Name: y-27632 enriches the yield of human melanocytes from adult skin tissues
Uploaded: 2020-07-08T13:00:00
Description: Watch this Scientific Journal Video about Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues at JoVE.com

这项工作得到了国家国家重点研究发展计划（2017YFA0104604）、国家自然科学基金总计划（81772093）、军事后勤研究项目（AWS17J005）、山东省自然科学基金重点项目（ZR2019ZD3）的支持。 6）和山东省重点研究发展计划（2019GSF108107）至X.W.广东基础与应用基础研究基金会（2019A1515110833）和山东大学基础研究基金（2019GN043）至J.M.

本协议中成人年金组织的使用已获人类研究伦理委员会（第2015120401号，日期：2015年5月12日）批准。
注：在无菌环境中执行以下所有程序，以防止细胞和培养物受到污染。
1. 准备工作
<ol><li>在含有 10 mL 磷酸盐缓冲液 （PBS） 的 15 mL 管中收集包皮手术中的新鲜成人包皮组织，并储存在 4 °C 中。 注：切除后24小时内将组织分离至下文详细操作。</li>
	<li>?...

<ol>
	<li>Costin, G. E., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skin+pigmentation:+melanocytes+modulate+skin+color+in+response+to+stress.">Human skin pigmentation: melanocytes modulate skin color in response to stress.</a> FASEB Journal. 21 (4), 976-994 (2007).</li><li>Battyani, Z., Xerri, L., Hassoun, J., Bonerandi, J. J., Grob, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosinase+gene+expression+in+human+tissues.">Tyrosinase gene expression in human tissues.</a> Pigment Cell Research. 6 (6), 400-405 (1993).</li><li>Michaloglou, C., 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=BRAFE600-associated+senescence-like+cell+cycle+arrest+of+human+naevi.">BRAFE600-associated senescence-like cell cycle arrest of human naevi.</a> Nature. 436 (7051), 720-724 (2005).</li><li>Hu, F., Staricco, R. J., Pinkus, H., Fosnaugh, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanocytes+in+tissue+culture.">Human melanocytes in tissue culture.</a> Journal of Investigative Dermatology. 28 (1), 15-32 (1957).</li><li>Eisinger, M., Marko, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+proliferation+of+normal+human+melanocytes+in+vitro+in+the+presence+of+phorbol+ester+and+cholera+toxin.">Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin.</a> Proceedings of the National Academy of Sciences of the United States of America. 79 (6), 2018-2022 (1982).</li><li>Hirobe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+keratinocyte-derived+factors+involved+in+regulating+the+proliferation+and+differentiation+of+mammalian+epidermal+melanocytes.">Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes.</a> Pigment Cell Research. 18 (1), 2-12 (2005).</li><li>Halaban, R., 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=Basic+fibroblast+growth+factor+from+human+keratinocytes+is+a+natural+mitogen+for+melanocytes.">Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes.</a> Journal of Cell Biology. 107 (4), 1611-1619 (1988).</li><li>Kunisada, 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=Keratinocyte+expression+of+transgenic+hepatocyte+growth+factor+affects+melanocyte+development,+leading+to+dermal+melanocytosis.">Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocytosis.</a> Mechanisms of Development. 94 (1-2), 67-78 (2000).</li><li>Hirobe, T., Shinpo, T., Higuchi, K., Sano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+human+melanocytes+is+regulated+by+endothelin-1+and+stem+cell+factor+in+synergy+with+cyclic+AMP+and+basic+fibroblast+growth+factor.">Life cycle of human melanocytes is regulated by endothelin-1 and stem cell factor in synergy with cyclic AMP and basic fibroblast growth factor.</a> Journal of Dermatological Science. 57 (2), 123-131 (2010).</li><li>Wen, J., Zu, T., Zhou, Q., Leng, X., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Y-27632+simplifies+the+isolation+procedure+of+human+primary+epidermal+cells+by+selectively+blocking+focal+adhesion+of+dermal+cells.">Y-27632 simplifies the isolation procedure of human primary epidermal cells by selectively blocking focal adhesion of dermal cells.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (2), 1251-1255 (2018).</li><li>Liu, Z., 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=A+simplified+and+efficient+method+to+isolate+primary+human+keratinocytes+from+adult+skin+tissue.">A simplified and efficient method to isolate primary human keratinocytes from adult skin tissue.</a> Journal of Visualized Experiments. (138), e7862 (2018).</li><li>Terunuma, A., Limgala, R. P., Park, C. J., Choudhary, I., Vogel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+procurement+of+epithelial+stem+cells+from+human+tissue+specimens+using+a+Rho-associated+protein+kinase+inhibitor+Y-27632.">Efficient procurement of epithelial stem cells from human tissue specimens using a Rho-associated protein kinase inhibitor Y-27632.</a> Tissue Engineering Part A. 16 (4), 1363-1368 (2010).</li><li>Cerqueira, M. T., Frias, A. M., Reis, R. L., Marques, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfollicular+epidermal+stem+cells:+boosting+and+rescuing+from+adult+skin.">Interfollicular epidermal stem cells: boosting and rescuing from adult skin.</a> Methods in Molecular Biology. 989, 1-9 (2013).</li><li>Zhou, 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=ROCK+inhibitor+Y-27632+increases+the+cloning+efficiency+of+limbal+stem/progenitor+cells+by+improving+their+adherence+and+ROS-scavenging+capacity.">ROCK inhibitor Y-27632 increases the cloning efficiency of limbal stem/progenitor cells by improving their adherence and ROS-scavenging capacity.</a> Tissue Engineering Part C, Methods. 19 (7), 531-537 (2013).</li><li>Yokoyama, S., 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=Pharmacologic+suppression+of+MITF+expression+via+HDAC+inhibitors+in+the+melanocyte+lineage.">Pharmacologic suppression of MITF expression via HDAC inhibitors in the melanocyte lineage.</a> Pigment Cell &amp; Melanoma Rresearch. 21 (4), 457-463 (2008).</li><li>Mi, 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=A+ROCK+inhibitor+promotes+keratinocyte+survival+and+paracrine+secretion,+enhancing+establishment+of+primary+human+melanocytes+and+melanocyte-keratinocyte+co-cultures.">A ROCK inhibitor promotes keratinocyte survival and paracrine secretion, enhancing establishment of primary human melanocytes and melanocyte-keratinocyte co-cultures.</a> Pigment Cell &amp; Melanoma Research. 33 (1), 16-19 (2020).</li></ol>

