Name: isolation and intravenous injection of murine bone marrow derived monocytes
Uploaded: 2014-12-27T15:00:00.000+00:00
Duration: 9 min 21 s
Description: Watch this Scientific Journal Video about Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes at JoVE.com

This work was supported by the DFG (Deutsche Forschungsgemeinschaft, German Research Foundation) SFB 854 (Sonderforschungsbereich, collaborative research center). 
Thanks to Hans-Holger G&#228;rtner, Audiovisuelles Medienzentrum, Otto-von-Guericke University Magdeburg, Magdeburg, Germany, for technical support.

与萨克森州和萨克森 - 安哈尔特州，Regierungspraesidium德累斯顿/哈勒的国家批准进行这项研究，根据德国法律的保护动物（24D-9168.11-1 / 2008-24）第8。 1细胞分离 1.1准备股骨和胫骨的<ol><li>用5％异氟烷浓度通过在一个封闭的仓汽化异氟烷麻醉小鼠。后的小鼠停止移动，持续3秒，执行颈椎脱位。 </li><li>消毒后肢用乙醇（96％）。去除皮肤和肌肉分开的骨头用无菌手术刀。 <ol><li>开始与腹股沟皮肤切口，并延伸朝向内踝切口并执行围绕下部小腿皮肤的圆形解剖。由近端脱皮彻底去除腿部的皮肤。 </li><li>从分离用锋利的手术刀股骨股四头肌，同时删除前腓骨和腓肠肌肌肉群并通过定位和解剖韧带disarticulate腿在髋关节。 </li><li>启动向前，继续关节周围小心地旋转腿和横断韧带，从而脱节的联合。 </li></ol></li><li>洗股骨和胫骨用96％的乙醇最少90秒，以保证无菌细胞制剂。继续洗涤过程中使用无菌PBS冲洗掉剩余的乙醇。 注意:所有的后续步骤必须是无菌的，以避免污染。 </li></ol> 1.2收获骨髓<ol><li>切割每个骨的近端和远端具有一对细剪刀来访问的股骨和胫骨的轴。小心这一步，因为这些骨头断裂非常容易。 </li><li>冲洗骨头用温介质（M199 + 10％胎牛血清（FCS），+ 1％青霉素/链霉素）。使用无菌28-G针，1ml注射器，和10-15毫升培养基每间骨冲洗。 </li><li>握住骨用细镊子和，直到它变成半透明一个冲洗之一。小心施加压力的注射器柱塞的端部，以减少细胞应激。 </li><li>通过70微米尼龙网过滤骨髓并收集滤液。以提取骨髓的最大数量，从所述远端和近端反复冲洗。 </li><li>冲洗用PBS筛，并收集滤液。小心撞出的碎片和细胞集团轻轻搅拌和移液。 </li></ol> 1.3栽培<ol><li>离心细胞悬浮液在250×g离心在室温10分钟。弃上清，悬浮细胞与大约25毫升介质。重复一次。 </li><li>第二清洗步骤之后重悬细胞在6ml培养基。混合50微升的细胞溶液与50μl的台盼蓝。计数细胞在计数室在光学显微镜下。计算C的数使用此公式中的溶液ELLS: <img alt="式（1）" fo:content-width="1.3in" src="/files/ftp_upload/52347/52347eq1.jpg" /></li><li>种子上6孔超低附着板表面的细胞，以防止永久附着在板的底部。使用每毫升10 6个细胞的浓度以每孔最多至6ml。 </li><li>补充悬架20毫微克/毫升的M-CSF，促进细胞分化。 </li><li>培养在37℃下将细胞5天，5％二氧化碳和每日观察。 </li></ol>细胞收获1.4 注意:这些步骤应在冰上进行。继续无论1.4.1或1.4.2相应。 <ol><li>在这一点上，收获整个培养，包括分化的巨噬细胞，将粘附到培养板，即使它们是超低附着表面板。 <ol><li>通过温和移液收获整个培养和4℃的PB中的溶液分离S，0.5％牛血清白蛋白（BSA）和2mM EDTA中。重复，直到这些板块都清楚剩余的贴壁细胞 - 确认在显微镜下，如果不能确定。 </li></ol></li><li>可替代地，通过收获与细胞在悬浮液中，其中主要含有单核细胞的上清液丢弃粘附的巨噬细胞。 <ol><li>收获非贴壁细胞用的​​无EDTA的PBS中的溶液和0.5％牛血清白蛋白。不适用为了避免撞出轻度粘连的巨噬细胞用力过猛。 </li></ol></li></ol> 1.5 FACS分析（可选） 注意:可以通过使用MACS的耗尽CD117 +茎和祖细胞的细胞悬浮液。使用制造商协议，在此过程中。 <ol><li>根据要么步骤1.4.1或1.4.2收获细胞。 </li><li>离心细胞，在250×g离心10分钟，在4℃，并再次重复该步骤。 </li><li>悬浮至少250,000个细胞在300微升FACS的Buffer每个探头。 </li><li>转移的细胞溶液到96孔板（30每孔微升）。 </li><li>离心细胞悬浮液在250×g离心5分钟，在4℃。 </li><li>除去上清液，并添加25微升抗体，每孔（使用制造商稀释度）。 </li><li>染色1.5.1-1.5.6将细胞与抗体（按照生产商协议后步骤的细胞表型。常用标记的单核细胞/巨噬细胞识别包括的CD11b，F4 / 80（ 图3），CD115（图2），和GR- 1。 注意:对于小区的解决方案的进一步的描述中，使用象CD4，CD8A，的CD11c，CD25，CD45，CD45R，CD62L，CD80，CD86，CD145，CD117，MHCⅡ，的Ly6C，Ly6G和CD192标记的建议。蜂窝特性进行先前描述的12。 </li><li>孵育探针20分钟，在4℃。 </li><li>离心机的探针，在250×g离心5分钟，在4℃下重悬用200μl的FACS缓冲液中。重复次是步两次。 </li><li>转移探讨FACS管并进行FACS分析12。 </li><li>执行在第0天，第3和5 FACS分析12来分析细胞的分化。 </li></ol> 2.尾静脉注射 2.1准备<ol><li>暖在加热板上的动物，在37℃进行约10分钟。观察动物，同时升温至识别过热的迹象。 </li><li>悬浮的单核细胞，分离的步骤1.1- 1.4，在150微升的NaCl，并加载细胞到1 ml胰岛素注射器用30G针。装载注射器，以确保所有的单核细胞被注射之前轻轻旋涡振荡溶液。总是处理冰上细胞，避免热介导的附着和激活单核细胞。 </li></ol> 2.2抑制<ol><li>避免在选择鼠标静脉注射干扰其他老鼠在笼子里。 </li><li>将鼠标放到限制器。避免过多公关essure和小心的后肢。 （ 图4） 注:另外，使用全身麻醉，以保证无压力的动物处理。 </li><li>确保鼠标有呼吸的空间关闭限制器之前。 </li></ol> 2.3注塑<ol><li>消毒注射部位以消毒剂。喷在小鼠的尾部，静置至少1分钟。 </li><li>通过应用温和的压力，侧尾侧注射部位以上，为更好的可视性静脉停止尾部静脉血流。 </li><li>打开尾90度，注射前。 （ 图5） </li><li>注入以45度角。慢慢的注入，也没有每克超过5微升，以免伤及动物。 </li><li>如果有一个泡罩，这是一个失败的注入的标志，立即停止和更近侧重复注射。 </li><li>通过应用柔和的P止血注射侧ressure约1分钟。 </li><li>在该过程结束时，打开该卷收器，并放置在鼠标在其笼中。 </li><li>观察小鼠20分钟，以确保动物没有被注射伤害和胸骨斜卧被保持。延长观察时间，直到鼠标重新获得足够的意识。返回鼠标给其他动物的公司只后它已完全恢复。 </li></ol>

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The authors declare that they have no competing financial interests.

