This work was supported by the NRF grant HUJ-CREATE - Cellular and Molecular Mechanisms of Inflammation.

小鼠饲养在无病原体条件符合机构的指导方针，直到安乐死（机构动物护理和使用委员会（IACUC）的指导方针，在新加坡国立大学和国家咨询委员会的实验动物研究（NACLAR）的指导方针）。 1.鼠标<ol><li>订购一个鼠标适当的遗传背景。这个协议是基于组织从一个C57BL / 6小鼠衍生的。 </li></ol> 2.准备完全培养基<ol><li> 10％胎牛血清（FCS），50μM2-巯基乙醇，100微米的天冬酰胺，2mM谷氨酰胺，1％青霉素，链霉素溶液:通过添加以下组件来罗斯威尔公园纪念研究所（RPMI）1640培养基准备完全培养基。 </li></ol> 3.准备酶溶液<ol><li>准备在一个15毫升的锥形底部管胶原酶D溶液。 <ol><li>称取10毫克胶原酶D.迪在4毫升ssolve胶原酶d完成网上平台。 </li></ol></li><li>准备在1.7 ml离心管链霉蛋白酶的解决方案。 <ol><li>称取10毫克链霉的。 </li><li>加入5微升的1M Tris缓冲液（pH 8.0）。加入1微升的0.5M EDTA（pH 8.0）中。 </li><li>加满494微升无菌水。 </li><li>孵育在37℃的水浴中的链霉蛋白酶溶液30分钟。 </li></ol></li></ol> 4.准备胶原酶D-链霉混合（≤2反面） 注意:请在无菌细胞培养罩随后的步骤。 <ol><li>加入250微升链霉蛋白酶溶液至4毫升胶原酶D溶液。 </li><li>通过0.2微米的针筒式过滤器的胶原酶D-链霉蛋白酶混合物到无菌的15毫升锥形底部管。 </li></ol> 5.提取成纤维细胞，从耳朵和尾巴组织<ol><li>根据相应的制度指引安乐死小鼠。</li><li>放置在细胞培养罩蒸压手术器械（剪刀和镊子）。 </li><li>加入10 mL完全培养基分为两10厘米细胞培养每个菜肴。 </li><li>切耳（〜1厘米半径）和5厘米的尾巴（从尾部的尖端）的小鼠用剪刀和在无菌50ml锥形底部管孵育5分钟在40毫升70％的乙醇。 </li><li>风干耳朵和尾巴通过将它们放置在一个开放的10个厘米细胞培养皿在引擎盖5分钟。一旦干燥，如在步骤5.3中所述转印耳和尾件两个培养皿贴上"耳朵"和含有10ml完全培养基"尾巴"。 </li><li>从耳朵和尾巴用剪刀取下头发。 </li><li>切耳朵和尾巴成片超过3毫米大小用剪刀小。 </li><li>转移切割组织进入标有"耳朵"和"尾"1.8毫升冻存管小瓶和补充足够的胶原D-链霉蛋白酶溶液的体积达到1.8 ml刻度的小瓶。 </li><li> P水平花边冻存管瓶在振荡器上，在37℃下摇晃样品以200rpm进行90分钟。 </li><li>经过90分钟孵化，请从摇床冻存管小瓶，并将小瓶在通风橱中。 </li><li>加入10 mL完全培养基分为两10厘米细胞培养皿，标有"耳朵"和"尾"每一个，并把一个70微米的细胞过滤的每一道菜。 </li><li>将被消化的耳朵和尾巴组织在70微米的细胞过滤在步骤5.11编制的相应标记菜肴和强制使用10毫升注射器推杆&gt; 5分钟研磨组织。偶尔摇动细胞过滤培养基中洗细胞出细胞滤网。 </li><li>移液器从每道菜的细胞悬液到标有"耳朵"和"尾"两个15毫升锥形底管。洗净的菜，过滤与另外10 ml完全介质和介质添加到相应的15毫升锥形底管。 </li><li>降速细胞悬浮液在580〜xg离心7分钟，并使用冷冻细胞离心机4℃。 </li><li>除去上清液，加入10 ml完全培养基将细胞沉淀在15毫升的锥形底部管和悬浮细胞。 </li><li>重复步骤5.14和5.15。 </li></ol> 6.培养细胞混合物的<ol><li>除去上清液。确保细胞沉淀未受干扰。 </li><li>重悬的细胞在10ml完全培养基和相应混合物添加到标记的"耳朵"和"尾"两个10厘米细胞培养皿。 </li><li>加10微升两性霉素B溶液（原液:250微克/毫升）的培养。 </li><li>孵育在湿润的5％CO 2培养箱细胞在37℃。 </li><li>在第三天，用含有10微升两性霉素B以除去碎片10 ml的新鲜完全培养基（ 图1和2）替代培养基。 </li></ol>成纤维细胞7次文化<ol><li> WHEN培养达到约70％汇合（3-4培养天）取出介质，并用5毫升无菌1×磷酸盐缓冲盐水（PBS）洗细胞。 </li><li>除去PBS中并加入2毫升无菌1×胰蛋白酶-EDTA溶液到细胞中。 </li><li>在湿润的5％CO 2培养箱孵育细胞5分钟，在37℃。 </li><li> 5分钟后，轻轻拍打培养皿，并添加6 ml完全培养基的细胞中。 </li><li>细胞悬液转移到15毫升锥形底部管和使用冷冻细胞离心机旋转离心管5分钟，在450〜XG和4℃。 </li><li>除去上清液，并轻轻重新悬浮在5ml完全培养基将细胞沉淀。 </li><li>种子2×10 5个细胞中有10厘米细胞培养皿并重复步骤7.1至7.6之前孵育细胞，在37℃在湿润的5％CO 2培养箱3-4天。 </li></ol> 8.准备耳朵装运注:执行牛逼他在无菌细胞培养罩后续步骤。 <ol><li>根据相应的制度指引安乐死小鼠。 </li><li>放置蒸压剪刀和镊子进入细胞培养罩。 </li><li>加入50 ml完全培养基到50毫升锥形底部管。 </li><li>切割的小鼠用剪刀耳（〜1厘米半径）。 </li><li>转移耳朵入50ml锥形底部管（如制备步骤3）使用镊子。 </li><li>在适当的箱海运在室温之前密封50毫升锥形筒底部用封口膜。 </li></ol>

