This work was supported by the University of Pennsylvania Center for Musculoskeletal Disorders (5 P30 AR050950-09), a grant from the AO Foundation (S-16-12A), the Philadelphia VA Medical Center Translational Musculoskeletal Research Center, and an intramural orthopaedic surgery departmental research development fund. JWA is supported by the University of Pennsylvania Postdoctoral Opportunities in Research and Teaching (PENN-PORT) fellowship funded by the National Institute of General Medical Sciences Institutional Research and Career Development Award (IRACDA; 5 K12 GM081259-08).

按照由宾夕法尼亚机构动物护理和使用委员会大学（IACUC）批准的方案进行所有涉及脊椎动物的研究。 1.文化传媒准备<ol><li>由最低必需培养基（MEM）粉末和1.9克次硫酸钠碳酸氢钠溶解在900ml 的 H 2ö制备的α-MEM和补充有2mM L-谷氨酰胺，100单位/ ml青霉素，100微克/毫升链霉素和100毫升热灭活的胎牛血清。消毒使用0.22微米的聚醚砜（PES）过滤器。 </li><li>通过用35纳克补充的α-MEM制备巨噬细胞培养基/ ml重组小鼠巨噬细胞集落刺激因子（M-CSF） </li><li>通过用100ng / ml的RANKL补充巨噬细胞培养基制备破骨细胞培养基</li></ol> 2.骨髓细胞分离<ol><li>填充1个10毫升注射器用每只小鼠一个25⅝号针用10毫升的α-MEM。 </li&gt; <li>以1.3升/分钟的流速安乐死经由10分钟暴露于CO 2小鼠。通过解剖继续之前以下的 CO 2曝光颈椎脱位确保动物死亡。 </li><li> 使用清洁技术，解剖并分离胫骨和股骨。一种制造切口沿着每一后肢的前表面，以暴露胫骨和股骨前消毒用70％乙醇的小鼠皮肤。 <ol><li>通过膝关节切割以释放股骨的远端，并且通过股骨尽可能靠近骨盆尽可能削减。取出股骨和清理尽可能多的软组织成为可能。要取出胫骨，通过脚踝削减和删除尽可能多的软组织成为可能。 </li></ol></li><li>握住镊子和冲洗骨髓骨成用2.5ml室温α-MEM从一个单一的鼠标结合骨髓冲成单管15毫升锥形管。 注:一个成功的骨髓冲洗会改变T的着色他从红骨米色。 </li><li>允许碎屑沉降到试管底部并转移上清至干净的15毫升锥形管中，并注意避免传输任何碎屑。 注:某些细胞也可以解决与骨髓杂物。如果需要的细胞的更大数目，骨髓刷新可以通过一个70微米的细胞滤网过滤到干净的15ml试管中。 </li><li>离心管（多个）在300×g离心5分钟，在室温下沉淀细胞。 </li><li>真空吸出上清液和重悬细胞沉淀在0.5ml氯化铵-钾（ACK）裂解缓冲液，并在37°C孵育3分钟以溶解红细胞。 </li><li>直接加入10毫升的PBS至ACK细胞溶液和离心机在300×g离心5分钟。 注意:先前的红色细胞团，现在应该是米色。 </li><li>真空吸上清，重新悬浮颗粒在10毫升α-MEM，和板100毫米的组织培养处理的菜。孵育细胞过夜，在37℃在加湿的组织培养的孵化器。 注:计数细胞是没有必要的这一步。在此期间，从骨髓的贴壁细胞将附着在培养表面上;非粘附骨髓细胞群将保持在悬浮液中。 MCSF此初始孵育期间不存在于培养基中。 </li></ol> 3.破骨细胞前体富集<ol><li>后骨髓潮红，包含非粘附细胞到50ml锥形管转移培养物上清液温育过夜。 注:培养皿以贴壁细胞，其包括骨髓间充质祖细胞，可以被丢弃或用新鲜α-MEM供给，如果需要，对于其它实验。 注:破骨细胞可以直接从这些非粘附骨髓细胞通过每隔一天中的破骨细胞培养基和清凉介质电镀它们到组织培养处理过的菜以4×10 5个细胞/ cm 2的密度5天分化。 </l我&gt; <li>的α-MEM，以非贴壁细胞溶液添加到18 ml的终体积，并添加M-CSF为35纳克/毫升的最终浓度。 </li><li>加入3 ml的每种细胞悬浮液至6 60毫米的悬浮液培养皿。 </li><li>在湿润的组织培养箱在37℃下孵育板过夜。在这段时间中，M-CSF将刺激巨噬细胞，其可以附着甚至非组织培养处理过的表面的分化。 </li><li>温育过夜用3ml新鲜巨噬细胞培养基，重新进料的破骨细胞的前体之后。这将删除而没有分化成巨噬细胞非贴壁细胞。 </li><li>继续培养破骨细胞前体在37℃和5％CO 2的2天或直到培养物达到大约70％汇合。 </li></ol> 4.破骨细胞分化<ol><li>在室温下洗破骨细胞的前体用5毫升的PBS和治疗的细胞用1毫升海洋起源的蛋白水解/胶原溶解酶，直到CELLS可以通过板轻轻拍打被抛下。 注意:如果他们是悬浮培养的菜破骨细胞前体只能解除;前体连接得太紧，以组织培养处理的表面得以解除。胰蛋白酶可激活破骨细胞的前体，应该避免。 </li><li> 4毫升的α-MEM，以每个培养皿转移细胞加至50ml锥形管中。算使用血球细胞和细胞稀释至5×10 4个细胞/毫升的密度，并添加M-CSF为35纳克/毫升和RANKL的终浓度为100毫微克/毫升的最终浓度。 </li><li>种子细胞的密度2.6×10 4个细胞/ cm 2的成组织培养处理板孵育在37℃下在湿润的组织培养箱中2天。分化典型地在24孔板，其中5×10 4个细胞接种到每个孔中进行。 </li><li>分化，再进给细胞通过用1ml新鲜osteoc更换培养基开始后第2天去年网上平台。 注:破骨细胞分化通常会在3天内完成。导致破骨细胞可以TRAP染色，便于量化和形态分析。 </li></ol> 5. Notch信号的连续刺激与Jagged1的涂覆表面<ol><li>暂停在PBS山羊抗人IgG Fc抗体，以10微克/ ml的浓度（1:1000稀释度的10mg / ml的股票）。以1.3微克/ cm 2的密度加上抗体溶液对培养表面，在室温下孵育1小时。在24孔板的情况下，每孔加入250微升（2.5微克）。 </li><li>真空抽吸孔和填充用1毫升PBS中。重复此步骤2次。 </li><li>暂停在PBS重组Jagged1的-Fc融合蛋白以10微克/ ml的浓度。添加Jagged1的-FC溶液到抗体包被的培养表面以1.3微克/ cm 2的密度，并在室温下孵育2小时。涂有抗体外核层培养表面y可以有用作对照。 </li><li>真空抽吸孔和填充用1毫升PBS中。重复此步骤2次。请PBS井直到刚好在细胞接种，以防止它们干燥。 </li><li>种子2.6×10 4的破骨细胞的前体/厘米2到涂覆的表面。 Notch信号将被不断刺激至少3天。 </li></ol> 6. Notch信号的刺激临时用Jagged1处理涂层珠<ol><li>结合以下在一个适当大小的管:0.5微克/厘米2 Jagged1的-Fc的，21微升/ cm 2的G蛋白琼脂糖珠，和PBS中至1.5 ml的终体积。以制备的Jagged1的珠粒足够数量为1以及一个24孔板的，结合1微克Jagged1的-Fc的，40微升G蛋白琼脂糖珠，和PBS 1.5毫升</li><li>在4旋转管孵育 °下2小时。 </li><li>在300×g离心1分钟，以沉淀珠离心管中。悬浮珠粒在1.5ml PBS中。重复此洗2次以上。 </li><li>悬浮珠Jagged1的130微升/厘米2培养基，并适用于培养细胞。 </li><li>孵育小珠的细胞在37℃和5％所需的时间的 CO 2。用PBS或培养基的几次洗涤除去珠子。 </li><li>通过Notch靶基因转录10的测量来验证Notch信号激活。 </li></ol>

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The authors declare that they have no competing financial interests.

该协议中的关键步骤文化与破骨细胞前体体外分化为破骨细胞和骨再生，骨量保存治疗靶点识别的分子机制研究一个有益的平台。当鼠标培养破骨细胞的前体，最关键的因素是一个天真的状态前体的维护。作为巨噬细胞样细胞，破骨细胞前体填装到细菌成分和促炎细胞因子响应。在激活时，破骨细胞前体将不再有效地分化成破骨细胞。前体活化的主要原因是炎性细...

