作者要感谢安妮·达龙和皮埃尔路易斯Loyher为编辑的协助下，PLATEFORME Imagerie萨伯特慈善（PICPS）与双光子显微镜和动物基金"NAC"和卡米尔Baudesson小鼠繁殖援助援助。这项研究导致这些结果已收到的资金来自欧盟第七框架计划（FP7 / 2007至2013年）根据赠款协议N°304810 - 的RAID和n°241440-Endostem，从INSERM，从UNIVERSITE皮埃尔与玛丽·居里"崛起"从LA"法甲驳乐毒瘤"，从"协会倒拉RECHERCHE河畔乐癌症"和"法新社国立德拉RECHERCHE"计划诞生日（ANR-EMMA-050）。 PH由LA"法甲驳乐癌症"的支持。

注:所有实验的协议批准了法国动物实验伦理委员会和一些A-75-2065验证了"服务保障等桑特阿尼马莱斯，环境公司"。样本大小被选择以确保该实验的再现性，并根据动物伦理规章的3R。 1.制备的小鼠<ol><li>麻醉<ol><li>用于成像的短时间（少于1小时），麻醉的小鼠腹膜内注射200微升含氯胺酮（100毫克/千克）和赛拉嗪（10毫克/千克）盐水溶液。 </li><li>可替代地，用于成像的较长时间（达4小时），麻醉的小鼠用异氟烷2.5％汽化在O 2 / N 2 O，70/30混合物通过一个适于掩模吸入。 </li><li>注:此方法提供了更稳定的无意识，避免了连续腹腔注射药物。 </l我&gt; <li>一旦老鼠被麻醉，检查无意识通过脚踏垫钳刺激。 </li></ol></li><li>血管染色/额外的染色<ol><li>血管染色:在尾静脉静脉内注射，将200μl罗丹明 - 葡聚糖（2-丙二醛，10毫克/毫升在盐水缓冲液）。 </li><li>对于嗜中性粒细胞染色:注射2微克Ly6G-PE在100微升生理盐水溶液静脉内的尾部静脉（克隆1A8）。 </li></ol></li><li>固定<ol><li>用于固定的，可使用适用于显微镜和麻醉气体吸入一定制立体支架。 注:立体定位支架是金属支撑具有两个金属板支架，1被固定，而另一个可移动的和适应性强，收紧动物的头。 </li><li>安装在保持板之间的鼠标的头部，以确保从呼吸运动隔离。拧紧固定板和头部伸展SK之间的耳朵英寸检查的稳定性和动物的呼吸福利。 </li></ol></li><li>去除头皮<ol><li>小心用70％乙醇润湿的头皮头发用无菌涂布器，避免与眼睛接触。 </li><li>切割用无菌剪刀和镊子取下皮肤完全消除来自后方的顶骨的头皮额骨。远离长达3毫米的眼睛和耳朵，以防止大量出血。取出疏松结缔组织覆盖在颅骨（骨膜）。洗出剩余的头发用温水PBS浸湿的纸巾。 </li></ol></li><li>浸没系统设置<ol><li>上直接使用小规格针，以控制分配颅骨粘贴直径18mm与手术胶水的橡胶环。胶的橡胶圈的整个周边，以防止泄漏（30微升应该足够）。 </li><li>与PBS浸泡过的纸巾下擂台清洁骷髅最后一次，然后加入37℃的PBS的完全浸入头骨。添加下降0.9％生理盐水溶液或特定眼药膏对鼠标的每只眼睛，避免眼睛干涩。拖动立体支架下的目标。 </li><li>通过直接传输采用显微镜目镜约定位领域。 </li></ol></li></ol> 2.双光子成像采集<ol><li>使用多光子显微镜加上的Ti:蓝宝石激光晶体，它提供了近红外光的140fs脉冲，选择性地调谐从680至1080纳米，和一个声光调制器，用于激光功率控制。