Name: obtaining primary osteocytes through murine calvarial fractionation of gfp-expressing osteocytes
Uploaded: 2020-06-02T15:00:00
Description: Watch this Scientific Journal Video about Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes at JoVE.com

这项工作得到了日本科学促进会的JSPS KAKENHI资助（香港第19K10397号和I.M.第18K09862号）的部分支持。

所有动物程序和动物护理均按照东北大学的规章制度进行。
1.解剖新生dmp1-黄玉小鼠颅骨
<ol><li>对于该协议，使用 6\u20127 天龄的 C57BL/6-Tg（Dmp1-Topaz）1Ikal/J 小鼠幼崽。用5%异氟醚吸入对小鼠实施安乐死，然后将幼崽转移到70%乙醇中。</li>
	<li>将安乐死的幼犬转移到未经处理的培养皿中。</li>
	<li>用剪刀和镊子抓住头骨底部的皮肤并做一个切口。</li>
	<li>以第一个切口为起点，在...

<ol>
	<li>Manolagas, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+and+death+of+bone+cells:+basic+regulatory+mechanisms+and+implications+for+the+pathogenesis+and+treatment+of+osteoporosis.">Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis.</a> Endocrine Reviews. 21 (2), 115-137 (2000).</li><li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocytes+as+Dynamic+Multifunctional+Cells.">Osteocytes as Dynamic Multifunctional Cells.</a> Annals of the New York Academy of Sciences. 1116 (1), 281-290 (2007).</li><li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensation+and+Transduction+in+Osteocytes.">Mechanosensation and Transduction in Osteocytes.</a> BoneKEy osteovision. 3 (10), 7-15 (2006).</li><li>Feng, J. Q., 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=Loss+of+DMP1+causes+rickets+and+osteomalacia+and+identifies+a+role+for+osteocytes+in+mineral+metabolism.">Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism.</a> Nature Genetics. 38 (11), 1310-1315 (2006).</li><li>Lane, N. E., 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=Glucocorticoid-Treated+Mice+Have+Localized+Changes+in+Trabecular+Bone+Material+Properties+and+Osteocyte+Lacunar+Size+That+Are+Not+Observed+in+Placebo-Treated+or+Estrogen-Deficient+Mice.">Glucocorticoid-Treated Mice Have Localized Changes in Trabecular Bone Material Properties and Osteocyte Lacunar Size That Are Not Observed in Placebo-Treated or Estrogen-Deficient Mice.</a> Journal of Bone and Mineral Research. 21 (3), 466-476 (2005).</li><li>Dallas, S. L., Prideaux, M., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Osteocyte:+An+Endocrine+Cell+...+and+More.">The Osteocyte: An Endocrine Cell ... and More.</a> Endocrine Reviews. 34 (5), 658-690 (2013).</li><li>van der Plas, A., Nijweide, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+osteocytes.">Isolation and purification of osteocytes.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 7 (4), 389-396 (1992).</li><li>Westbroek, I., De Rooij, K. E., Nijweide, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocyte-specific+monoclonal+antibody+MAb+OB7.3+is+directed+against+Phex+protein.">Osteocyte-specific monoclonal antibody MAb OB7.3 is directed against Phex protein.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 17 (5), 845-853 (2002).</li><li>Gu, G., Nars, M., Hentunen, T. A., Metsikk&#246;, K., V&#228;&#228;n&#228;nen, H. 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 and Tissue Research. 323 (2), 263-271 (2006).</li><li>Kramer, I., 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=Osteocyte+Wnt/&#946;-Catenin+Signaling+Is+Required+for+Normal+Bone+Homeostasis.">Osteocyte Wnt/&#946;-Catenin Signaling Is Required for Normal Bone Homeostasis.</a> Molecular and Cellular Biology. 30 (12), 3071-3085 (2010).</li><li>Kato, Y., Windle, J. J., Koop, B. A., Mundy, G. R., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+Osteocyte-like+Cell+Line,+MLO-Y4.">Establishment of an Osteocyte-like Cell Line, MLO-Y4.</a> Journal of Bone and Mineral Research. 12 (12), 2014-2023 (1997).</li><li>Kato, Y., 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=Establishment+of+an+osteoid+preosteocyte-like+cell+MLO-A5+that+spontaneously+mineralizes+in+culture.">Establishment of an osteoid preosteocyte-like cell MLO-A5 that spontaneously mineralizes in culture.