这项工作是由美国国立卫生研究院资助R01 GM087609（到HPL），由英格丽·莱和比尔舒纪念安东尼·舒（到HPL），美国国立卫生研究院资助U01 HL099776（以MTL）的礼品，在Hagey实验室的资助部分支持小儿再生医学和橡树基金会（至MTL，GCG和HPL）。 GGW是由斯坦福大学医学院，斯坦福大学医学院的科学家培训项目的支持，并NIGMS培训资助GM07365。 ZNM是由整形外科基金会研究奖学金资助和Hagey家族基金的支持。 MSH是由美国加州再生医学研究所（CIRM）临床研究员的培训补助TG2-01159，颌面外科医生协会（ASMS）/颌面外科基金会（MSF）研究基金奖，并在移植与组织工程研究金支持。

该协议遵循经对实验动物的斯坦福大学行政专家组的方法。 1.消化鼠真皮<ol><li>安乐死小鼠麻醉后颈椎脱位与腹膜内注射氯胺酮100mg / kg的甲苯噻嗪+ 20mg / kg的+乙酰丙嗪3毫克/公斤。 注意:各种年龄和背景都可以使用。 </li><li>刮脸和脱毛的背部皮肤。大约100,000细胞可以从一块背部皮肤的60毫米×100毫米的分离。 </li><li>淹没鼠标在70％的乙醇和地点上的无尘，无菌的表面干燥。 </li><li>立即用无菌解剖剪刀收获背小鼠皮肤。在雌性小鼠，避免包括乳腺组织。 </li><li>开始尾根部，使用镊子帐篷向上皮肤和沿超筋膜平面解剖之前进行横向切割。 </li><li>小心避免，同时包括任何收获皮下脂肪皮。检查皮肤收获任何皮下脂肪，仔细用解剖刀的钝边刮它。 </li><li>冲洗在优碘收获的皮肤后跟五倍PBS洗涤在冰上。 注意:保持皮肤接近无菌尽可能避免污染是非常重要的。 </li><li>使用剃刀刀片和解剖在无菌培养皿剪刀直至样品是用2-3毫米件的均匀一致性剁碎皮肤。 </li><li>制备50个含20ml胶原酶IV锥形管以1毫克/毫升的DMEM的浓度。划分真皮成每管五只小鼠的基础上的管中。 </li><li>搅拌样品，在37℃剧烈而培养1小时在任一水浴或烘箱。 </li><li>从培养箱中取出样品，并通过10ml的无针注射器3-5x在无菌罩。 </li><li>将样品放回培养箱中在37℃，剧烈摇晃另外30分钟。 </li><li>在无菌罩，使用10毫升吸管吸取样品上下3-5x。通过一个100微米的过滤器将样品吸取到一个新的50ml锥形管中。 </li><li>通过将20ml的10％FBS的DMEM通过相同的过滤器，以最大限度地提高细胞产量和使总体积至40ml。离心机以300g于4℃下8分钟。 </li><li>用无菌玻璃吸管时，注意先删除之前，剩余的上清液上层脂肪层去除上清。 注意:该步骤是关键的，以减少脂肪细胞污染。 </li><li>重悬粒料在20毫升10％FBS的DMEM。 </li><li>通过一个70微米的过滤器通过细胞/ DMEM悬液。 </li><li>冲洗过滤器，用10毫升10％FBS的DMEM和离心机的过滤悬浮液以300g于4℃下8分钟。 </li><li>用无菌玻璃吸管，再次小心地第一除去任何剩余的脂肪层除去上清液。 </li><li>如果有显著红细胞污染（颗粒是明显红），重新悬浮小球在20毫升的ACK裂解缓冲液孵育5分钟，在室温。否则，跳到步骤24。 </li><li>加的FACS缓冲液（PBS，10％胎牛血清，0.1％叠氮化钠）等体积（20毫升）中，然后混合，并保持5毫升一份留作未染色对照。离心剩余的样品以300g于4℃下8分钟。 </li><li>除去上清液，把沉淀在冰上。细胞可以被冷冻向下此时如果FACS时间不可用。 </li></ol>通过流式细胞仪2.隔离成纤维细胞<ol><li>使500微升血统抗体孵育组合的每个颗粒。 ，CD45（1:200），Tie2的（1:50），泰尔:通过第一加法475微升FACS缓冲液含有DNA酶（10微克/毫升）至管，然后加入荧光团共轭的CD31（100 1）这样做-119（1:200），和EpCAM的（1:100）的抗体，以实现各稀释为每个抗体。 </li><li>重悬在500μl的谱系抗体孵育混合物每个颗粒孵育该悬浮液在冰上20分钟。 </li><li>添加5毫升FACS缓冲液含有DNA酶（10微克/毫升）的样品并轻轻混匀。离心机以300g于4℃下8分钟。 </li><li>去除上清，洗涤细胞沉淀用5ml FACS缓冲液含有DNA酶（10微克/毫升），并使用相同的条件离心机如在步骤26。 </li><li>重悬在含有DNA酶（10微克/毫升）加入500μlFACS缓冲液中的颗粒和放下作为生存力染料控制一个50微升等分试样。 </li><li>选择的生存力染料添加到剩余的样品中为所选择的染料指示的浓度。 </li><li>进行FACS分析31和用于存活力染料- / CD31 - / CD45 - / Tie2- /泰尔-119- / EpCAM-细胞（参见图2A）排序。排序直接进入流式细胞仪缓冲。 </li></ol>

<ol>
	<li>Sorrell, J. M., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts-a+diverse+population+at+the+center+of+it+all.">Fibroblasts-a diverse population at the center of it all.</a> International review of cell and molecular biology. 276, 161-214 (2009).</li><li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+fibrosis.">Cellular and molecular mechanisms of fibrosis.</a> The Journal of pathology. 214, 199-210 (2008).</li><li>Powell, D. W., 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=Myofibroblasts.+I.+Paracrine+cells+important+in+health+and+disease.">Myofibroblasts. I. Paracrine cells important in health and disease.</a> The American journal of physiology. 277, C1-C9 (1999).</li><li>Wilson, M. S., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+fibrosis:+pathogenesis,+etiology+and+regulation.">Pulmonary fibrosis: pathogenesis, etiology and regulation.