Name: application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts
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Description: Watch this Scientific Journal Video about Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts at JoVE.com

资金由“波捷特一个concorso基金会IRCCS Ospedale Maggiore的Policlinico的2009至2010年”，以医师和的Associazione巴比诺Nefropatico荷兰ONLUS，米兰提供

所有动物实验均根据国家和有关用于科学目的，并审查和批准米兰大学的伦理委员会动物保护欧洲现行法规执行。 1，分离成骨细胞的该方法中，用小的修改，如下通过多迪格等[14]描述的过程。 <ol><li>制备消化培养基中，加入0.1％胶原酶P和0.05％的胰蛋白酶（2.5％胰蛋白酶10X，无酚红的开始），以HBSS培养基（Hanks平衡盐溶液?...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+primary+osteocytes+express+functional+gap+junctions+in+vitro.">Isolated primary osteocytes express functional gap junctions in vitro.</a> Cell Tissue Res. 323 (2), 263-271 (2006).</li><li>Boukhechba, F., 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=Human+primary+osteocyte+differentiation+in+a+3D+culture+system.">Human primary osteocyte differentiation in a 3D culture system.</a> J Bone Miner Res. 24 (11), 1927-1935 (2009).</li><li>Krishnan, V., Dhurjati, R., Vogler, E. A., Mastro, A. 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在过去几年中的骨细胞已成为在骨中最核心的细胞。研究进展，正逐步显露出一些以前没有料到的或未经证实的骨细胞特性，这是巨大的价值，新颖的设计和更好的治疗各种骨疾病。然而，骨细胞生物学的研究已患体外模型的有限到这样的程度，成骨细胞已被用来作为替代细胞经过一段长时间的骨细胞样细胞系MLO-Y4被提供之前。最近，条件永生化细胞系是由同一组16，它可以分化成...

骨细胞，最丰富的骨细胞类型，是终末分化，高度分枝细胞深部的骨架内。成熟的骨细胞的细胞体都包含在骨陷窝，并有不同的形状;骨细胞与细长的细胞体存在于皮质骨，而圆形骨细胞更频繁的骨小梁1。树突分支从细胞体延伸，居住在狭小的通道，称为泪小管，形成一个复杂的网络，使多个联系人不仅与其他骨细胞，也可以与其他骨细胞类型，骨髓，血液和血管周细胞相关。通过包含在陷窝及小管间质液，骨细胞也最终连接到循环系统，因此，他们可以影响不仅本地而且系统性事件，反之亦然他们的行为可以通过局部和全身变化2进行调节。 该骨细胞的研究最近势头，得益于几个技术进步，如产生组织和细胞特异性的转基因动物，利用强大的显微镜技术和高通量分子筛选3,4。然而，这些细胞的知识仍然是不完整的，主要是由于足够的体外模型的相对稀缺性。实际上，骨细胞一直总是很难获得，因为在深的位置，并在培养维持和增殖为特征的这种分化的细胞类型的低水平。 沿着这些年来，已经开发了许多方法来分离主骨细胞5-7，尽管它们通常产生低细胞产量和携带，即使一些污染性的成纤维细胞将迅速长满骨细胞8的风险。由于这个原因，大多数实验在体外的工作已经进行迄今在行之有效的骨细胞细胞升国家统计局MLO-Y4 9。 额外的体外方法可提高研究这些细胞的可能性，并能改善骨细胞生物学和生理学的分析。被大量采用，这样的方法应该可以很容易地复制，并且不需要特殊的仪器或非常长的时间，以达到细胞成熟。重要的是，如果适用于原代细胞，它们将使得有可能采取的转基因动物的优势。最近，我们描述了治疗与视黄酸主要的成骨细胞和成骨细胞株MC3T3-E1的诱导快速的表型变化，导致支链的细胞骨轴承10的形态学和分子特征的均匀种群的发展。 全反式维甲酸（ATRA）是维生素A的活性代谢产物，通过结合核维甲酸受体（维甲酸受体）调节基因转录。维甲酸受体与DNA结合的异源二聚体与类视黄醇X受体（RXR的），最终导致视黄酸（RA）响应性的靶基因11的调制。全反式维甲酸已经显示调节分化几种类型细胞的分化和成熟，其中其它支链的细胞，如神经元细胞12和足细胞13。 这里所描述的方法是基于除了ATRA的初级成骨细胞。是有效的，全反式维甲酸具有对细胞接种在限定的密度准确成熟阶段加入;在我们的经验，这些条件是至关重要的，以获得表型转化的成骨细胞向骨细胞。

