Name: osteoclast derivation from mouse bone marrow
Uploaded: 2014-11-06T14:00:00
Description: Watch this Scientific Journal Video about Osteoclast Derivation from Mouse Bone Marrow at JoVE.com

我们承认美国国立卫生研究院拨款R01 DE021683，R01 DE019434，U01 HL099776，橡树基金会和Hagey实验室小儿再生医学的支持。

注：伦理声明：在批准的斯坦福大学行政专家组对实验室动物护理（APLAC）按照协议进行的所有涉及脊椎动物的研究。 1.准备<ol><li>允许10毫升市售的密度梯度的细胞分离介质（包含多聚蔗糖和泛影酸钠，调整至10.77克/毫升的密度）来RT在50ml锥形管中。 </li><li>制备流式细胞术（FACS）缓冲液用1×磷酸盐缓冲盐水（PBS）和2％胎牛血清（FCS）中。取50毫升的等分试样，并且在密度梯度的细胞分离?...

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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=Hyperglycemia+induced+and+intrinsic+alterations+in+type+2+diabetes-derived+osteoclast+function.">Hyperglycemia induced and intrinsic alterations in type 2 diabetes-derived osteoclast function.</a> Oral diseases. 19, 303-312 (2013).</li><li>Schueler, 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=Intratibial+injection+of+human+multiple+myeloma+cells+in+NOD/SCID+IL-2Rgamma(null)+mice+mimics+human+myeloma+and+serves+as+a+valuable+tool+for+the+development+of+anticancer+strategies.">Intratibial injection of human multiple myeloma cells in NOD/SCID IL-2Rgamma(null) mice mimics human myeloma and serves as a valuable tool for the development of anticancer strategies.</a> PloS one. 8, (2013).</li><li>Xing, L., Boyce, B. 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G., Marchetti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+disruptors+and+bone+metabolism.">Endocrine disruptors and bone metabolism.</a> Archives of toxicology. 87, 735-751 (2013).</li><li>Manolagas, S. C., O'Brien, C. A., Almeida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+estrogen+and+androgen+receptors+in+bone+health+and+disease.">The role of estrogen and androgen receptors in bone health and disease.</a> Nature Reviews Endocrinology. 9, 699-712 (2013).</li><li>Martin, T. J., Udagawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+regulation+of+osteoclast+function.">Hormonal regulation of osteoclast function.</a> Trends in endocrinology and metabolism. 9, 6-12 (1998).</li><li>Nakamura, 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=Estrogen+prevents+bone+loss+via+estrogen+receptor+alpha+and+induction+of+Fas+ligand+in+osteoclasts.">Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts.</a> Cell. 130, 811-823 (2007).</li><li>Bellido, 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=Regulation+of+interleukin-6,+osteoclastogenesis,+and+bone+mass+by+androgens.+The+role+of+the+androgen+receptor.">Regulation of interleukin-6, osteoclastogenesis, and bone mass by androgens. 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一个很容易分离，培养大量的破骨细胞在体外的能力一直负责帮助推动骨生物学和破骨细胞介导的疾病的认识。这是RANKL的识别，导致此，当它最近被鉴定为破骨细胞的形成，分化和存活16-18的主要调节剂。 据我们的经验是， 在体外培养骨髓破骨细胞在很大程度上依赖于接种密度。我们观察到，该协议得到的破骨细胞的失败通常是由于一个不理想的接种?...