我们描述了一个简单而具有成本效益的方法，以分离大量骨髓的小鼠单核细胞。相较于使用外周血其他协议，其中获得的单核细胞产率5 1.4×10 6，我们能够从一个单一的供体小鼠获得的11×10 6±3×10 6单核细胞更高的产量。 当考虑使用这种方法的挑战，它在非无菌条件下工作时，提供潜在的污染是重要的。如果冲洗和不遵守以下洗涤步骤正确，污染的细菌和真菌的可能性较大。此外，骨髓由异质细胞悬浮液，包括粒细胞，淋巴细胞和红细胞。通常这些细胞2天和第3天培养的间消失，由于缺乏生长因子。后生长因子从动分化，巨噬细胞是污染的主要来源。为了使吨他数的巨噬细胞的低，并尽量减少单核细胞向巨噬细胞的分化，使用超低附着板和上述的洗涤步骤是关键的。 所以很难确定一个标记物只单核细胞特异性的。标记之间的干扰必须使用一个以上的标志物，并有可变性，在粒度和细胞的大小在分析细胞悬浮液。标记与图1-3中所示的描述是对于在培养中有代表性的细胞类型良好的替代品。它可以是难以区分的各种细胞类型，特别是在5天所以关键是要使用其他标记物像CD115和F4 / 80。 考虑骨髓细胞的粘附无的条件下膨胀，巨噬细胞分化，可以延长，未分化的单核细胞可以积累。巨噬细胞分化表明污染;然而，杜E要即使在超低附着培养表面的粘合剂的性质，它们可以容易地丢弃，而只有从培养收获非粘着细胞。在以前的实验1,3，污染被巨噬细胞并没有导致严重的临床副作用。器官和细胞移植后的小鼠的肌肉组织学后处理表明，注入的巨噬细胞吸收在诸如脾，肝或肺器官。在这些器官的巨噬细胞的同化证明在小鼠中没有观察到的临床效果。这些巨噬细胞引发的分子机制可用于进一步调查有趣的问题。 当全身应用的单核细胞以及巨噬细胞的影响动脉中的外周动脉疾病，特别是。临床副作用，如栓塞或块状系统性炎症无法观察到甚至注射超过250万的单核细胞后，但这些细胞可引发炎症和Potentially造成严重的系统性影响。在人体试验中，这些影响可能有临床意义。动脉的触发可影响肿瘤生长或糖尿病性视网膜病变，并注射细胞溶液应当在未来的研究探讨的这些可能的全身效应。 以前的出版物9描述的外围血-和骨髓来源的单核细胞，这解释了为什么从骨髓单核细胞的特性可以从那些从外周血细胞中分离的不同的特性之间的差异。 隔离的方法是临床感兴趣的为好。抽血，而不是提取人骨髓是临床医学的背景下更实用。照顾动物福利后，如果需要，以应用镇痛是很重要的。用于静脉内注射，它实行同时处理动物和注射器在同一时间是至关重要的。如果一个ANIMAL正在经历多次注射，静脉的质量和尾部的皮肤受罪。 尽管有这些缺点，这种方法是非常有用的，研究的行为和单核细胞的特性。在动脉粥样硬化，免疫或炎症的现场实验可以受益于这种方法。仅30分钟，需要从骨髓提取到细胞接种在6孔超低附着表面板。伦理问题被最小化，因为高收率此协议的最小化需要作为细胞供体动物的数量。这种新方法将成为许多研究涉及单核细胞有帮助的。 我们一直专注于侧支血管生长通过单核细胞移植治疗增强。这个过程的多因素的性质可以解释为什么单因素的方法动脉的增强都产生不同的结果13,14。这导致了在王爱平细胞为基础的治疗。自体骨髓衍生的干细胞和祖细胞已经被确定为潜在的移植细胞，但仅中度临床益处已报道15。除了对剂量，分离方法，和给药途径的研究，进一步的研究仍然需要确定最佳的细胞类型。自体细胞移植的缺点可能是其不能激活先天和/或适应性免疫应答者，都为动脉的关键。增加局部炎症可能代表动脉16的最好刺激。 静脉内注射的单核细胞是内小鼠模型细胞移植的常用方法，如果全身给药是理想的。由于系统性影响的，副作用可能会出现为好。确保静脉是针对注射。是很常见的动脉和静脉，尤其是当注入大量色素麦克风混淆即动脉注射可引起血栓，从而导致流通中的系统性问题。

