作者要感谢在大学健康网络高级光学显微镜基金（www.aomf.ca），与显微镜的帮助，并从玛嘉烈医院癌症中心的机械车间贾森·埃利斯先生制造WC和成像阶段。我们还要感谢光圈Kulbatski博士稿件编辑。

注:所有动物的工作是在协议＃2615由大学健康网络机构动物护理和使用委员会批准进行。 1.手术鼠的制备<ol><li>在手术前，消毒所有手术器械和窗口室（WC）通过高压灭菌。用50毫克/公斤体手术前2天重量阿莫西林补充饮用水。注入小鼠与手术前腹膜内4小时0.1毫克/千克体重丁丙诺啡。 </li><li>麻醉由350-400微升生理盐水腹腔注射无胸腺裸鼠含80毫克/千克氯胺酮和13毫克/公斤甲苯噻嗪（注:这是由机构动物护理委员会批准，我们还没有与不利影响的任何经验）。通过测试足部反射确认适当的麻醉。继续观察的过程中呼吸模式，粘膜颜色和足部反射。 </li><li>执行手术PR生物安全柜下ocedures保持手术过程中无菌条件。将鼠标上覆盖有外科被单的手术过程中，保持体温的电加热垫。在立体显微镜下进行所有的外科手术。 </li><li>应用眼膏在手术过程中，保持眼睛湿润。必要时重复进行的外科手术过程中。通过用7.5％聚维酮 - 碘，70％异丙醇和10％的聚维酮碘擦拭连续消毒鼠标的操作区，并重复此过程两次。 </li><li>使用手术刀用刀片（＃15）10毫米纵向切开皮肤，从侧面接近股骨。使用镊子和止血，以保证肌肉功能钝性分离暴露屈肌和伸肌的股骨。 </li><li>插入骨下的U形杆，并安装WC（ 图1）。安全使用两个螺母窗口栏，并拧紧与螺母牛肝菌。填充骨和牙科用粘固了WC之间的空间。 </li><li>磨得在一个6×2 mm 2的区域中的骨皮质用微钻头获得对骨髓腔的光接入。 注意:仔细检查骨皮质变薄，在显微镜下，而执行此过程。离开骨完整，几乎透明的骨膜层。 </li><li>将8mm的玻璃罩到窗口，并用卡环固定玻璃罩，以保持它在的地方。 </li><li>通过使用镊子，以保持其功能的手术后，重新调整屈伸回原来的位置的肌肉。缝合使用5-0缝合和针保持器真皮。 </li><li>监测小鼠进行恢复，直到它是有意识的，并能自由移动。把动物进入一个新的笼子里回过神来的时候。 </li><li>恢复后，执行上的当天或随后几天的动物图像采集（见第2节）。监控physi鼠标校准状态每天。 注:监控物理条件，如负重肢体，拱起，快速或呼吸浅，后肢肿胀，自残，蜂窝组织炎，重度皮炎。 </li><li>得到的0.1毫克/千克体重丁丙诺啡腹腔注射3天，和1毫克/公斤体美洛昔康的5天的手术治疗手术后疼痛和炎症以下重量。继续5天提供阿莫西林补充水。 </li><li>在实验终点，用CO安乐死动物2麻醉，随后由次级方法（ 例如 ，颈脱位）。 </li></ol> 2.图像采集使用组合多光子和共聚焦显微镜<ol><li>成像之前，由含有80毫克/千克氯胺酮和13毫克/千克甲苯噻嗪一个350-400微升盐溶液的腹膜内注射麻醉小鼠。另外，尽可能使用吸入麻醉剂。应用眼膏。 </li><li>注入用于标记脉管的血管内染料（0.65毫克2-丙二醛荧光素（FITC） - 葡聚糖）和嗜中性粒细胞（4微克Allophycocyanine（APC的） - 抗 - Ly6G抗体）的混合物。 </li><li>使用一个直立组合后的多光子和共聚焦显微镜配备多光子激光从705-980纳米为双光子激发，以及单光子激光器提供激发波长从蓝色至红色。 </li><li>设置一个定制的成像舞台上镜（ 图1B）。