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 16 (9), 1622-1633 (2001).</li><li>Zhang, C., Bakker, A. D., Klein-Nulend, J., Bravenboer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+Osteocytes+in+Their+3D+Native+Matrix+Versus+2D+In+Vitro+Models.">Studies on Osteocytes in Their 3D Native Matrix Versus 2D In Vitro Models.</a> Current Osteoporosis Reports. 17 (4), 207-216 (2019).</li><li>Woo, S. M., Rosser, J., Dusevich, V., Kalajzic, I., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+line+IDG-SW3+replicates+osteoblast-to-late-osteocyte+differentiation+in+vitro+and+accelerates+bone+formation+in+vivo.">Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 26 (11), 2634-2646 (2011).</li><li>Spatz, J. M., 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=The+Wnt+Inhibitor+Sclerostin+Is+Up-regulated+by+Mechanical+Unloading+in+Osteocytes+in+Vitro.">The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro.</a> The Journal of biological chemistry. 290 (27), 16744-16758 (2015).</li><li>Gillet, J. P., Varma, S., Gottesman, M. 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+clinical+relevance+of+cancer+cell+lines.">The clinical relevance of cancer cell lines.</a> Journal of the National Cancer Institute. 105 (7), 452-458 (2013).</li><li>Sandberg, R., Ernberg, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+tumor+characteristic+gene+expression+in+cell+lines+using+a+tissue+similarity+index+(TSI).">Assessment of tumor characteristic gene expression in cell lines using a tissue similarity index (TSI).</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (6), 2052-2057 (2005).</li><li>Kalajzic, I., 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=Dentin+matrix+protein+1+expression+during+osteoblastic+differentiation,+generation+of+an+osteocyte+GFP-transgene.">Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene.</a> Bone. 35 (1), 74-82 (2004).</li><li>Paic, 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=Identification+of+differentially+expressed+genes+between+osteoblasts+and+osteocytes.">Identification of differentially expressed genes between osteoblasts and osteocytes.</a> Bone. 45 (4), 682-692 (2009).</li><li>Nakashima, 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=Evidence+for+osteocyte+regulation+of+bone+homeostasis+through+RANKL+expression.">Evidence for osteocyte regulation of bone homeostasis through RANKL expression.</a> Nature Medicine. 17 (10), 1231-1234 (2011).</li><li>Kalajzic, I., 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=In+vitro+and+in+vivo+approaches+to+study+osteocyte+biology.">In vitro and in vivo approaches to study osteocyte biology.</a> Bone. 54 (2), 296-306 (2013).</li><li>Chia, L. Y., Walsh, N. C., Martin, T. J., Sims, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+gene+expression+of+haematopoietic-cell-free+preparations+of+highly+purified+murine+osteocytes.">Isolation and gene expression of haematopoietic-cell-free preparations of highly purified murine osteocytes.</a> Bone. 72, 34-42 (2015).</li><li>Halleux, C., Kramer, I., Allard, C., Kneissel, M., Helfrich, M. H., Ralston, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bone Research Protocols. , 55-66 (2012).</li><li>Bernhardt, A., Wolf, S., Weiser, E., Vater, C., Gelinsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+to+isolate+primary+human+osteocytes+from+bone.">An improved method to isolate primary human osteocytes from bone.</a> Biomedizinische Technik (Berlin). 65 (1), 107-111 (2020).</li></ol>

第一个分离的骨细胞来自使用（OB7.3）或PHEX的禽变种分离的鸡颅骨7 ;然而，这种方法受到可行抗体可用性的限制，因为必须制造具有特异性的骨细胞特异性抗体。研究人员使用顺序酶促过程的不同修饰从小鼠和大鼠长骨中获得骨细胞;据报道，这些培养物的纯度设定为约70%9。cre小鼠模型的发展允许工程骨细胞，其在其表面表达GFP。该小鼠模型与FACS一起用于获得?...

