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，在耳朵前面颅骨的两侧切开，去除皮肤，露出颅骨。</li>
	<li>握住鼻梁的头部，沿着λ样缝合线切开颅骨。沿着顶骨的侧边缘切割，并将其延伸至包括额部。</li>
	<li>将颅骨与下面的脑组织分开。 注意：为确保清洁的骨性颅骨和最少的软组织附着，请勿深入骨骼;作为参考，人们应该能够看到骨头下面剪刀的影子。</li>
	<li>将钙质转移到磷酸盐缓冲盐水中（PBS，整个方案中的pH 7.4）。确保凹面朝上。</li>
</ol>2.新生小鼠颅骨的分馏
<ol><li>将多达 5 个颅骨转移到含有 5 mL 2 mg/mL 胶原酶溶液的 50 mL 锥形管中。使用前准备的新鲜胶原酶溶液。<ol><li>要制备新鲜的胶原酶溶液，将胶原酶粉末加入分离缓冲液（70 mM NaCl、10 mM NaHCO 3、60 mM 山梨糖醇、3 mMK2 HPO4、1 mM CaCl 2、1 mg/mL 牛血清白蛋白 （BSA）、5 mg/mL 葡萄糖和 25 mM HEPES）中，浓度为2 mg/mL。将胶原酶粉末溶解在磁力搅拌器上。</li>
		<li>将胶原酶溶液通过0.22μm无菌过滤单元运行到50 mL管中，并在整个方案中将其保持在冰上。</li>
	</ol></li>
	<li>将Calvaria在37°C下以300rpm在摇床上孵育20分钟以获得馏分1。</li>
	<li>丢弃馏分 1 的消化物，用 5 mL 磷酸盐缓冲盐水 （PBS） 洗涤钙质并丢弃洗涤液。</li>
	<li>将钙瓦里亚在含有1mg / mL BSA的5mM乙二胺四乙酸（EDTA）中的PBS中孵育，PH 7.4在300rpm的振荡器上孵育15分钟。</li>
	<li>将消化物收集在 50 mL 锥形管中。用 5 mL PBS 洗涤颅骨，并将洗涤溶液添加到消化液中。 注意：在收集消化物和洗涤的每个点，将骨头移入其溶液中以将细胞与骨分离并防止双峰和细胞聚集。</li>
	<li>为了获得馏分2，将消化物在4°C下以300× g 离心5分钟。弃去上清液，将沉淀重悬于含有 10% 胎牛血清 （FBS）、100 IU/mL 青霉素 G 和 100 μg/mL 链霉素的 8 mL α最低必需培养基 （MEM） 中。在10厘米培养皿上播种。将馏分2在37°C下在5%CO2下孵育。</li>
	<li>将Calvaria在37°C的5mL胶原酶溶液中以300rpm在摇床上孵育20分钟。</li>
	<li>要收集分数 3，请重复步骤 2.5-6。将级分3在37°C下在5%CO2下孵育。 注意：此时，骨骼会变小，并减少到碎片。为确保不会意外吸入消化物中的骨头，从而导致在此过程中细胞丢失，请使用小型吸头移液器收集消化物（1000\u2012200 μL 移液器吸头）。</li>
	<li>将Calvaria在37°C的5mL胶原酶溶液中以300rpm在摇床上孵育20分钟。</li>
	<li>要收集分数 4，请重复步骤 2.5-6。将级分4在37°C下在5%CO2下孵育。</li>
	<li>将Calvaria在5mL含有1mg / mL BSA的5mM EDTA中的PBS（pH 7.4）在37°C下以300rpm的振荡器孵育15分钟。</li>
	<li>要收集分数 5，请重复步骤 2.5-6。将级分5在37°C下在5%CO2下孵育。骨细胞可以在同一天准备分选，也可以在分选前培养长达24小时。</li>
</ol>3. 用于荧光活化细胞分选的骨细胞制备
<ol><li>吸出每个细胞级分的培养基，并用 10 mL PBS 轻轻洗涤两次。</li>
	<li>在PBS中加入5mL的0.5%胰蛋白酶-EDTA，并将细胞在37°C孵育5分钟。 加入 5 mL 10% FBS α-MEM 并移液器以轻轻分离细胞。通过将 40 μm 细胞过滤器筛入 50 mL 锥形离心管中，将每个级分中的细胞合并。</li>
	<li>用 10 mL PBS 洗涤细胞，并通过 40 μm 细胞过滤器筛入 50 mL 锥形离心管中收集。将细胞在4°C下以300× g 离心5分钟。</li>
	<li>吸出上清液并向沉淀中加入10%FBS α-MEM。将细胞浓度调节至约 1 x 107 个细胞/mL。</li>
	<li>在35μm尼龙网盖管中过滤细胞。这些牢房现已准备好进行FACS。</li>
	<li>对于此协议，请使用100μm尺寸的喷嘴。收集液应由10%的FBS α-MEM组成。保持正在搅拌分选的样品，如果可能的话，在整个分选过程中保持在4°C。</li>
	<li>在排序之前，优化门控，通过调整侧向散射 （SSC） 区域和前向散射 （FSC） 区域来去除伪影和细胞。通过调整 SSC 宽度与 SSC 高度和 FSC 面积与 FSC 宽度来消除双峰。GFP阴性骨细胞应用作对照，以调整分选仪器的参数。</li>
	<li>细胞收集完成后，将细胞在4°C下以300× g 离心10分钟。用PBS清洗细胞。将细胞在4°C下以300× g 离心5分钟。</li>
	<li>重新悬浮细胞并使用10%FBS α-MEM根据需要调整数量。</li>
</ol>

<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>

作者没有什么可透露的。

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 ...

obtaining-primary-osteocytes-through-murine-calvarial-fractionation

Research

JoVE Journal

Biology

2.0K 次观看.  Tohoku University Graduate School of Dentistry. 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。

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

Obtaining Eggs from Xenopus laevis Females

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, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

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, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

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

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

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. 

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 inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

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

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

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

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

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

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

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 ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

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

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

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

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

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

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 the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

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 ...