哺乳动物Notch信号传导途径是同源的果蝇的相同的途径和由四个跨膜Notch受体（Notch1-4）和铁血（JAG1＆JAG2）和Delta样（DLL1，3-五膜结合配体，及4）家庭1。当在接收单元Notch受体被配体结合在一传送单元2 Notch信号被启动。在此反式激活，膜结合Notch配体上产生的膜结合Notch受体3,4的拉伸力。配体结合的拉伸力诱导的Notch受体由肿瘤坏死因子α促进受体的胞外切割位转换酶（TACE），随后通过含早老-γ分泌复合物（γ分泌）介导的细胞内切割事件的构象变化。 γ分泌释放缺口INTracellular域（NICD），该易位进入它形成具有CBF -1-苏（H）-Lag-1（CSL），主脑样（MAML）一个转录激活复合物的核和细胞类型特异性的因素来驱动表达的靶基因5。 的Notch信号传导的激活结果中，需要在体外 Notch途径激活的独特方法的机械元件。可溶性Notch配体可以结合Notch受体，但不能产生拉伸所需NICD释放力，而在同一时间竞争性地抑制细胞相关的Notch配体的结合。因此，除了可溶性Notch配体到培养基可以减轻正常Notch信号6,7。幸运的是，Notch配体可诱导NICD释放，如果他们被固定到合适的刚性基板5，8，9，10。上涂覆的配体培养基材播种细胞或施加涂覆配体珠的细胞可以既激活Notch信号，以及它们之间的选择主要取决于缺口刺激期望的定时。立即，临时Notch信号传导的激活，如将一个功能或分化测定的中点期间需要，Notch配体可以结合到琼脂糖珠，施加到培养的细胞，并在任何时间洗掉。为从培养期开始时更持续的Notch信号，组织培养板可涂之前细胞接种配体。 对于此协议的目的，方法被进行了使用小鼠的破骨细胞的前体，但在这里所描述的方法的方法和变体适用于各种各样的细胞类型6，11，12，13， </SUP&gt; 14。破骨细胞是终末分化的造血行数的细胞，它们负责骨组织的再吸收，并且它们在骨损失15的多个障碍有关。因此， 在他们的单核/巨噬细胞系细胞前体破骨细胞和分子机制控制它们的功能是为了更好地理解破骨细胞和新骨再生疗法的发展至关重要分化的体外研究。而它现在普遍接受的是Notch信号起着破骨细胞的分化和功能的积极作用，在这两种Notch信号传导的刺激和破骨细胞的前体培养和分化的变化导致最初矛盾的发现16，17，18，19。在方法的差异进一步检查和利用遗传模型已经大大澄清Notch信号在破骨细胞的作用，但标准化的缺口刺激和培养方法的应用程序可以防止这类争议在其他类型的细胞20，21，22，23 Notch信号的未来研究。 有用于培养和分化小鼠破骨细胞前体的多种方法，并且，与用于刺激Notch信号改变方法，最好的方法将取决于实验问题。这里，我们的优选培养来自小鼠长骨冲洗骨髓细胞的粘附和非粘附的级分的方法将提交。这种方法有本质上不需要专门的设备并生产了适用于各种分化方法的细胞的优点。

<table><tbody><tr><td>Recombinant mouse M-CSF</td><td>Biolegend</td><td>576402</td><td>Available from multiple suppliers, test activity before experiments</td></tr><tr><td>Recombinant mouse RANKL</td><td>Shenandoah Biotechnology</td><td>200-04</td><td>Available from multiple suppliers, test activity before experiments</td></tr><tr><td>Recombinant human Jagged1-Fc</td><td>R&amp;amp;D Systems</td><td>1277-JG-050</td><td>Available from multiple suppliers, test activity before experiments</td></tr><tr><td>Protein G agarose beads</td><td>InvivoGen</td><td>gel-agg-2</td><td></td></tr><tr><td>Goat anti-human IgG Fc</td><td>Jackson ImmunoResearch</td><td>109-001-008</td><td></td></tr><tr><td>Minimum Essential Medium powder</td><td>Sigma-Aldrich</td><td>M0894</td><td></td></tr><tr><td>Accutase cell dissociation reagent</td><td>ThermoFisher</td><td>A1110501</td><td>Used to lift osteoclast precursors</td></tr><tr><td>Acid Phosphatase, Leukocyte (TRAP) Kit</td><td>Sigma-Aldrich</td><td>387A-1KT</td><td>Used to stain differentiated osteoclasts</td></tr></tbody></table>

stimulation of notch signaling in mouse osteoclast precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