The isolation of monocytes is important and critical for many in vitro and in vivo studies. These cells are targets for diseases such as peripheral arterial disease, coronary heart disease, or other ischemic diseases, since collateral vessel growth is strongly driven by local inflammation. Inflammatory responses include endothelial activation and local recruitment of leukocytes, mainly monocytes, which then mature to macrophages and create a highly arteriogenic environment by secreting multiple growth factors to induce the remodeling of an arteriole into a functional collateral artery1-3. Monocytes also mature to dendritic cells, which are frequently used for immunological studies4,5 and cancer research6,7.
Problematic in the approach for monocyte isolation from peripheral blood8 is the high number of donor animals needed to produce a sufficient amount of monocytes for most analyses. Former protocols describe methods such as density gradient centrifugation and cell depletion via MACS9 when isolating monocytes; however, these techniques can alter the characteristics and functionality of monocytes which can lead to difficulties in interpretation10,11. Moreover, these methods are difficult and can reduce experimental reproducibility.
Our aim with this protocol is to provide a simple and cost effective method to generate large amounts of bone marrow-derived monocytes. Due to the high cell yield of 11 x 106 &#177; 3 x 106 cells obtained by this protocol, we can substantially reduce the number of mice required during the isolation of bone marrow-derived monocytes. The procedure can be completed within a minimal amount of time, and without using expensive and complicated techniques as referenced above. Here, we extract monocytes from native bone marrow suspension of donor mice, cultivate the suspension on ultralow attachment plates, and supplement the solution with 20 ng/ml M-CFS. On day 5 of incubation, cells are harvested and characterized to confirm functional and phenotypic properties.
For experiments in the field of arteriogenesis, intravenous transplantation of these bone marrow-derived monocytes into mice is an effective method of systemic drug delivery, which can be combined with femoral artery ligation in common peripheral arterial disease models.