骨细胞是来自成骨细胞祖细胞的终末分化细胞，嵌入其分泌基质1。它们是骨细胞群中最丰富和最长寿的细胞。它们位于腔隙内，具有特征性的星状形态，树突通过称为小管的通道延伸，形成与周围环境和骨表面的广泛沟通和代谢交换网络2。骨细胞在骨重塑中的作用是成骨细胞和破骨细胞，它们是赋予适应机械负荷的主要机械感觉细胞3，参与磷酸盐稳态4和骨基质矿化5，并与腔隙小管系统一起充当内分泌器官，向远处组织发出信号6。
骨细胞位于矿化基质内，这限制了可及性并使其分离具有挑战性，从而阻碍了体外研究。最早的分离方法之一描述了使用骨细胞特异性单克隆抗体（OB7.3）7从鸡颅骨中分离出骨细胞，该抗体后来被称为PHEX（PHosphate调节基因与X染色体上的内肽酶同源）的禽变体8。其他研究人员使用大鼠9或小鼠10长骨的顺序消化来获得富含骨细胞的部分，其中骨细胞纯度据报道约为70%9。该程序的局限性包括被骨细胞以外的其他细胞类型污染的培养物纯度欠佳，并且由于骨细胞已失去分裂能力，骨细胞可能被培养物中的其他细胞过度生长。这些挑战限制了长期文化的可用性。
为了克服这些限制，开发了不同的骨细胞系。MLO-Y4细胞系11和MLO-A5细胞系12是研究最广泛的细胞系，可用于研究早期骨细胞;然而，它们对于研究成熟骨细胞信号传导不太有用，因为它们表达低水平的硬化素和FGF2313，两者都是成熟的骨细胞标志物。其他细胞系包括IDG-SW3 14和Ocy45415，表达高水平的硬化素和FGF23，可用于研究晚期骨细胞阶段。细胞系被证明是有用的研究工具;然而，它们并非没有限制，因为它们不能完全代表原代细胞的生物学。不同的细胞系代表骨细胞成熟谱的不同发育阶段，细胞系不能代表原代骨细胞的异质性16,17。
为了获得原代骨细胞的纯培养物，研究人员利用了CRE小鼠模型，其中8kb dmp1启动子用于驱动骨细胞中的绿色荧光蛋白（GFP）表达18,19。Paic等人的双转基因小鼠（pOB-Col 2.3-GFP-青色和DMP1-GFP-托帕石）19和Nakashima等人的dmp1-黄玉转基因小鼠已被用于获得骨细胞群。其中他们采用表达GFP的骨细胞的顺序消化和FACS来获得原代骨细胞的培养物19,20。激活tdTomato蛋白的10 kb dmp1报告小鼠Ai9中的cre方向被证明存在于骨髓内的骨细胞，成骨细胞，肌肉和细胞中。8 kb dmp1启动子具有相同的表达模式，但只有一部分成骨细胞和骨髓细胞表达该蛋白，这表明8 kb dmp1启动子更具特异性21。尽管如此，应谨慎解释使用8 kb dmp1启动子获得的结果，并且应常规使用骨细胞与成骨细胞特异性标记进行基因表达谱，以确保获得的群体具有足够高的纯度。
破骨细胞标志物OSCAR和Dcstamp在造血未耗尽与缺氧骨细胞群体中发现，这一发现使作者得出结论，从8 kb dmp1-黄玉新生儿颅骨分馏和GFP分选中获得的消化物被造血细胞污染。造血细胞的污染可以通过收紧GFP分选门来减轻，因为GFP阳性造血细胞的GFP强度比GFP阳性间充质细胞（骨细胞）低22。
体外研究骨细胞的方法为最近关于骨细胞生物学的丰富信息做出了贡献。然而，骨细胞分离仍然是一个劳动密集型和漫长的过程，细胞产量低。所描述的使用胶原酶和EDTA的骨消化方法通常高达823级分，需要几个小时，其中骨细胞活力被征税。研究人员报告使用级分（2\u20125）进行细胞分选20，并表明与骨细胞相关的基因与成骨细胞相关的基因的表达谱证实了分离纯骨细胞群的成功20。在本文中，我们描述了获得分数（2\u20125）的过程，并比较了从分数1到8开始的每个分数的骨细胞产量，以确定每个分数中骨细胞的返回。我们还描述了使用胶原酶和乙二胺四乙酸（EDTA）解剖新生dmp1-topaz小鼠颅骨和颅骨消化，以及制备FACS细胞。