由WC的两头鳄鱼夹夹紧固定在舞台上的鼠标。 注意:舞台的关键组件是:二鳄鱼夹持有了WC，和一个加热元件，以在成像期间保持鼠标生理温度（37℃）。 </li><li>打开双光子激光和共聚焦系统，并通过点击"开始系统"启动图像采集的软件。有4个选项卡的窗口（定位，采集，处理和维护）将打开。 </li><li>建立了成像通道。点击"智能设置"取样选项卡下，选择将用于成像染料:FITC（488 nm激发，493-588 nm发射）和APC（633纳米激发和638-743 nm发射）。选择"最佳的信号"，然后点击"应用"。 </li><li>手动添加二次谐波（SHG）图像采集渠道。打开双光子激光下的采集选项卡中的"激光"窗口。根据通道窗口中，点击"+"号添加曲目。 </li><li>建立了SHG轨道。使用向上和向下箭头的波长变化至840纳米的下通道窗口，选择检测范围是根据使用滑块栏中的"光路"窗口415-425纳米。 </li><li>点击"在线目镜"的定位选项卡下。点击过滤器设置下为FITC FITC查看感兴趣的区域，并转动T手动调整焦距他调焦旋钮显微镜的一面。当感兴趣的区域的位置，单击该选项卡中找到的"目镜离线"。 </li><li>点击采集标签。选择在通道窗口FITC轨道，然后点击"实时"预览FITC葡聚糖具有5X客观的形象。如有必要，调整焦距，然后单击"停止"收购选项卡下停止预览。 </li><li>通过移动镜头上方的轮手动打开20倍水的目标。再次查看图像使用20X客观FITC葡聚糖和渠道窗下调整主增益和针孔，以达到最佳的信号和对比度。重复的通道窗口下选择APC和SHG轨道等渠道。 </li><li>选择的摄像参数。点击"采集模式"，以打开其​​窗口。设置帧大小为1,024×1,024像素，和4X的平均化，以获得高质量的图像。 </li><li>获取二维快照。选择所有通道下通道取胜道指和点击"捕捉"收购选项卡下。检查图像的质量，并在必要时更改成像参数。 </li><li>获取2D时间推移图像。 <ol><li>检查关"时间序列"中的采集标签打开时间序列窗口。输入0的时间间隔下，和40扫描的总数采取连续40张无时间间隔。 注:40扫描产生大约5分钟长的时间序列与7.5秒/帧的扫描速度和600微米×600微米的图像尺寸。调整间隔，并根据在显微镜，图像采集设置，摄像机的帧速率的扫描次数。固定时间间隔（&gt; 0秒）可用于比较在不同时间点获取的时间推移的图像。较长的时间推移成像持续时间可以根据在实验条件下的中性粒细胞（或其它感兴趣的细胞）的预期平均速度是必需的。 </li></ol></li><li>获得3D图像。<ol><li>检查关"的Z-Stack"，在采集选项卡打开Z-Stack窗口。启动FITC葡聚糖"实时"预览模式。专注于样品的顶部，然后单击"设置第一"，并专注于样品的另一端，并单击Z堆栈窗口"设置最后"。 </li><li>设置间隔为Z-Stack窗口在10微米。取决于第一和最后一个光学片在上一步中选择，这将产生10至20片，与90-180微米的总光学厚度。 注意:Z堆叠间隔是依赖于针孔，它决定了光切片的厚度的大小。将间隔设置为小于光学切片的厚度。 </li><li>点击采集选项卡下的"开始实验"开始采集图像。 </li></ol></li><li>获得3D时间推移图像。 <ol><li>检查关"时间序列"和"Z堆栈"中的采集标签打开时间序列和Z堆叠的窗户。 </li><li>在步骤2.14和2.15方法设定时间序列和Z堆栈采集参数。