使用包括3个外部非退扫描探测器（NDD）（即能够同时记录第3荧光通道）中的2分色镜（565纳米和690纳米），610分之565和500/550的带通滤波器的组合的结构，并且一485短通滤波器，并计划复消色差透镜×20（NA = 1）水浸泡的目的。 </li><li>设置在加热腔室，以允许anaes良好homeothermythetized鼠标。， 注:温度可能设置1小时成像会话更好的稳定性之前。在我们的情况下，32℃下已被选定，以获得麻醉小鼠的最佳体温（36-37℃）和被保持长达4小时。该温度可以适合于该系统中使用（数字探针可用于检查这点）。此外，所使用的加热室是暗/黑和覆盖系统（目标和机动板），以避免外界的光污染的一部分。 </li><li>打开系统<ol><li>打开钛:蓝宝石激光，那么整个显微镜系统。启动采集软件。激光的波长范围设定为870纳米。记录选择合适的NDD。 注:在本例中，二次谐波（SHG）19和青色荧光蛋白（ECFP）的485短通滤波器后的NDD检测。 ECFP也由NDD检测的500/550带通滤波器和罗丹明 - 葡聚糖为t检测后他NDD后610分之565带通滤波器。 </li></ol></li><li>调整收购设置<ol><li>使用该软件的"实时采集"成像命令，并使用显微镜方向控制，调整焦距与ECFP和罗丹明信号的区域。设置激光功率为最小，以获得最佳的信噪比以最小的光损伤。 注:所用的功率设置根据感兴趣的区域的深度而变化。通常AOM的设置被设置在20％左右，这代表一个激光功率超过约15毫瓦的目标。这是更好地提高功率增益的光电倍增管的每个NDD，以减少光漂白和光损伤。典型的收购设置与1.58μs的像素停留时间的单扫描，以及512×512像素，1.5倍的分辨率。 </li></ol></li><li>感兴趣选择地区<ol><li>一旦记录参数设定，再次使用"活"收购指令，和调查的组织，以选择所期望的（X，Y，Z）场中实时进行监控。 注意:通常情况下，我们选择了包括血管和脑实质的区域的字段。 </li><li>寄存器字段用"位置"的软件命令。 注:此命令记忆的x，y，不同的远野Z坐标。 </li><li>选择并注册的兴趣（最多3）使用相同的步骤附加字段。 </li><li>根据与所选择的放大倍率所需要的卷上的第一个记忆的位置设置了3D Z堆栈与"Z堆栈"的软件命令。记忆更深的位置，然后再在最高位置。正常化激光功率与深度。 注:对于多个伴随场成像，我们获得了5片由3微米的z步骤分离为每个字段获得的12微米厚的体积。这些设置可以适于研究细胞的类型。 </li></ol></li><li>启动收购<ol><li>选择220;时间序列"软件命令，并选择时间间隔的持续时间和周期数。根据通过选择"开始实验"软件的命令所要求的时间流逝和持续时间开始的时间推移成像。 注:在本例中，我们在60次代表30分钟的实时采集一个卷各30秒。较小的时间推移可被执行以在血管跟踪高速小区轧制。在这种情况下更薄的Z堆叠并且不超过2个不同的字段可以记录在同一时间。 </li><li>定期监测的设置。 <ol><li>随时监控动物，以确保它是无意识的。 注意:摇晃晶须或腿的动物是复苏的指标。在情况下，动物的呼吸是生涩，异氟醚的量可以稍微降低。