这里描述的协议是基于最近的出版物16。应注意以下关键步骤，以便采用新方法获得最佳效果。首先，成功分离表皮和真皮至关重要。使用手术刀刀片将成人的外皮组织切成 3-4 mm 宽的条，使脱毛工作更彻底、更容易地将表皮与真皮分离。其次，在分离这两层时，应避免真皮污染，因为皮肤纤维细胞也可以生长在黑色素细胞培养基中。第三，三是消化步骤应小于30分钟;否则，它会...

本研究的目标是开发一种简单有效的方案，将黑色素细胞从成人皮肤的表皮中分离，用于生物研究和临床应用。黑色素细胞位于皮肤和毛囊的基底表皮中，通过产生黑色素1，在皮肤和头发色素沉着中发挥重要作用。表皮黑色素细胞产生的皮肤色素沉着作为紫外线辐射过滤器，减少/防止DNA损伤皮肤基础细胞2。黑色素细胞在皮肤中的异常增殖是很常见的，例如在良性neve（摩尔）的形成中，其中黑色素细胞有可能转化为致癌生长，然后是细胞衰老3。
自1957年以来，人类原发性黑色素细胞的分离和随后的培养已经成为可能4，但自1982年以来，才有一种有效的方法，可以从表皮5中重现人类黑色素细胞的培养。将原发性黑色素细胞与表皮分离的传统方法涉及两步酶消化。简言之，皮肤最初被消化与脱皮，以分离表皮从真皮，然后表皮消化与三普辛产生悬浮液含有黑色素细胞和角蛋白细胞，然后可以有选择地生长在不同的介质。目前，初始培养通常需要大约3至4周的时间，黑色素细胞使用传统方法达到汇合，这很可能是由于它们的分离效率低。因此，增加成人皮肤组织最初生产的黑色素细胞对实验室研究和临床应用都很有帮助。
许多生长因子，其中多数已被证明由角蛋白细胞分泌（例如，+-MSH，ACTH，bFGF，NGF，ET-1，GM-CSF，HGF，LIF和SCF）5-8，调节哺乳动物黑色5-8素细胞的分化和增殖。在没有喂食层的情况下，缺乏这些生长因子导致黑色素细胞的增殖减少，其凋亡增加9。角蛋白细胞作为馈生细胞和黑色素细胞的共培养可导致黑色素细胞增殖加速和凋亡减少。然而，共培养方法不仅需要更多的皮肤组织来准备角蛋白细胞，这是不实际的，但也不有效工作，因为黑色素细胞所需的培养条件不偏向角蛋白细胞生长，反之亦然。
先前的研究已经报道，将Y-27632，一种 ROCK抑制剂，加入生长介质，可以提高人类原发性表皮细胞从皮肤组织10-14-的产量。因此，测试人类原发性黑色素细胞的分离是否会受益于Y-27632的存在是很有趣的。事实上，Y-27632加入接种TIVA介质15，其中含有TPA，IBMX，Na3VO4和3dbcAMP，显著提高了4原发性黑色素细胞在初始培养体16中从包皮组织分离的产量。与传统协议相比，这种新协议在提高黑色素细胞16的产量方面效率显著提高。因此，本协议在此前的研究16的基础上，详细描述了使用成人皮肤组织的新方法，以促进其在基础和应用生物学研究中的应用。