点击采集选项卡下的"开始实验"开始采集图像。 </li></ol></li><li>在成像过程中监视鼠标，以确保它是稳定的和无意识的。如果鼠标成像期间不稳定和/或意识，从台上移除小鼠，腹膜内注入额外100-150微升麻醉剂溶液中，并继续成像。 注意:不稳定的迹象包括呼吸急促和鼠标，它可以被看作是在获取的图像移位的工件运动。 </li><li>图像采集后，将鼠标离开舞台，并使其返回到笼子里。使用加热灯来保持温暖，并监测小鼠，直到它是有意识的。 </li></ol> 3.图像分析和量化<ol><li>使用图像分析软件来分析三维和时间序列数据。打开软件和将其拖动到打开的画面，这就是所谓的"竞技场"视图中添加图像。在图像文件打开双击它;图像将在"超越"视图中打开。 </li><li>创建一个2D / 3D图像。 <ol><li>从右上角找到"其他"栏，点击"编辑/显示显示调整"，打开"显示调整"窗口。通过调整阈值，为每个通道优化荧光显示屏，并采取快照保存图像。 </li></ol></li><li>生成使用时间推移图像对象轨道。 <ol><li>打开图像下的"超越"的观点进行分析。点击橙色小点的图像，发现下面的"超越"图标上。这将打开一个新的"点"窗口。 </li><li>勾选"追踪斑点（随时间变化）"，并点击蓝色箭头进入下一个步骤。选择包含​​对象的频道的"源通道"进行跟踪，并设置估计对象diamet根据"估计XY直径"通过键入一些呃，点击蓝色箭头进入下一步。 注意:对于中性粒细胞的跟踪，选择包含APC荧光中性粒细胞的通道，并设置基于中性粒细胞已知大小的物体直径12微米。 </li><li>在窗口中，找到并点击向下箭头，选择"质量"过滤器。观察由软件定义的斑点和手动调整阈值包含或排除斑点。一旦阈值定义，点击蓝色箭头转到"跟踪"的步骤。 </li><li>在算法中，找到并点击向下箭头，选择"自回归运动算法"。键入10微米的"最大距离"和3"最大间隙大小"下的参数找到。勾选"装满所有检测到的对象的空白"，开始跟踪，然后点击蓝色箭头进入下一步。 注:"自回归运动算法"用于˚F或已经指示运动，因为它基于从先前的运动预测位置的轨道的对象。 "最大距离"和"最大间隙尺寸"可以根据预期的尺寸和物体的速度被追踪，以及隔时图像的总持续时间来调整。 </li><li>在过滤器类型，请选择"跟踪时间"过滤器。点击绿色箭头来完成跟踪，检查生成的轨道。返回到前面的步骤，如果必要的磁道手动排序。 </li></ol></li><li>如果生成隔时图像漂移校正平移漂移。 <ol><li>点击图标用铅笔的图像。这是打开"编辑曲目"窗口中编辑标签。检查隔时图像，并找到一个参考点不应该移动的所有图像。 </li><li>在屏幕的左上角，找到"指针"窗口，勾选"选择"激活立方米rsor用于选择一个点。点击参考点。 </li><li>在"编辑曲目"窗口中点击"正确漂"。检查关"转化漂"。该软件将自动生成校正图像系列。 </li></ol></li><li>通过测量物体在轨道时间推移图像定义细胞运动。 <ol><li>在"编辑曲目"窗口，勾选"曲目"，并选择代表一个细胞的运动轨迹。所选曲目将在黄色中的时间序列图像中突出显示。 </li><li>点击图标与图的图像打开"统计"窗口（统计标签）。 </li><li>在"选择"，通过点击向下箭头，产生轨道速度意味着所选的曲目选择"跟踪速度平均值"。选择不同的轨道，并重复步骤测量的轨道速度意味着所有感兴趣的细胞。 </li></ol></li></ol>