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			<td height="20" style="height:20px;width:283px;">Name</td>
			<td style="width:239px;">Company</td>
			<td style="width:219px;">Cat #</td>
			<td style="width:183px;">comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td>Roche Applied Science</td>
			<td>11213857001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco, Life Technologies</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco, Life Technologies</td>
			<td>14175129</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alpha-MEM</td>
			<td>Invitrogen, Life Technologies</td>
			<td>22571038</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin/Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol 2-phosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATRA</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Protect ATRA from light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Dissolve in PBS and filter before use. Work always under a chemical hood.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>32670</td>
			<td>Can be added to the secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Janus Green</td>
			<td>Sigma-Aldrich</td>
			<td>201677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>176745</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorsave</td>
			<td>Calbiochem - Merck</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #7, 0.40 x 0.5mm tips</td>
			<td>World Precision Instruments</td>
			<td>501981</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #4, 0.40 x 0.45mm tips</td>
			<td>World Precision Instruments</td>
			<td>501978</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors, straight,10cm curved</td>
			<td>World Precision Instruments</td>
			<td>14394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Scissors, 14 cm, straight, S/S</td>
			<td>World Precision Instruments</td>
			<td>501218</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture flasks</td>
			<td>Corning</td>
			<td>430168</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Well-plates</td>
			<td>Corning</td>
			<td>3335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermanox coverslips</td>
			<td>Thermo Scientific Nunc</td>
			<td>12-565-27</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microscope</td>
			<td>Zeiss Apotome</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">spectrometer</td>
			<td>Safas Xenius</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts

<html>需要进行骨细胞培养物是众所周知的骨研究者的社区;原发性骨细胞的分离是困难的，产生低细胞数量。因此，在最广泛使用的蜂窝系​​统是骨细胞样MLO-Y4细胞系。 这里描述的方法是指利用视黄酸生成支链的细胞形态学和分子骨细胞功能的均质群体。 来自小鼠颅盖骨的成骨细胞的分离后，全反式维甲酸（ATRA）加入到细胞培养基中，细胞监测在倒置显微镜下每天进行。第一形态的变化是可检测后第2天治疗和分化的一般是完整的5天，用树突，以产生细胞外基质的能力丧失，下调成骨细胞标志物和逐步发展上调骨细胞特异性分子。 内容“&gt;日小区监控是必要的，因为初级细胞的固有变异性，并且该协议可以适于与变化最小，以从不同的小鼠品系得到，并应用到转基因模型中的细胞。 该方法易于进行，且不需要特殊的仪器，它是高度可重复的，并迅速产生一个成熟的骨细胞种群中完全不存在的细胞外基质，允许使用这些细胞的无限生物学应用。

得到的结果是5〜10个独立的实验。 细胞形态 AA / GP治疗原发性细胞具有多鹅卵石样的功能，成熟的成骨细胞的特性。穿插分枝状细胞（红色箭头， 如图1A所示），可以发现，这可能代表了一些骨细胞。 全反式维甲酸治疗，细胞迅速开始显示后果，其中后2天，一般观察。 如图1B所示 ，支链的细胞需要空间来扩展他...

Watch this Scientific Journal Video about Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts at JoVE.com

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

维甲酸的应用获取从初级小鼠成骨细胞骨细胞培养

原代小鼠成骨细胞与维甲酸治疗产生分枝细胞轴承骨细胞的形态学和分子特征的同质人口。该方法克服了获得和保持主骨细胞在培养的难度，并且可以是有利的，研究从转基因模型中的细胞。

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Research

JoVE Journal

Biology

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

科研

生物学

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

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 ...

Establishing Primary Adult Fibroblast Cultures From Rodents

建立从小学成人成纤维细胞培养鼠害

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Primary Cell Cultures from Drosophila Gastrula Embryos

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

从小鼠心脏中分离主黑素细胞样细胞

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

NaÃƒÆ’Ã‚Â¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...