<table><tbody><tr><td>6-well-ultra-low-attachment plate</td><td>Corning Incorporated, NY, USA</td><td></td><td>6-well-ultra-low-attachment plate, with cap, sterile</td></tr><tr><td>8- 12 week old, male, balb/c mice&amp;nbsp;</td><td>Charles River, Sulzfeld, Germany</td><td></td><td></td></tr><tr><td>96-well-plate</td><td>Greiner bio one GmbH, Frickenhausen, Germany</td><td></td><td></td></tr><tr><td>Blue dead cell stain</td><td>Life technologies GmbH, Darmstadt, Germany</td><td></td><td></td></tr><tr><td>Bovine serum albumine</td><td>GE Healthcare, Freiburg, Germany</td><td></td><td>Fraction V, pH 7.0</td></tr><tr><td>Canules</td><td>B. Braun, Melsungen AG, Melsungen, Germany</td><td></td><td>28G, 30G</td></tr><tr><td>CD115</td><td>eBioscience, San Diego, USA</td><td>12-1152</td><td></td></tr><tr><td>CD11b</td><td>eBioscience, San Diego, USA</td><td>53-0112</td><td></td></tr><tr><td>Cell culture dish</td><td>Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td>With cap, steril</td></tr><tr><td>Centrifuge</td><td>Beckman Coulter GmbH, Krefeld, Germany</td><td></td><td>Allegra&amp;reg; X-15R centrifuge</td></tr><tr><td>Depilatory cream</td><td>Veet, Mannheim, Germany</td><td></td><td></td></tr><tr><td>Disinfection agent</td><td>Sch&amp;uuml;lke&amp;amp;Mayr GmbH, Norderstedt, Germany</td><td></td><td>Kodan Tinktur forte</td></tr><tr><td>Disposable scalpel No.10&amp;nbsp;</td><td>Feather safety razor Co.Ltd, Osaka, Japan&amp;nbsp;</td><td></td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Ethanol 96%&amp;nbsp;</td><td>Otto Fischar GmbH und Co KG, Saarbr&amp;uuml;cken, Germany</td><td></td><td></td></tr><tr><td>Extraction unit Pipetus</td><td>Hirschmann Laborger&amp;auml;te GmbH &amp;amp; Co.KG, Eberstadt, Germany</td><td></td><td></td></tr><tr><td>F4/80</td><td>AbD Serotec, D&amp;uuml;sseldorf, Germany</td><td>MCA497APC</td><td></td></tr><tr><td>FACS buffer&amp;nbsp;</td><td>Manufactured by our group with single components</td><td></td><td>PBS, 0.5% BSA, 0.1% NaN&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>FACS device</td><td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td><td></td><td>BD FACS Canto II</td></tr><tr><td>FACS tubes&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td><td></td><td></td></tr><tr><td>Falcon&amp;reg; pipette</td><td>Becton Dickenson Labware, NY, USA</td><td></td><td></td></tr><tr><td>Fetal calf serum</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Fine forceps</td><td>Rubis, Stabio, Switzerland</td><td></td><td></td></tr><tr><td>Gloves</td><td>R&amp;ouml;sner-Matby Meditrade GmbH, Kiefersfelden, Germany</td><td></td><td></td></tr><tr><td>Gr1</td><td>eBioscience, San Diego, USA</td><td>53-5931</td><td></td></tr><tr><td>Heating plate&amp;nbsp;</td><td>Labotect GmbH, G&amp;ouml;ttingen, Germany&amp;nbsp;</td><td></td><td>Hot Plate 062</td></tr><tr><td>Incubator</td><td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td><td></td><td>Incu safe</td></tr><tr><td>Isofluran</td><td>Baxter Deutschland GmbH, Unterschlei&amp;szlig;heim, Germany</td><td></td><td></td></tr><tr><td>Light microscope</td><td>Carl Zeiss SMT GmbH, Oberkochen, Germany</td><td></td><td>Axiovert 40 &amp;deg;C</td></tr><tr><td>Macrophage-Colony Stimulating Factor</td><td>Sigma Aldrich, Hamburg, Germany</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/srp3110?lang=de&amp;amp;region=DE&quot;&gt;SRP3110&amp;nbsp;&lt;/a&gt;</td><td></td></tr><tr><td>Mechanical shaker</td><td>IKA, Staufen, Germany</td><td></td><td>ms2 minishaker</td></tr><tr><td>Medium 199</td><td>PAA Laboratories GmbH, Pasching, Austria</td><td></td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Micro test tubes</td><td>Eppendorf AG, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Microbiological work bench</td><td>Thermo Electron, LED GmbH, Langenselbold, Germany</td><td></td><td>Hera safe</td></tr><tr><td>Monocyte wash buffer&amp;nbsp;</td><td>Manufactured by our group with single components</td><td></td><td>PBS, 0.5% BSA, 2 mM EDTA</td></tr><tr><td>Mouse restrainer</td><td>Various</td><td></td><td></td></tr><tr><td>NaCl</td><td>Berlin Chemie AG, Berlin, Germany</td><td></td><td></td></tr><tr><td>NaN&lt;sub&gt;3 &lt;/sub&gt;(sodium acide)</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Neubauer counting chamber</td><td>Paul Marienfeld GmbH und Co.KG, Lauda-K&amp;ouml;nigshofen, Germany</td><td></td><td></td></tr><tr><td>Nylon cellsieve</td><td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td><td></td><td>Cell strainer, 70 &amp;micro;m mesh size</td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Life technologies GmbH, Darmstadt, Germany</td><td></td><td>pH 7.4, sterile</td></tr><tr><td>Pipettes</td><td>Eppendorf AG, Hamburg, Germany</td><td></td><td>10&amp;micro;l/100&amp;micro;l/200&amp;micro;l/1,000&amp;micro;l</td></tr><tr><td>Pipetting heads</td><td>Eppendorf AG, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Serological pipette</td><td>Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td>Cellstar 5 ml, 10 ml</td></tr><tr><td>Suction unit</td><td>Integra bioscience, Fernwald, Germany</td><td></td><td>Vacusafe comfort</td></tr><tr><td>Surgical scissors</td><td>Word Precision Instruments, Inc., Sarasota, USA</td><td></td><td></td></tr><tr><td>Syringe</td><td>B. Braun, Melsungen AG, Melsungen, Germany</td><td></td><td>1 ml Omnifix&lt;sup&gt;&amp;reg; &lt;/sup&gt;-F insuline syringe</td></tr><tr><td>Tubes with cap</td><td>Greiner bio one GmbH, Frickenhausen, Germany</td><td></td><td>15 ml/50 ml Cellstar tubes</td></tr><tr><td>Warm water bath</td><td>Julabo Labortechnik GmbH, Seelbach, Germany</td><td></td><td>Julabo SW22</td></tr></tbody></table>