<ol>
	<li>Fitzpatrick, L. E., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+matrices+for+tissue+engineering+and+regenerative+medicine+applications.">Cell-derived matrices for tissue engineering and regenerative medicine applications.</a> Biomater Sci. 3 (1), 12-24 (2015).</li><li>Elenbaas, B., 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+breast+cancer+cells+generated+by+oncogenic+transformation+of+primary+epithelial+cells.">Human breast cancer cells generated by oncogenic transformation of primary epithelial cells.</a> Genes Dev. 15 (1), 50-65 (2001).</li><li>Stansley, B., Post, J., Hensley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+review+of+cell+culture+systems+for+the+study+of+microglial+biology+in+Alzheimer's+disease.">A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease.</a> J Neuroinflammation. 9 (1), 115 (2012).</li><li>Lim, J., Dobson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+transfection+of+HUVEC+and+MEF+cells+using+DNA+complexes+with+magnetic+nanoparticles+in+an+oscillating+field.">Improved transfection of HUVEC and MEF cells using DNA complexes with magnetic nanoparticles in an oscillating field.</a> J Genet. 91 (2), 223-227 (2012).</li><li>Li, 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=High-efficiency+transduction+of+fibroblasts+and+mesenchymal+stem+cells+by+tyrosine-mutant+AAV2+vectors+for+their+potential+use+in+cellular+therapy.">High-efficiency transduction of fibroblasts and mesenchymal stem cells by tyrosine-mutant AAV2 vectors for their potential use in cellular therapy.</a> Hum Gene Ther. 21 (11), 1527-1543 (2010).</li><li>Patel, M., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+reprogramming+somatic+cells+to+induced+pluripotent+stem+cells.">Advances in reprogramming somatic cells to induced pluripotent stem cells.</a> Stem Cell Rev. 6 (3), 367-380 (2010).</li><li>Newman, A. C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+for+fibroblasts+in+angiogenesis:+fibroblast-derived+matrix+proteins+are+essential+for+endothelial+cell+luman+formation.">The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell luman formation.</a> Mol Biol Cell. 22 (20), 3791-3800 (2011).</li><li>Ohlund, D., Elyada, E., Tuveson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+heterogeneity+in+the+cancer+wound.">Fibroblast heterogeneity in the cancer wound.</a> J Exp Med. 211 (8), 1503-1523 (2014).</li><li>Guo, S., Dipietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+wound+healing.">Factors affecting wound healing.</a> J Dent Res. 89 (3), 219-229 (2010).</li><li>Werner, S., Krieg, T., Smola, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte-fibroblast+interactions+in+wound+healing.">Keratinocyte-fibroblast interactions in wound healing.</a> J Invest Dermatol. 127 (5), 998-1008 (2007).</li><li>Shen, Y. 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=Genome-derived+cytosolic+DNA+mediates+type+I+interferon-dependent+rejection+of+B+cell+lymphoma+cells.">Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells.</a> Cell Rep. 11 (3), 460-473 (2015).</li><li>Seluanov, A., Vaidya, A., Gorbunova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+primary+adult+fibroblast+cultures+from+rodents.">Establishing primary adult fibroblast cultures from rodents.</a> J Vis Exp. (44), (2010).</li><li>Baglole, C. 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=Isolation+and+phenotypic+characterization+of+lung+fibroblasts.">Isolation and phenotypic characterization of lung fibroblasts.</a> Methods Mol Med. 117, 115-127 (2005).</li><li>Alt, 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=Fibroblasts+share+mesenchymal+phenotypes+with+stem+cells,+but+lack+their+differentiation+and+colony-forming+potential.">Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential.</a> Biol Cell. 103 (4), 197-208 (2011).</li><li>Kuilman, T., Michaloglou, C., Mooi, W. J., Peeper, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essence+of+senescence.">The essence of senescence.</a> Genes Dev. 24 (22), 2463-2479 (2010).</li><li>Lander, M. R., Moll, B., Rowe, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+culture+of+cells+from+mouse+tail+biopsies:+brief+communication.">A procedure for culture of cells from mouse tail biopsies: brief communication.</a> J Natl Cancer Inst. 60 (2), 477-478 (1978).</li><li>Moore, C. B., Allen, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ear+fibroblast+derivation+from+mice.">Primary ear fibroblast derivation from mice.</a> Methods Mol Biol. (1031), 65-70 (2013).</li><li>Liu, 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=Generation+of+stable+pluripotent+stem+cells+from+NOD+mouse+tail-tip+fibroblasts.">Generation of stable pluripotent stem cells from NOD mouse tail-tip fibroblasts.</a> Diabetes. 60 (5), 1393-1398 (2011).</li></ol>