<table>
	<tbody>
		<tr>
			<td>BD FACSDiva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Data aquisition and analysis</td>
		</tr>
		<tr>
			<td>BD Falcon Tube</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>12 x 75 mm Tube with Cell Strainer Cap, 35 &#956;m nylon mesh.</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Wako, Osaka, Japan</td>
			<td>034-22363</td>
			<td>0.2% (w/v), crude collagenase mix sourced from C. histolyticum.</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Dojindo, Kumamoto, Japan</td>
			<td></td>
			<td>5mM EDTA prepared with 0.1% BSA</td>
		</tr>
		<tr>
			<td>FACSAriaTM II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biowest, Nuaill&#233;, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolation buffer</td>
			<td></td>
			<td></td>
			<td>70mM NaCl, 10mM NaHCO, 60mM sorbitol, 3mM K2HPO4, 1mM CaCl2, 0.1% (w/v) BSA, 0.5% (w/v) glucose and 25 mM HEPES</td>
		</tr>
		<tr>
			<td>Millex Sterile Filter Unit</td>
			<td>Merck Millipore, Ireland</td>
			<td>SLGV033RS</td>
			<td>0.22&#956;m</td>
		</tr>
		<tr>
			<td>Nylon cell strainer</td>
			<td>FALCON, NY, USA</td>
			<td></td>
			<td>40&#956;m</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies, NY, USA</td>
			<td></td>
			<td>0.5% x10. diluted to x1 in PBS</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Wako, Osaka, Japan</td>
			<td></td>
			<td>Containing 10% fetal bovine serum, 100 IU/ml penicillin G, and 100 &#956;g/ml streptomycin</td>
		</tr>
	</tbody>
</table>

obtaining primary osteocytes through murine calvarial fractionation of gfp-expressing osteocytes

骨细胞曾经被认为是骨骼的被动驻留，具有感知机械负荷的后台功能，现在被带到聚光灯下，并已被证明具有多种主要功能，例如主动修改细胞外基质并与腔隙小管系统形成内分泌器官，该系统包围它向远处发送信息。由于可以从分离原代骨细胞到骨细胞样细胞系的体外测试骨细胞的方法，骨细胞现在正经历着巨大的兴趣和对结构和功能的知识激增。骨细胞生物学的许多方面以及与其他分子成分的相互作用尚未被发现。在该协议中，我们详细描述了从dmp1-topaz新生小鼠颅骨中有效分离原代骨细胞，其通过细胞分离并随后通过FACS获得原代骨细胞的培养物在骨细胞中表达绿色荧光蛋白。

该协议的目的是通过分离过程从dmp1-topaz新生小鼠颅骨中获得原代骨细胞培养物的过程，该过程使用胶原酶降解胶原基质和EDTA进行钙螯合，之后准备用于FACS的细胞以将骨细胞与其他细胞群分离。
从新生小鼠颅骨获得原代骨细胞的方法通常描述使用馏分（1\u20128）进行分类23。为了测试该方法的效率，我们比较了从组分1到8开始的从dmp1-topaz小鼠颅骨中获得的骨?...

Watch this Scientific Journal Video about Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes at JoVE.com

Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

该协议描述了新生儿dmp1-黄玉小鼠颅骨的解剖和通过细胞消化和分级分离表达绿色荧光蛋白的骨细胞，以及用于荧光活化细胞分选（FACS）的骨细胞制备。

通过表达GFP的骨细胞的小鼠颅骨分割获得原代骨细胞

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the ...