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.

来自鼠骨髓中提取的细胞溶液包含各种细胞类型。主要的细胞类型是淋巴细胞，粒细胞和单核细胞。细胞类型可以通过尺寸和粒度，其示于图1中为5天后分化的收获两个天然悬浮液和细胞来估计。注意培养过程中的换档细胞组成。然而，人口的准确分类必须依靠细胞标记鲜明的表达。 用于识别的MPS-系统的细胞中最重要的标记是CD115，其功能是作为对M-CSF受体和专门对单核细胞祖细胞，单核细胞和巨噬细胞表达。因此，它可能不被用于识别独特的MPS-子集，但能可靠地估计细胞驱入单核细胞/巨噬细胞分化的绝对量。参见图2 CD115的表达在不同的时间安排entiation。 成熟标记F4 / 80是有用的区分少年单核细胞，成熟的单核细胞和巨噬细胞。巨噬细胞表现出强劲的F4 / 80的表达相比，少年单核细胞的祖细胞，它们是负的标记。成熟的单核细胞显示出的F4 / 80的中间表现。因此CD115和F4 / 80标记的组合表示用于定量和监测细胞分化（ 见图3）一个可行的方法。 CD115和F4 / 80的结合是足够的常规培养观察和质量控制应用之前的细胞。在培养的第5天的骨髓来源的单核细胞的更详细的检查证实了他们的结构和功能特性的一致性的那些的外周血单核细胞12。 它小心固定鼠标在阻挡器，避免张力尾巴上是重要的。限制freedo米运动尽可能不妨碍呼吸，以方便注射。 <img alt="图1" src="/files/ftp_upload/52347/52347fig1highres.jpg" /> 图1.左面板:细胞类型在培养第1天的组合物的细胞类型，包括淋巴细胞，单核细胞和粒细胞。右侧面板:注意培养过程中的换档细胞组成。细胞群包括单核细胞和巨噬细胞。 <a href="https://www.jove.com/files/ftp_upload/52347/52347fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52347/52347fig2highres.jpg" /> 图2.时间轴分化过程中CD115的表达。注意，&gt; 95％的细胞是CD115 5天后分化的阳性，这表明广大<html>大部分细胞要么单核细胞或巨噬细胞。分类可以根据细胞大小和粒度，但特异性细胞标志物更加可靠。 <a href="https://www.jove.com/files/ftp_upload/52347/52347fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52347/52347fig3highres.jpg" /> 图3.增加单核细胞/巨噬细胞的成熟标记F4 / 80 5天的表达。注意与中间的F4 / 80的表达，这与单核细胞的表型相一致的人口的外观。第7天:注意右移F4 / 80的表达，与巨噬细胞成熟一致的<a href="https://www.jove.com/files/ftp_upload/52347/52347fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> E 4"SRC ="/文件/ ftp_upload / 52347 / 52347fig4highres.jpg"/&gt; 图4.鼠标固定在阻挡器尾静脉注射。在认真抑制鼠标，旋转尾部90度，直至脉的背侧可见。 <a href="https://www.jove.com/files/ftp_upload/52347/52347fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52347/52347fig5highres.jpg" /> 的小鼠尾部图5的截面示意图。该图说明中的鼠尾船只的位置。动脉（红色）位于腹侧和背侧，和静脉都在尾部的侧面。 <a href="https://www.jove.com/files/ftp_upload/52347/52347fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes at JoVE.com