骨重建是动态的，并且涉及骨形成与骨吸收1的耦合。这个严格调节的过程是负责维持正常的体内平衡中的骨架，并响应于损伤和疾病。 破骨细胞是独特的，多核细胞，其能够再吸收二者的有机和无机骨基质。破骨细胞是来源于骨髓的2-5的单核细胞/巨噬细胞系。在破骨细胞的功能，或形成的异常可导致多种临床病症，包括骨质疏松症象常见的情况的。 在体外产生的破骨细胞的能力已经允许在我们的骨生物学6的理解显著进步。其结果是，新的治疗剂的新兴治疗破骨细胞有关的疾病，这是负责显著发病率和死亡率7 </sUP&gt;。稳态维持骨量和骨强度需要骨形成的成骨细胞和骨吸收破骨细胞8,9的一致行动人。骨骼平衡被改变中的一些疾病，包括绝经后骨质疏松症，其中增加的破骨细胞活性导致骨质量和密度10的致病损失。随着人类疾病的转基因小鼠模型的可用性，有更多的机会来破译人骨疾病11-13的破骨细胞的作用。 对破骨细胞的培养技术大量协议，出现在文献中，有许多变型描述9,12,14。性和同事描述了类似的方法，以如下所述，在小鼠骨髓细胞中破骨测定中对它们的描述的协议。然而，释放以下长骨收获骨髓细胞，性等冲洗骨髓腔的α-MEM完全培养基14。Catalfamo检查高血糖对破骨细胞功能的影响，并描述了其中的所有细胞动员骨髓潮红的方法进行培养24小时，在该点处的非贴壁细胞被丢弃12，一种技术也可用于由Boyle 等人的9，这些先前公布的协议必要冲洗骨髓，一个繁琐的实践中，其中还引入了针刺伤害和有价值的骨髓损失的风险，因为一个人必须切骨的两端部的做法。的协议中，我们描述了，实现了使用研钵和研杵的分离的破骨细胞，其类似于由Weischenfeldt 等人所述的巨噬细胞分离的方法。15 我们的经验，但是，是使用在破骨细胞的生产方面先前公布的技术的结果变量的结果即破骨细胞的分离和体外培养，常常导致在无法培养的破骨细胞。因此，我们设计了一个协议，该协议允许对小鼠骨髓的一贯隔离，以产生大量的体外多核破骨细胞，具有细胞最初镀形成的巨噬细胞和破骨细胞随后的70-80％的产率约，在存在破骨细胞诱导培养基。

<table border="0" cellpadding="0" cellspacing="0" width="1017">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:503px;">Name of the Material/Equipment</td>
			<td style="width:165px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM, no glutamine, no phenol red</td>
			<td>Gibco</td>
			<td>51200-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-CSF, recombinant mouse</td>
			<td>Gibco</td>
			<td>PMC2044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Mouse TRANCE/RANK L/TNFSF11 (E. coli expressed)</td>
			<td>R&#38;D Systems</td>
			<td>462-TEC-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prostaglandin E2</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque-1077</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid Phosphatase, Lekocyte (TRAP) kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Osteoassay bone resorption plates, 24 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3987</td>
			<td></td>
		</tr>
	</tbody>
</table>

osteoclast derivation from mouse bone marrow

<html>破骨细胞是从骨髓中的单核细胞/巨噬细胞系衍生的高度特化的细胞。其重吸收两者的有机和无机骨基质的独特能力意味着它们在调节骨骼重塑中发挥关键作用。在一起，成骨细胞和破骨细胞是负责动态耦合的过程，涉及既骨吸收和骨形成的共同行动，以维持健康和疾病过程中的正常骨架。 如在体内主要骨吸收细胞，改变破骨细胞的分化或功能可能导致在体内产生深远的影响。具有改变破骨细胞功能有关的疾病的范围可以在严重程度从致命新生儿疾病由于不能形成对造血骨髓的空间，更通常观察到的疾病，如骨质疏松症，其中过度的破骨细胞骨吸收易患断裂形成。 ENT“&gt;一个孤立的破骨细胞的数量高在体外的能力也允许在骨重建周期的理解显著的进展，并铺平了道路，以应对这些疾病的新的治疗策略的发现。 在这里，我们描述了一个协议，分离培养小鼠骨髓，将产生大量的破骨细胞的破骨细胞。

<html>该方法的目的是要在一个星期容易分离大量破骨细胞的体外培养 ，典型。大量破骨细胞的成功分离用抗酒石酸酸性磷酸酶染色（ 图1A）中得到证实。大破骨细胞被可视化为紫色大细胞细胞核多个（通常≥3核）。使用该协议，它是常见的，分离的破骨细胞与破骨细胞每多达30细胞核（ 图1B）。 使用矿化的表面被认为是评估在体外破骨...

Watch this Scientific Journal Video about Osteoclast Derivation from Mouse Bone Marrow at JoVE.com

Osteoclast Derivation from Mouse Bone Marrow

破骨细胞是体内的主要骨吸收细胞。一个分离的破骨细胞中大量的能力已经导致破骨细胞生物学的理解显著进步。在这个协议中，我们描述了一种用于分离，培养和定量破骨细胞的体外活性。

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 ...

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从小鼠胚胎干细胞的造血干细胞的推导

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文化髓系树突状细胞从骨髓前体

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主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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归巢的骨髓造血细胞

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小鼠胚胎生殖细胞的分离和推导

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诱导多能干细胞来源于小鼠的生成

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

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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) ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

大鼠骨髓破骨衣原体的分离、纯化及鉴别

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

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小鼠新鲜骨髓外植体的丙酸盐形成动力学

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...