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	<li>Zhao, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+and+the+control+of+immunity.">Bone marrow and the control of immunity.</a> Cell Mol Immunol. 9 (1), 11-19 (2012).</li><li>Borregaard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils,+from+marrow+to+microbes.">Neutrophils, from marrow to microbes.</a> Immunity. 33 (5), 657-670 (2010).</li><li>Boxio, R., Bossenmeyer-Pourie, C., Steinckwich, N., Dournon, C., Nusse, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+bone+marrow+contains+large+numbers+of+functionally+competent+neutrophils.">Mouse bone marrow contains large numbers of functionally competent neutrophils.</a> J Leukoc Biol. 75 (4), 604-611 (2004).</li><li>Fridlender, Z. G., Albelda, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+neutrophils:+friend+or+foe?.">Tumor-associated neutrophils: friend or foe?.</a> Carcinogenesis. , (2012).</li><li>Fridlender, Z. G., 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=Polarization+of+tumor-associated+neutrophil+phenotype+by+TGF-beta:+'N1'+versus+'N2+TAN.">Polarization of tumor-associated neutrophil phenotype by TGF-beta: 'N1' versus 'N2 TAN.</a> Cancer Cell. 16 (3), 183-194 (2009).</li><li>Coffelt, S. 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=IL-17-producing+[ggr][dgr]+T+cells+and+neutrophils+conspire+to+promote+breast+cancer+metastasis.">IL-17-producing [ggr][dgr] T cells and neutrophils conspire to promote breast cancer metastasis.</a> Nature. 522 (7556), 345-348 (2015).</li><li>Kitamura, T., Qian, B. Z., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+promotion+of+metastasis.">Immune cell promotion of metastasis.</a> Nat Rev Immunol. 15 (2), 73-86 (2015).</li><li>Granot, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+entrained+neutrophils+inhibit+seeding+in+the+premetastatic+lung.">Tumor entrained neutrophils inhibit seeding in the premetastatic lung.</a> Cancer Cell. 20 (3), 300-314 (2011).</li><li>Travlos, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+structure,+function,+and+histology+of+the+bone+marrow.">Normal structure, function, and histology of the bone marrow.</a> Toxicol Pathol. 34 (5), 548-565 (2006).</li><li>Pittet, M. J., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Imaging.">Intravital Imaging.</a> Cell. 147 (5), 983-991 (2011).</li><li>Alieva, M., Ritsma, L., Giedt, R. J., Weissleder, R., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+windows+for+long-term+intravital+imaging.">Imaging windows for long-term intravital imaging.</a> IntraVital. 3 (2), e29917 (2014).</li><li>Hamon, P., Rodero, M. P., Combadiere, C., Boissonnas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+mouse+bone+marrow+monocytes+in+vivo.">Tracking mouse bone marrow monocytes in vivo.</a> J Vis Exp. (96), e52476 (2015).</li><li>Mazo, I. 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=Hematopoietic+Progenitor+Cell+Rolling+in+Bone+Marrow+Microvessels:+Parallel+Contributions+by+Endothelial+Selectins+and+Vascular+Cell+Adhesion+Molecule+1.">Hematopoietic Progenitor Cell Rolling in Bone Marrow Microvessels: Parallel Contributions by Endothelial Selectins and Vascular Cell Adhesion Molecule 1.</a> J. Exp. Med. 188 (3), 465-474 (1998).</li><li>Scott, M. K., Akinduro, O., Lo Celso, C. <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+4-Dimensional+Tracking+of+Hematopoietic+Stem+and+Progenitor+Cells+in+Adult+Mouse+Calvarial+Bone+Marrow.">In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow.</a> J. Vis. Exp. (91), e51683 (2014).</li><li>Lassailly, F., Foster, K., Lopez-Onieva, L., Currie, E., Bonnet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+imaging+reveals+structural+and+functional+heterogeneity+in+different+bone+marrow+compartments:+functional+implications+on+hematopoietic+stem+cells.">Multimodal imaging reveals structural and functional heterogeneity in different bone marrow compartments: functional implications on hematopoietic stem cells.</a> Blood. 122 (10), 1730-1740 (2013).</li><li>Kohler, A., Geiger, H., Gunzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+hematopoietic+stem+cells+in+the+marrow+of+long+bones+in+vivo.">Imaging hematopoietic stem cells in the marrow of long bones in vivo.</a> Methods Mol Biol. 750, 215-224 (2011).