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

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 ...

isolation-intravenous-injection-murine-bone-marrow-derived

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

23.6K 次观看.  Otto von Guericke University Magdeburg. 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).

视频: Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

科研

免疫与感染

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 ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

小鼠外周动脉疾病模型中单核细胞归巢和肿瘤相关血管生成的活体显微镜观察

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

医学

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

骨髓来源巨噬细胞的分离和 体外 培养用于NO-氧化还原生物学研究

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...

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

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

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

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

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

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

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

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

从小鼠中分离和培养骨髓来源的巨噬细胞

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

新生儿骨髓来源巨噬细胞的吞噬试验评价 

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

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

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

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

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

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

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and development of bones are mainly regulated by osteoblasts and osteoclasts. It has been widely accepted that osteoclasts are derived from bone marrow monocyte-macrophage cells (BMMs). BMMs and other hematopoietic stem cells are located in the bone marrow cavity. Therefore, isolating single stable BMMs from different and heterogeneous cell populations is a huge challenge. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. Adherent cells cultured for 24-48 h in primary culture were collected. Flow cytometric analysis showed that approximately 37.94% of the cells were CD11b/c+ (monocyte-macrophage surface antigen). Tartrate resistant acid phosphatase (TRAP) staining and western blot analysis demonstrated that BMMs could differentiate into osteoclasts in vitro. The above findings suggested that the secondary adherence cells could be considered as a suitable cellular model for osteoclast differentiation research.

Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.

隔离和小鼠骨髓的静脉注射单核细胞衍生

摘要

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此视频中的章节

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

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

小鼠外周动脉疾病模型中单核细胞归巢和肿瘤相关血管生成的活体显微镜观察

骨髓来源巨噬细胞的分离和体外培养用于NO-氧化还原生物学研究

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

从小鼠中分离和培养骨髓来源的巨噬细胞

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

通过二级粘附法从大鼠骨骼中分离单核细胞 - 巨噬细胞谱系细胞

隔离和小鼠骨髓的静脉注射单核细胞衍生

摘要

探索更多视频

此视频中的章节

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

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

小鼠外周动脉疾病模型中单核细胞归巢和肿瘤相关血管生成的活体显微镜观察

骨髓来源巨噬细胞的分离和 体外 培养用于NO-氧化还原生物学研究

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

从小鼠中分离和培养骨髓来源的巨噬细胞

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

通过二级粘附法从大鼠骨骼中分离单核细胞 - 巨噬细胞谱系细胞

骨髓来源巨噬细胞的分离和体外培养用于NO-氧化还原生物学研究