The authors declare no conflict of financial interests.

在这里，我们提供了一种简单，廉价且快速的实验步骤建立从耳朵和小鼠的尾巴初级成纤维细胞培养物。提取应导致粘附和迅速分裂成纤维细胞内后3天的隔离组织。原代细胞的一个重要的限制是衰老，一个永久生长停滞15。使用的协议，成纤维细胞培养可传代5-6次之前的成纤维细胞变得衰老，通过细胞的扁平化表示，尺寸增加（2-3倍增加）和故障扩大。 当执行的成纤...

主细胞来源于体外条件下活组织进行培养。它通常假设原代细胞更接近生理状态和其原始比永生化或肿瘤细胞系1的组织的遗传背景。出于这个原因，原代细胞代表用于研究生物学问题2,3的有用模型。然而，不同于无限增长建立的细胞系，原代细胞最终经历衰老中培养并需要经常重新建立。 常用的原代细胞包括成纤维细胞，上皮细胞，内皮细胞，T细胞，B细胞，骨髓衍生的巨噬细胞（BMDM）和骨髓来源的树突细胞（BMDC）。成纤维细胞通常用作原代细胞培养模型。它们提供超过其他初级细胞关键优势。细胞培养物容易建立，容易地维持和不需要purifica用前，培养细胞。他们有快速的初始扩散和专门的媒体或活化协议没有要求。成纤维细胞可以使用的生物，化学和物理协议4,5-有效地转染。有一种可能性，以存储耳朵之前建立的细胞培养物达10天室温。成纤维细胞培养有利于胞质突起的可视化和适于重新编程成诱导多能干细胞（iPS）6。 成纤维细胞是结缔组织的重要细胞分泌胶原蛋白和细胞外基质7。它们所提供的结构框架在许多组织8和播放在伤口愈合和组织修复9,10-必不可少的作用。 在这里，我们描述了一个简单而快速（&lt;4小时）协议来建立从耳朵成纤维细胞培养和小鼠11尾。该协议需要最少的鼠标经验收获组织（相对于其它协议12,13），并且可以用于建立从存储在介质在RT至10天耳朵培养物。