该方法的目的是培养和刺激Notch信号中的破骨细胞的前体。当适当培养，破骨细胞的前体显示出具有光滑细胞质（ 图1A）一个主要的细长纺锤形的形态。应当小心，以避免破骨细胞前体的免疫激活。在激活时，前体扩散并成为泡沫状细胞质（ 图1B）平坦化。这些"煎鸡蛋"细胞对RANK信号耐不能有效地分化为成熟的破骨细胞。下正确培养和分化的条件?...

Watch this Scientific Journal Video about Stimulation of Notch Signaling in Mouse Osteoclast Precursors at JoVE.com

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

在鼠标破骨细胞前体Notch信号的刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

stimulation-of-notch-signaling-in-mouse-osteoclast-precursors

Research

JoVE Journal

Developmental Biology

7.8K 次观看.  Perelman School of Medicine, University of Pennsylvania. Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

视频: Stimulation of Notch Signaling in Mouse Osteoclast Precursors

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

科研

发育生物学

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

一种新型的<em&gt;在体内</em&gt;基因转移技术和<em&gt;在体外</em&gt;细胞分析为肌肉骨骼疾病骨质流失的研究

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

医学

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

从小鼠面部过程和培养的腭间充质细胞中分离全细胞蛋白质裂解物以进行磷酸化蛋白分析

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

破骨细胞与人诱导多能干细胞的分化和表征

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

生物学

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

一种简单的坑式检测方案，用于可视化和定量体外碎屑病再吸收

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases ...

分析牙槽骨重塑以探索骨骼力学生物学

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial process of alveolar bone remodeling in response to mechanical force, but the underlying mechanism remains elusive. Previous studies have been unable to reveal the precise mechanism of bone remodeling in any time and space due to animal model-related restrictions. The signal transducer and activator of transcription 3 (STAT3) is important in bone metabolism, but its role in osteoblasts during OTM is unclear. To provide in vivo evidence that STAT3 participates in OTM at specific time points and in particular cells during OTM, we generated a tamoxifen-inducible osteoblast lineage-specific Stat3 knockout mouse model, applied orthodontic force, and analyzed the alveolar bone phenotype. 
Micro-computed tomography (Micro-CT) and stereo microscopy were used to access OTM distance. Histological analysis selected the area located within three roots of the first molar (M1) in the cross-section of the maxillary bone as the region of interest (ROI) to evaluate the metabolic activity of osteoblasts and osteoclasts, indicating the effect of orthodontic force on alveolar bone. In short, we provide a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describe methods for analyzing alveolar bone remodeling during OTM, thus shedding new light on skeletal mechanical biology.

作者聚焦：比较牙槽和长骨重塑以探索 OTM 模型的潜力 

This study provides a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describes methods for analyzing alveolar bone remodeling during orthodontic tooth movement, thus shedding light on skeletal mechanical biology.

使用诱导型成骨细胞谱系特异性 Stat3 敲除小鼠研究正畸牙齿移动过程中的牙槽骨重塑

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial ...

在鼠标破骨细胞前体Notch信号的刺激

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RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

一种新型的

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

从小鼠面部过程和培养的腭间充质细胞中分离全细胞蛋白质裂解物以进行磷酸化蛋白分析

破骨细胞与人诱导多能干细胞的分化和表征

小鼠骨髓破骨细胞的推导

一种简单的坑式检测方案，用于可视化和定量体外碎屑病再吸收

使用诱导型成骨细胞谱系特异性 Stat3 敲除小鼠研究正畸牙齿移动过程中的牙槽骨重塑

使用诱导型成骨细胞谱系特异性 Stat3 敲除小鼠研究正畸牙齿移动过程中的牙槽骨重塑

使用诱导型成骨细胞谱系特异性 Stat3 敲除小鼠研究正畸牙齿移动过程中的牙槽骨重塑