采集过程中强有力的组织漂移鼠标复兴的一个指标。 </li><li>确保目标是正确的浸泡。更新37℃PBS between两个收购（30分钟）。 注:信号的采集过程中逐步消失是PBS泄漏指标。 </li></ol></li></ol></li><li>程序结束<ol><li>一旦所需的视频被记录，快速复苏之前安乐死的动物通过颈椎脱位。 注:伤口愈合导致痂在24至48小时，这限制了进行纵向成像的可能性的形成。 </li></ol></li></ol> 3.数据分析<ol><li>漂移校正<ol><li>使用了Imaris位平面软件，三维自动跟踪。 </li><li>上打开了Imaris 3D图像序列。选择"点"的命令，和用于最小的4个不同的定位点产生手动跟踪（鼠标点击+偏移）的所有时间点，如果有可能均匀地分布在该领域。 </li><li>在"编辑曲目"选项卡中的"正确漂"命令应用漂移校正。 </li><li>检查吨他修正在x，y和特别是在z与质量，以避免假象游移。 注意:如果修正不理想，尝试其他锚点或从分析中排除的视频。 </li></ol></li><li>细胞追踪<ol><li>使用"添加新景点"命令，然后选择"跟踪点随着时间的推移"算法设置。进入下一步骤。 </li><li>应用自动细胞跟踪过程无论是在关注使用"源通道"命令，整个通道。对象集（即，小区）直径10微米。进入下一步骤。 注:选择将取决于对象的测定信号和特异性之间的区别。总之，在整个信道的自动跟踪是可能的，只有检测到的信号是特定的感兴趣对象。因为无论SHG和ECFP由NDD 485短通滤波器后发现，跟踪将更加具体和有效的使用后5记录的NDD的ECFP信号00/550带通滤波器。群集或密集的包装对象将需要手动选择类似于步骤3.1.2单个对象。 </li><li>选择"质量"过滤器类型，设置门槛，以排除非特异性的对象跟踪。进入下一步骤。 </li><li>选择"布朗运动算法"两个点之间的"最大间距"和"间隙尺寸"的准确参数。验证轨迹生成。 </li><li>一旦轨道被自动计算，选择"跟踪时间"过滤器类型，设置门槛，以消除轨道小于120秒（相当于4个时间点）。 </li><li> 强制性:经过自动跟踪，检查铁轨的质量。 <ol><li>展开时间条来检查对象跟随光道路径。如果没有，使用跟踪点校正的可用选项中的"编辑"选项卡:删除虚假或不正当的轨道与"删除"命令（删除任何轨迹Point单独使用"断开连接"命令）。如果跟踪路径是不连续的，加上缺少时间点（鼠标点击+移动），选择一个特定的轨道上的所有点，并使用"编辑曲目"选项卡"连接"命令。 </li></ol></li></ol></li><li>量化参数<ol><li>选择"首选项"命令中的"统计"选项卡中，选择列表中的权益的动态参数。选择"导出所有提交的统计数据"命令并保存生成的Excel文件。 注意:几个动态参数是直接从软件获得，例如轨道的长度，瞬时速度，平均速度，和跟踪直度。运动系数6,20可以被确定，但是不直接由软件提供。 </li><li>对于每个小区，计算出位移为每个周期的时间（ΔT）的平均值（对于30秒的时间间隔的电影，ΔT为30，60，90秒，等等）。绘制方位移的平均值作为时间间隔的函数。通过计算该曲线的线性部分的斜率获得的蠕动系数。 </li></ol></li></ol>