</li><li>Balan, M., Kiefer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+for+ectopic,+chronic,+intravital+multiphoton+imaging+of+bone+marrow+vasculature+and+architecture+in+split+femurs.">A novel model for ectopic, chronic, intravital multiphoton imaging of bone marrow vasculature and architecture in split femurs.</a> IntraVital. 4 (2), e1066949 (2015).</li><li>Hansen-Algenstaedt, 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=Femur+window--a+new+approach+to+microcirculation+of+living+bone+in+situ.">Femur window--a new approach to microcirculation of living bone in situ.</a> J Orthop Res. 23 (5), 1073-1082 (2005).</li><li>Xu, N., Lei, X., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+Neutrophil+Intraluminal+Crawling,+Transendothelial+Migration+and+Chemotaxis+in+Tissue+by+Intravital+Video+Microscopy.">Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy.</a> J. Vis. Exp. (55), e3296 (2011).</li><li>Mizuno, 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=In+vivo+imaging+reveals+PKA+regulation+of+ERK+activity+during+neutrophil+recruitment+to+inflamed+intestines.">In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines.</a> J. Exp. Med. 211 (6), 1123-1136 (2014).</li><li>Kreisel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+two-photon+imaging+reveals+monocyte-dependent+neutrophil+extravasation+during+pulmonary+inflammation.">In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation.</a> Proc. Natl. Acad. Sci. 107 (42), 18073-18078 (2010).</li><li>Yipp, B. G., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+against+neutrophil+LY6G+do+not+inhibit+leukocyte+recruitment+in+mice+in+vivo.">Antibodies against neutrophil LY6G do not inhibit leukocyte recruitment in mice in vivo.</a> Blood. 121 (1), 241-242 (2013).</li><li>Bucher, 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=Fluorescent+Ly6G+antibodies+determine+macrophage+phagocytosis+of+neutrophils+and+alter+the+retrieval+of+neutrophils+in+mice.">Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice.</a> J Leukoc Biol. 98 (3), 365-372 (2015).</li><li>Lammermann, 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=Neutrophil+swarms+require+LTB4+and+integrins+at+sites+of+cell+death+in+vivo.">Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo.</a> Nature. 498 (7454), 371-375 (2013).</li><li>Progatzky, F., Dallman, M. J., Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+seeing+to+believing:+labelling+strategies+for+in+vivo+cell-tracking+experiments.">From seeing to believing: labelling strategies for in vivo cell-tracking experiments.</a> Interface Focus. 3 (3), (2013).</li><li>Koewler, N. 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=Effects+of+a+Monoclonal+Antibody+Raised+Against+Nerve+Growth+Factor+on+Skeletal+Pain+and+Bone+Healing+After+Fracture+of+the+C57BL/6J+Mouse+Femur.">Effects of a Monoclonal Antibody Raised Against Nerve Growth Factor on Skeletal Pain and Bone Healing After Fracture of the C57BL/6J Mouse Femur.</a> J. Bone Miner Res. 22 (11), 1732-1742 (2007).</li><li>Garcia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+animal+models+of+delayed+bone+healing+and+non-union+formation:+a+comprehensive+review.">Rodent animal models of delayed bone healing and non-union formation: a comprehensive review.</a> Eur Cell Mater. 26, 1-12 (2013).</li><li>Liu, 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=A+murine+femoral+segmental+defect+model+for+bone+tissue+engineering+using+a+novel+rigid+internal+fixation+system.">A murine femoral segmental defect model for bone tissue engineering using a novel rigid internal fixation system.</a> J Surg Res. 183 (2), 493-502 (2013).</li><li>Morrison, S. J., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bone+marrow+niche+for+haematopoietic+stem+cells.">The bone marrow niche for haematopoietic stem cells.</a> Nature. 505 (7483), 327-334 (2014).</li><li>Ellis, S. 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=The+relationship+between+bone,+hemopoietic+stem+cells,+and+vasculature.">The relationship between bone, hemopoietic stem cells, and vasculature.</a> Blood. 118 (6), 1516-1524 (2011).</li><li>Vacaru, A., Vitale, J., Nieves, J., Baron, M., Singh, S. R., Coppola, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Genetics.">Mouse Genetics.</a> Methods in Molecular Biology. 1194, 289-312 (2014).</li></ol>

