>734-0004</td>
 <td></td>
 </tr>
 <tr>
 <td>NMR tubes </td>
 <td>VWR, Germany</td>
 <td>634-0461</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Dissection tools</td>
 <td>FST</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CO2 incubator </td>
 <td>Binder</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Small animal MR system</td>
 <td>Bruker Biospin</td>
 <td></td>
 <td>9.4T BioSpec 94/20 USR, ParaVision Acquisition and Processing Software</td>
 </tr>
 <tr>
 <td>1H/19F dual-tunable volume RF coil </td>
 <td>Rapid Biomed, W&uuml;rzburg, Germany</td>
 <td></td>
 <td>35 mm inner diameter, 50 mm length</td>
 </tr>
 <tr>
 <td>19F spectroscopy coil </td>
 <td>in-house</td>
 <td></td>
 <td>tune/match loop coil, 4 turns, inner diameter 5 mm, 10 mm long, two capacitors for tuning and matching</td>
 </tr>
 <tr>
 <td>Isoflurane inhalation system</td>
 <td>F&ouml;hr Medical Instruments GmbH</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Animal monitoring system Model 1025</td>
 <td>SA Instruments Inc., New York, USA</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

monitoring dendritic cell migration using 19f / 1h magnetic resonance imaging

如磁共振成像（MRI）的非侵入性的成像方式的不断进步，极大地提高了我们的学习能力，在生物体的生理或病理过程。 MRI也证明是一个有价值的工具捕捉移植细胞在体内 。初始小区标记策略用于MRI使用的造影剂的影响的MR弛豫时间（T1，T2，T2 *）和引起增强（T1）或耗尽（T2 *）标记的细胞存在的信号。 T2 *增强剂，如超小型氧化铁剂（USPIO）已经被用来研究细胞迁移和一些人也已被FDA批准用于临床应用。 T2 *剂的缺点是难以区分从其他文物，如血块，微出血或气泡标记细胞的信号灭绝创建的。在这篇文章中，我们描述了一个新兴的技术跟踪细胞在体内基于氟（第19楼）丰富的颗粒细胞标记。这些颗粒的制备是通过乳化的全氟化碳（PFC）的化合物，然后用的标签细胞，其后，可以通过19 F核磁共振成像成像。的PFCs的重要的优点包括（i） 在体内的碳结合的19 F 在体内 ，然后通过19 F磁共振波谱产生背景图像和量化的细胞信号的完整的细胞选择性强（ⅱ）的可能性的情况下进行细胞跟踪。

18〜24小时后，皮内的应用程序，19 F-标记的树突状细胞（DC）迁移到排水的腘淋巴结。可以理解的DC，通过到排水politeal的淋巴结的淋巴管的运动，通过叠加的1 H 19 F DC图像（ 图2A）的解剖图像。我们以前曾报道这些细胞在体内的迁移，以及19 F-粒度对DC免疫生物学，包括吸收效率11的影响。为了量化的DC迁移到淋巴结的程度，我们提取引流淋巴结?...

Watch this Scientific Journal Video about Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging at JoVE.com

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

细胞利用磁共振成像跟踪，在过去几年里取得了显着的关注。本协议描述标签的树突状细胞与氟（<sup&gt; 19</sup&gt; F）的丰富的颗粒，这些细胞在体内的应用程序，和监测他们迁移到引流淋巴结的程度<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H MRI和<sup&gt; 19</sup&gt; F刘健。

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

monitoring-dendritic-cell-migration-using-19f-1h-magnetic-resonance

Research

JoVE Journal

Immunology and Infection

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

11.7K 次观看.  A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine. 细胞利用磁共振成像跟踪，在过去几年里取得了显着的关注。本协议描述标签的树突状细胞与氟（ 19 F）的丰富的颗粒，这些细胞在体内的应用程序，和监测他们迁移到引流淋巴结的程度 19 F / 1 H MRI和 19 F刘健。

视频: Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

白三烯B实时成像 4介导的细胞迁移和BLT1与β- arrestin的相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

竞争的原点实验研究肠嗜T细胞迁移

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

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 ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染<em&gt;牛分枝杆菌</em&gt;卡介苗

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

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 ...