<table>
	<tbody>
		<tr>
			<td>Alexa fluor-594 donkey anti-mouse IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21203</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Alexa fluor-594 donkey anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37119</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Eppendorf</td>
			<td>30702115</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Corning incorporated</td>
			<td>431792</td>
			<td>Cell filtration</td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifuge</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifuge</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026333</td>
			<td>For cell incubating</td>
		</tr>
		<tr>
			<td>Constant Temperature Shaker</td>
			<td>Shanghai Boxun</td>
			<td>150036</td>
			<td>For water bath</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>For melanocyte isolation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>C11995500</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biological Industries</td>
			<td>04-001-1AC5</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>5E44316</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>MCE</td>
			<td>HY-15371</td>
			<td>Induce pigmentation</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Thermo Scientific</td>
			<td>11330032</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>5C42258</td>
			<td>For cell microscopic observation</td>
		</tr>
		<tr>
			<td>3-isobutyl-1-methyl xanthine (IBMX)</td>
			<td>Sigma</td>
			<td>17018</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>mouse anti-human MITF</td>
			<td>Abcam</td>
			<td>ab12039</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td>melanocytes culture medium</td>
		</tr>
		<tr>
			<td>N6,2&#39;-O-dibutyryladeno-sine 3&#39;,5&#39;-cyclic monophosphate (dbcAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>12-O-tetradecanoyl phorbol-13-acetate (TPA&#65289;</td>
			<td>Sigma</td>
			<td>79346</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>rabbit anti-human Ki-67</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution</td>
			<td>Solarbio Life Science</td>
			<td>P1020-500</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>Sorvall ST 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004380</td>
			<td>Cell centrifugation</td>
		</tr>
		<tr>
			<td>TC20TM automated cell counter</td>
			<td>Bio-Rad</td>
			<td>1450102</td>
			<td>Automatic cell counting</td>
		</tr>
		<tr>
			<td>0.05% Trypsin</td>
			<td>Life Technologies</td>
			<td>25300-062</td>
			<td>For melanocyte dissociation</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>For melanocyte isolation</td>
		</tr>
	</tbody>
</table>

y-27632 enriches the yield of human melanocytes from adult skin tissues

原发性黑色素细胞与皮肤组织的分离和培养在生物研究中非常重要，已广泛应用于临床应用。通过传统方法从皮肤组织中隔离原发性黑色素细胞通常需要大约 3 到 4 周才能充分通过。更重要的是，使用的组织通常是新生儿前皮，从成人组织中有效分离原发性黑色素细胞仍然是一个挑战。我们最近开发了一种新的黑色素细胞分离方法，将Y-27632（一种Rho激酶抑制剂）添加到初始培养基48小时。 与常规方案相比，这种新方法显著提高了黑色素细胞的产量，缩短了从前皮组织中分离黑色素细胞的时间。我们现在更详细地描述这种新方法，使用成人表皮来有效地培养原发性黑色素细胞。重要的是，我们表明，从这种新方法编写的成人组织中获得的黑色素细胞可以正常工作。这一新协议将显著有利于研究色素沉着缺陷黑色素瘤使用原发性黑色素细胞准备从容易访问的成人皮肤组织。

图 1显示了比较传统方法和新方法的示意图。新方法的组织制备和消化过程与传统方法的程序相同，唯一的区别是分离的细胞在10μM Y-27632存在的情况下在TIVA培养基10 mL中重新产生。播种两天后，用不带 Y-27632 的正常 TIVA 介质替换介质。将原发性黑色素细胞与皮肤组织分离的传统方法通常需要大约3至4周，黑色素细胞生长在足够数量通过，但更重要的是，组织通常来自新生儿?...