作者有没有竞争经济利益。

实时的，在骨髓动态细胞过程的串行成像提供以其它方式挑战，得到使用常规技术，如组织学和总血细胞计数信息。这里所描述的股骨厕所模型提供随时间调查在骨髓细胞和结构改变了独特的机会。虽然股骨WC模型先前已经报道​​，我们的新颖设计提供的视图和较小的整体WC尺寸较大的摄像视场，这是在成年小鼠使用更兼容。股骨厕所模型可在各种小鼠品系，包括免疫活性和/或转基因报告小鼠?...

骨髓是参与造血和免疫调节的重要器官。它由含有造血干细胞和祖细胞（骨髓干细胞）造血成分和包含于间充质细胞1产生非造血祖细胞的基质组分。造血活动的三分之二是专门为骨髓细胞2的产生。特别是，在骨髓中产生了大量的中性粒细胞，以在正常的成人每天2生成1-2×10 11个细胞。中性粒细胞是抗微生物感染的防御的第一线，在骨髓大多保留，直到触发器压力的动员，以补充外周血中性1,3。除了它们的抗微生物作用，最近的研究表明在癌症生物学中性粒细胞的重要作用，同时具有亲和抗肿瘤发生的表型取决于转化生长因子β（TGF-β）在肿瘤微环境4,5-信令。此外，研究表明，在原发肿瘤积聚的中性粒细胞产生通过抑制T细胞6,7-的细胞毒性作用的亲致瘤性和转移性的效果，而在循环嗜中性粒细胞产生细胞毒性，抗转移效应8。因此，造血细胞在骨髓中，特别是嗜中性粒细胞，调查是在免疫和肿瘤调节阐明它们的作用是至关重要的。 组织病理学和完整的外周血计数通常用于在骨髓9来评估细胞和结构改变。然而，这些方法仅提供了不同的细胞群或组织中微观结构的静态信息。纵向的体内成像可在结合使用的标准方法来评估多个蜂窝，血管和基质组分的动力学以及细胞到CELL相互作用以纵向方式。活体显微镜（IVM），定义为在显微镜的分辨率10活体动物成像，是对于相同的试样中随时间评估动态的细胞过程，减少了所需的实验动物的数量是特别有用的。 IVM通常与慢性移植窗口室（WC）数周的持续时间，以访问感兴趣的器官成像到几个月组合。颅骨和背皮褶WC车型有使用可以追溯到20世纪90年代中期的历史最长。最近，其他器官特异性厕所模型，例如那些乳腺脂肪垫和各种腹部器官已经开发11。 用于体内成像骨髓典型的方法有小鼠的颅盖，其中所述变薄的骨能以最少的手术干预12-14单个细胞的直接可视化的主要涉及曝光。然而，颅骨骨髓可以bE从其他骨骼，如长骨的不同，这表现在在颅盖的下数骨髓干细胞和缺氧细胞的，这表示降低骨髓干细胞15的维护和发展。因此，评估在长骨细胞成分的替代方法进行了研究。这些措施包括股骨骨髓16的直接暴露和在背皮褶WC 17分股骨移植异位。然而，前者是不允许蜂窝，结构和功能的改变的跟踪在更长的时间段的终端程序，后者可能是由于股骨移植向背侧皮褶厕所内异位位点扰乱正常骨髓的功能。使股骨骨髓原位串行成像随时间的另一种方法是在股骨骨使用WC的。一前一报告显示微循环的长期成像在股骨骨髓使用在小鼠18股骨WC。此外，作者证明肿瘤细胞在股骨的可视化，这表明它在监测骨髓转移工具。然而，这种厕所设计是由它的大尺寸（1.2厘米直径的）和相对小的成像区域（4毫米直径的），这是仅适用于大鼠（26-34克，3-6个月的年龄）的限制，从而使方法不切实际常规使用。 因此，一个新的WC具有较小的整体尺寸，以及较大的内部成像区域被设计为这项研究的目的。本研究的目标是提供用于在股骨骨髓成像各种细胞类型的方法。股骨WC模型内部开发的，并用于在3D血管网内可视化和跟踪中性粒细胞。利用该模型，骨髓IVM可以连续40天进行。这种方法可以适用于各种领域的阐明造血的方法中，免疫调节第二肿瘤的发展。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>NRCNU-F athymic nude mice</td>
			<td>Taconic</td>
			<td>Ncr nude</td>
			<td>8-10 weeks old, female</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Baxter</td>
			<td>JB1302P</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Bioniche Animal Health Canada, Inc.&#160;</td>
			<td>DIN 01989529</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer HealthCare, Bayer Inc.</td>
			<td>DIN 02169592</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical drape</td>
			<td>Proxima</td>
			<td>DYNJP2405</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric heating pad</td>
			<td>Life Brand</td>
			<td>57800827375</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>Leica M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment (tear gel)</td>
			<td>Novartis&#160;</td>
			<td>T296/2</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5% betadine</td>
			<td>Purdue Frederick Co</td>
			<td>67618-151-16</td>
			<td></td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol</td>
			<td>GreenField</td>
			<td>P010IP7P</td>
			<td></td>
		</tr>
		<tr>
			<td>10% betadine</td>
			<td>Purdue Frederick Co</td>
			<td>67618-150-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle (#3)</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade (#15)</td>
			<td>VWR</td>
			<td>89176-368</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors curved</td>
			<td>Fine science Tools</td>
			<td>15023-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Baby-Mixter Hemostat</td>
			<td>Fine science Tools</td>
			<td>13013-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine science Tools</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps</td>
			<td>Fine science Tools</td>
			<td>11151-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsted-Mosquito Hemostats</td>
			<td>Fine science Tools</td>
			<td>13008-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-drill</td>
			<td>Harvard Apparaus</td>