Primary osteocyte isolation is a lengthy procedure that often leaves out in low cell areas. This protocol provides efficient methods for isolating high numbers of primary GFP-expressing osteocytes. This protocol reduces the handling time and allows a higher number of osteocytes to be obtained in a single procedure with minimal contamination from hematopoietic or mesenchymal cells.For the isolation of GFP-expressing osteocytes, place euthanized, postnatal day six to seven, transgenic mouse pups into a non-treated culture dish, and use scissors and tweezers to grasp the skin at the base of one's skull. Use the scissors to make an initial incision in the lifted skin, followed by cuts along both sides of the skull in front of the ears. Remove the skin to expose the calvaria and holding the head from the nasal bridge, cut the calvaria along the lamboid suture and along the edges of the parietal bones to the frontal part of the tissue.Separate the calvaria from the underlying brain tissue and place the bones concave side up in PBS. To obtain fraction one, pool up to five calvaria per 50-milliliter tube in five milliliters of freshly prepared two-milligram per milliliter collagenase, and incubate the tissues at 37 degrees Celsius and 300 revolutions per minute for 20 minutes. At the end of the incubation, discard the digest from fraction one and wash the calvaria with five milliliters of PBS.Replace the wash with five milliliters of five-millimolar EDTA supplemented with one milligram per milliliter of bovine serum albumin in PBS for 15 minutes at 37 degrees Celsius with shaking. At the end of the incubation, transfer the digest to a 50-milliliter conical tube and wash the calveria with five milliliters of PBS. Then transfer the wash solution to the digest.To obtain fraction two, place the digest for centrifugation and discard the supernatant. Re-suspend the pellet in eight milliliters of alpha minimum essential medium containing 10%fetal bovine serum, or FBS, and antibiotics, and seed the cells onto a 10-centimeter culture dish. Then place the fraction two cells into the cell culture incubator.After collecting and culturing fractions three and four as just demonstrated. Incubate the calveria in five milliliters of five-millimolar EDTA supplemented with one milligram per milliliter of bovine serum albumin in PBS for 15 minutes at 37 degrees Celsius with shaking. At the end of the incubation, collect and culture fraction five as demonstrated for fractions two, three, and four.Immediately after fractionation, collect the cells for sorting. Alternatively, collect the cells after 24 hours of culture by aspirating the medium and gently wash the cells two times with 10 milliliters of PBS per dish per wash. After the second wash, treat the cells with five milliliters of 0.5%tripsin EDTA in PBS per plate.After five minutes at 37 degrees Celsius, add five milliliters of complete alpha minimum essential medium to each plate and gently detach the cells with pipetting. To combine the cells in each fraction, filter the cell suspensions through a 40-micron cell strainer into a single 50-milliliter conical centrifuge tube. After washing the cells with 10 milliliters of PBS, add them to the tube.Filter the cells through a strainer and collect the cells by centrifugation. Once the cells are pelleted down, re-suspend the pellet in complete minimal essential medium to a one times 10 to the seventh cells per milliliter of medium concentration and filter the cells through a 35-micron nylon mesh capped tube. To test the efficiency of this method, fractions were separately sorted and cultured for 24 hours to determine the percentage and yield of osteocytes from each fraction.The density of the osteocytes in fractions two to five were higher than that observed for fraction one and decreased remarkably in fractions six, seven, and eight. Although the percentage of osteocytes obtained between all of the fractions is not statistically significant, the density of the osteocytes differs dramatically between fractions. Here the gating strategy for isolating GFP-positive osteocytes from their GFP-negative counterparts by fluorescence-activated cell sorting is shown.After sorting, osteocytes can be analyzed for the expression of osteocyte markers of interest. In this analysis, the osteocyte marker genes were expressed at higher levels in the isolated GFP-positive osteocytes compared to presort fraction two, which is known as a high osteoblast fraction. Here the morphology of a GFP-positive osteocyte retaining a stellate shape with dendrites extending from the cell body after 24 hours of culture on a plastic culture dish, post sort, can be observed.Pay attention when aspirating the digests as the calveria will be reduced to minute pellets. Remember to use a small tip aspirator to avoid cell loss. Osteocytes have been enigmatic because they are entombed in bone.This technique has paved the way to studying osteocyte molecular interactions and their control over osteoclasts and osteoblasts.

Research

JoVE Journal

Biology

Obtaining Eggs from Xenopus laevis Females

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

科研

生物学

Preparation and Fractionation of Xenopus laevis Egg Extracts

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

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 ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

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

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

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

原代培养小鼠肠嗜铬细胞的全细胞电生理

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

来自肠道密码的穆林原结肠上皮单层的生成

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

揭开炎症性关节炎的面纱：探索滑膜细胞的功能

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

作者聚焦：从小鼠关节炎组织中分离和培养原代滑膜巨噬细胞和成纤维细胞  

小鼠关节炎组织中原代滑膜巨噬细胞和成纤维细胞的分离和培养

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...