<table border="0" cellpadding="0" cellspacing="0" width="642">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI-1640</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SH30027.01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Calf Serum</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Asparagine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:140px;">Merck Millipore</td>
			<td style="width:119px;">107017</td>
			<td>Absolute for analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:140px;">Roche Diagnostics</td>
			<td style="width:119px;">11088866001</td>
			<td>From Clostridium histolyticum, lyophilized, non-sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pronase protease</td>
			<td style="width:140px;">Merck Millipore</td>
			<td style="width:119px;">53702</td>
			<td>From Streptomyces griseus&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris buffer (pH 8)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">1415</td>
			<td>Ultra pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5M EDTA (pH 8)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">BUF-1053</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10X Phosphate Buffered Saline (PBS)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">BUF-2040-10X4L</td>
			<td>Ultra pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA solution 10X</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">59418C-100ML</td>
			<td>0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amphotericin B</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2492-20ml</td>
			<td>250 &#956;g/ml in deionized water, sterile-filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:140px;">Aesculap</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forcep</td>
			<td style="width:140px;">Aesculap</td>
			<td style="width:119px;">AE-BD312R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2 &#956;M syringe filter</td>
			<td style="width:140px;">Sartorius Stedim</td>
			<td style="width:119px;">16534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 &#956;M cell strainer</td>
			<td style="width:140px;">SPL</td>
			<td style="width:119px;">93070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe plunger</td>
			<td style="width:140px;">Terumo</td>
			<td style="width:119px;">SS+10L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryovial tube</td>
			<td style="width:140px;">NUNC</td>
			<td style="width:119px;">368362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.7 ml microcentrifuge tube</td>
			<td style="width:140px;">Axygen</td>
			<td style="width:119px;">MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 cm cell culture dish</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">664160</td>
			<td>Cell culture treated dish&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml conical bottom tube</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml conical bottom tube</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:140px;">GFL</td>
			<td style="width:119px;">1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:140px;">Eppendorf</td>
			<td style="width:119px;">5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubation shaker</td>
			<td style="width:140px;">Sartorius Stedim</td>
			<td style="width:119px;"></td>
			<td>Certomat-BS1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axiovert 25 light microscope</td>
			<td style="width:140px;">Carl Zeiss AG</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

generating primary fibroblast cultures from mouse ear and tail tissues

原代细胞是直接从组织来源和被认为是更具有代表性的细胞在体内比建立的细胞系的生理状态。然而，原代细胞培养，通常具有有限的寿命，需要频繁重新建立。成纤维细胞是原代细胞的一个方便的来源。在这里，我们讨论一个简单而快速的实验程序，以建立从耳朵和老鼠尾巴初级成纤维细胞培养。该协议可以用于建立初级成纤维细胞培养从存储在RT至10天耳朵。当协议是经过精心接着，污染物是不太可能发生尽管使用储存延长的时间在某些情况下，非无菌的组织。成纤维细胞增殖迅速在培养和接受复制衰老之前可扩展到相当数量。

来自组织的结果的成纤维细胞的一个显著量组织碎片的提取（ 图1）。在对比组织碎片，纤维母细胞附着到1天，3培养的间组织培养塑料表面。成纤维细胞培养物的培养基可以安全地变更上培养，其中应显著减少在培养（图2）的碎片本水平的3天。的成纤维细胞显示的细长形态和一个清晰可见的细胞质（图1和2）。有丝分裂的细胞应该从培养的第3?...

Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

我们描述用于产生从耳朵和小鼠的尾部的成纤维细胞初级一个简单而快速的实验程序。该过程不需要特殊的动物训练和可用于成纤维细胞培养的产生从存储在RT至10天耳朵。

从鼠耳和尾组织生成主成纤维细胞培养

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

generating-primary-fibroblast-cultures-from-mouse-ear-and-tail-tissues

Research

JoVE Journal

Developmental Biology

33.6K 次观看.  Yong Loo Lin School of Medicine, National University of Singapore. 我们描述用于产生从耳朵和小鼠的尾部的成纤维细胞初级一个简单而快速的实验程序。该过程不需要特殊的动物训练和可用于成纤维细胞培养的产生从存储在RT至10天耳朵。 

视频: Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

科研

发育生物学

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

隔离和脂肪干细胞分化的猪从皮下脂肪组织

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

使用主神经球文化研究纤毛

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

成人皮肤角质形成细胞的生成与培养

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

小鼠颅面组织和未切除骨骼的组织制备及免疫染色

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

小鼠原发性颅神经冠细胞的解剖、培养与分析，用于神经冠细胞脱角和迁移研究

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...