			<td>72-6065</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-drill burrs</td>
			<td>Fine Science Tools</td>
			<td>19007-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Femur window chamber</td>
			<td>PMCC machine shop</td>
			<td>custom design</td>
			<td>9.1mm- 8.5mm- 7.5 mm (outer to inner diameter), 2.16 mm (radius of two holes), 13.9mm (distance between two holes), 0.7mm (thickness)</td>
		</tr>
		<tr>
			<td>U-shaped bar</td>
			<td>PMCC machine shop</td>
			<td>custom design</td>
			<td>13.8mm (length), 1.6 mm (width), 3.7mm (height)</td>
		</tr>
		<tr>
			<td>Coverglass (8mm)</td>
			<td>Warner Instruments&#160;</td>
			<td>HBIO 64-0701 CS-8R</td>
			<td></td>
		</tr>
		<tr>
			<td>Retaining ring (8mm)</td>
			<td>ACKLANDS GRAINGER</td>
			<td>UNSPSC # 31163202</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuts (hexagon stainless steel)</td>
			<td>Fastenal</td>
			<td>70701</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>3M</td>
			<td>RelyX U200</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture (5-0 Monosof black)</td>
			<td>Covioien</td>
			<td>SN-5698</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey needle holder</td>
			<td>Fine Science Tools</td>
			<td>12501-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic)</td>
			<td>Reckitt Benckiser</td>
			<td>DIN 0281251</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Metacam)</td>
			<td>Boehringer Ingelheim</td>
			<td>DIN 02240463</td>
			<td></td>
		</tr>
		<tr>
			<td>Amoxicillin (Clamavox)</td>
			<td>Pfizer</td>
			<td>DIN 02027879</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD2000S</td>
			<td></td>
		</tr>
		<tr>
			<td>APC- Anti-Mouse Ly-6G (Gr-1)&#160;</td>
			<td>eBioscience</td>
			<td>17-9668</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon microscope LSM 710</td>
			<td>Carl Zeiss</td>
			<td>Zeiss LSM 710 NLO</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging stage</td>
			<td>PMCC machine shop</td>
			<td>custom design</td>
			<td>15.9cm (length), 11cm (width), 0,9cm (height)</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td>Imaris 8.0</td>
			<td>Image analysis software described in Section 3 of the Protocol&#160;</td>
		</tr>
		<tr>
			<td>Zen 2012</td>
			<td>Zeiss</td>
			<td>Zen 2012</td>
			<td>Image acqusition software described in Section 2 of the Protocol&#160;</td>
		</tr>
	</tbody>
</table>

femur window chamber model for in vivo cell tracking in the murine bone marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

鼠股骨骨髓是使用了WC启用个人中性粒细胞和血管网络的可视化成功访问。 图1示出了WC工具和描述了外科手术，其涉及股骨骨和骨皮质变薄的曝光以获得内部的光接入骨。该手术是很好的耐受性小鼠;他们进行了评估不良生理反应，如后肢浮肿，非负重肢体，自残，蜂窝组织炎和皮炎，以及损害了WC 5天手术后，无痛苦的报告。值得注意的是，窗口仍然IVM光学透...

Watch this Scientific Journal Video about Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow at JoVE.com

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

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JoVE Journal

Immunology and Infection

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

8.1K 次观看.  Princess Margaret Cancer Centre. The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

视频: Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

科研

免疫与感染

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 ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

鼠后肢长骨解剖和骨髓隔离

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

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 ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

果蝇蛹中炎症细胞动力学的长期体内追踪

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...