Watch this Scientific Journal Video about Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues at JoVE.com

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

本文报告，在TIVA培养基中加入Y-27632可以显著提高成人皮肤组织中黑色素细胞的产生。

Y-27632 丰富成人皮肤组织中人类黑色素细胞的产量

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

基于细胞的协议的一般目标是分离和培养人类黑色素细胞，改善传统方法的缺点。从新方法中，我们可以在相对较短的时间内，基于材料和设备获得更多的原发性黑色素细胞。本协议中使用的人体组织是新鲜成人皮肤组织丢弃从割礼手术在医院和处理根据指南的机构的人研究伦理委员会编号2015120401，日期2015年5月12日。这是所有必需试剂的显示。提前准备无菌工具和材料，将组织放入盘中，用 75%乙醇清洗组织 30 秒，用洗涤溶液将组织切成方便的碎片，并用手术刀片将组织刮掉以去除脂肪层。将真皮侧向下的组织压平，每毫升添加 2.5 毫克的空间溶液到皮肤组织。一夜之间在四摄氏度的温度下盖住并存放这些碎片。第二天，从真皮中剥离表皮，立即将表皮转移到另外三个盘子中，用洗涤溶液将0.05%的三辛在37摄氏度的水浴中孵育30分钟。添加中和溶液的相等体积。上下移液 10 到 15 次。通过 100 微米混搭过滤器传递溶液。在200倍g下离心5分钟，将上流液重新悬浮在10毫升的tiva介质中。加入 10 微米 Y-27632。在显微镜下标记信息，这是视野。它显示了在37摄氏度的培养箱中漂浮在培养基的培养中。此视图显示第二天单元格开始连接菜底的视图。第九天的观点显示，黑色素细胞存在明显的沉积。取出上一液洗涤细胞用PBS添加两毫米0.05%trypsin孵育细胞两分钟添加两毫升的中和溶液。在200次g下离心5分钟。用 10 毫米的 tiva 介质慢慢重新悬挂上一提液。每两天更换一次介质。这些图片显示了一个示意图的通过视图，因此在图中介绍了新方法和传统方法。组织制备和新方法消化的过程与传统方法相同。唯一的区别是，分离的脂肪细胞在她的tiva介质中接种了10分钟，存在10微米Y-27632。两天后更换介质与 tiva 介质没有补充在 Y-27632.这个面板显示了由传统方法顶排和新方法底部行准备的黑色素细胞的代表性图像，在第三天、第六天和第8天，我们在第三天、第六天和第八天调用黑色素细胞的数量，我们发现新方法使黑色素细胞的产量增加了大约五倍。八天后，我们做了打，并计算每道菜的黑色素细胞的数量。结果表明，新方法分离的黑色素细胞数量比传统方法多。Ki67站确认，用新方法分离的黑色素细胞通常可以增殖，更重要的是，我们用新方法获得了黑色素细胞的正常功能。我们从隔离区最初通过后检查了黑色素细胞的纯度。一段黑色素细胞代表MITF表达，只表示在黑色素细胞。我们发现近100%细胞对MITF呈阳性，表明在第一次通过后获得的纯黑色素细胞。测试与新方法隔离的黑色素细胞可以正常工作。我们在体外用力冷却了超过90个黑色素细胞，并观察到力冷却可以诱导黑色素细胞的色素沉着。将这些数据放在一起表明，从新方法中分离出的黑色素细胞通常会产生色素沉着。如上述皮肤所述，原发性黑色素细胞在培养中扩展，已很容易用于实验室研究和临床应用。在两天前将ID Y-27632报告为初始培养基后，可显著增加黑色素细胞的外培养。总之，我们成功地建立了一种新的简单和高效的方法，从成人组织中分离纯原发性黑色素细胞。

Research

JoVE Journal

Biology

Dissection of Organs from the Adult Zebrafish

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

科研

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

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 and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...