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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=Impaired+neutrophil+extracellular+trap+(NET)+formation:+a+novel+innate+immune+deficiency+of+human+neonates.">Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates.</a> Blood. 113, 6419-6427 (2009).</li><li>Devi, 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=Multiphoton+imaging+reveals+a+new+leukocyte+recruitment+paradigm+in+the+glomerulus.">Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus.</a> Nat Med. 19, 107-112 (2013).</li></ol>

The authors have nothing to disclose.

体内成像方法的关键点是，以确保以最大限度成像的持续时间，并尽量减少细菌污染和炎症，这可能会影响炎性细胞的动力学的风险的焦点的稳定性。颅骨骨髓成像如下这些目标为手术执行以访问骨髓是最小的。利用无菌材料和防腐剂的必须限制感染可能诱导扰动在细胞稳态的风险。 立体定向持有人的发展可能是具有挑战性的，并要求（客观的和机动阶段显微镜...

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.
Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14. 
Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset. 
The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

<table><tbody><tr><td>Ketamin</td><td>Merial</td><td></td><td>100 mg/ml, anesthetic</td></tr><tr><td>Xylazin</td><td>Bayer HealthCare</td><td></td><td>10 mg/ml, anesthetic</td></tr><tr><td>Isofluran</td><td>Baxter</td><td></td><td>2.5%, anesthetic</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;/NO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>70/30 mixture, anesthetic</td></tr><tr><td>Rhodamin-Dextran</td><td>Invitrogen</td><td></td><td>2 MDa, 10 mg/ml, Vascular staining</td></tr><tr><td>Ly6G-PE</td><td>Becton-Dickinson</td><td></td><td>clone 1A8, neutrophils staining</td></tr><tr><td>Stereotactic holder</td><td>Home made</td><td></td><td>surgery</td></tr><tr><td>Ethanol 70%</td><td></td><td></td><td>surgery</td></tr><tr><td>Sterile scissors and nippers</td><td></td><td></td><td>surgery</td></tr><tr><td>Rubber ring</td><td></td><td></td><td>18 mm diameter, surgery</td></tr><tr><td>Glubran 2</td><td>Queryo Medical</td><td></td><td>Surgical Glue, rubber ring fixation</td></tr><tr><td>Small gauge needles</td><td>Terumo</td><td></td><td>surgery</td></tr><tr><td>Zeiss LSM 710 NLO multiphoton microscope&amp;nbsp;</td><td>Carl Zeiss</td><td></td><td>Microscope</td></tr><tr><td>Ti:Sapphire crystal laser&amp;nbsp;</td><td>Coherent Chameleon Ultra</td><td></td><td>140 fsec pulses of NIR light</td></tr><tr><td>Zen 2010</td><td>Carl Zeiss</td><td></td><td>Acquistion Software</td></tr><tr><td>Imaris Bitplane&amp;nbsp;</td><td>Bitplane</td><td></td><td>Analysis Software, 3D automatic tracking</td></tr><tr><td>PBS 1X</td><td>D. Dutscher</td><td></td><td>surgery</td></tr><tr><td>Thermostated chamber</td><td>Carl Zeiss</td><td></td><td>intravital imaging</td></tr></tbody></table>

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.

鼠标的头骨结构提供了活体成像研究骨髓生理学的好机会。骨可绕前顶区薄，能够获得访问骨髓龛未经骨的磨损。 图1表示一个MacBlue转基因小鼠的头骨的宽2D场。骨基质主要是由胶原蛋白的我很容易被SHG 19检测。注射罗丹明-葡聚糖的污渍骨髓的血管网并允许对骨基质和容器14之间实质骨髓龛的鉴定。 ECFP细胞分布于骨髓的两个隔室，但是从骨基质缺席。单核细胞是根据...

Watch this Scientific Journal Video about Tracking Mouse Bone Marrow Monocytes In Vivo at JoVE.com

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 ...

tracking-mouse-bone-marrow-monocytes-in-vivo

Research

JoVE Journal

Immunology and Infection

Tracking Mouse Bone Marrow Monocytes In Vivo

9.5K 次观看.  Sorbonne Universités, UPMC Univ Paris 06, CR7. 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. 

视频: Tracking Mouse Bone Marrow Monocytes In Vivo

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

科研

免疫与感染

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 ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

股骨骨髓的愿望在活体小鼠

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

医学

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

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.

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, ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

生物学

<meta charset="utf8"></head><body>股骨内注射用于活小鼠骨髓研究

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

<meta charset="utf8"></head><body>使用活小鼠进行简化的股骨内注射允许连续骨髓分析

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

使用活小鼠进行简化的股骨内注射允许连续骨髓分析

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

跟踪小鼠骨髓单核细胞

摘要

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此视频中的章节

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

隔离和小鼠骨髓的静脉注射单核细胞衍生

股骨骨髓的愿望在活体小鼠

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

股骨窗口商会模型

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

使用活小鼠进行简化的股骨内注射允许连续骨髓分析

使用活小鼠进行简化的股骨内注射允许连续骨髓分析