</a> Mucosal immunology. 2, 103-121 (2009).</li><li>Hinz, 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=The+myofibroblast:+one+function,+multiple+origins.">The myofibroblast: one function, multiple origins.</a> The American journal of pathology. 170, 1807-1816 (2007).</li><li>Li, B., Wang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+and+myofibroblasts+in+wound+healing:+force+generation+and+measurement.">Fibroblasts and myofibroblasts in wound healing: force generation and measurement.</a> J Tissue Viability. 20, 108-120 (2011).</li><li>Wong, J. W., 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=Wound+healing+in+oral+mucosa+results+in+reduced+scar+formation+as+compared+with+skin:+evidence+from+the+red+Duroc+pig+model+and+humans.">Wound healing in oral mucosa results in reduced scar formation as compared with skin: evidence from the red Duroc pig model and humans.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 17, 717-729 (2009).</li><li>Szpaderska, A. M., Zuckerman, J. D., 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=Differential+injury+responses+in+oral+mucosal+and+cutaneous+wounds.">Differential injury responses in oral mucosal and cutaneous wounds.</a> Journal of dental research. 82, 621-626 (2003).</li><li>Hantash, B. M., Zhao, L., Knowles, J. A., Lorenz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+and+fetal+wound+healing.">Adult and fetal wound healing.</a> Frontiers in bioscience : a journal and virtual library. 13, 51-61 (2008).</li><li>Buchanan, E. P., Longaker, M. T., Lorenz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+skin+wound+healing.">Fetal skin wound healing.</a> Advances in clinical chemistry. 48, 137-161 (2009).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors:+wounds+that+do+not+heal.+Similarities+between+tumor+stroma+generation+and+wound+healing.">Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing.</a> The New England journal of medicine. 315, 1650-1659 (1986).</li><li>Dumont, N., 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=Breast+fibroblasts+modulate+early+dissemination,+tumorigenesis,+and+metastasis+through+alteration+of+extracellular+matrix+characteristics.">Breast fibroblasts modulate early dissemination, tumorigenesis, and metastasis through alteration of extracellular matrix characteristics.</a> Neoplasia. 15, 249-262 (2013).</li><li>Servais, C., Erez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+sentinel+cells+to+inflammatory+culprits:+cancer-associated+fibroblasts+in+tumour-related+inflammation.">From sentinel cells to inflammatory culprits: cancer-associated fibroblasts in tumour-related inflammation.</a> The Journal of pathology. 229, 198-207 (2013).</li><li>Orimo, A., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer:+a+novel+tumor-promoting+cell+type.">Stromal fibroblasts in cancer: a novel tumor-promoting cell type.</a> Cell cycle. 5, 1597-1601 (2006).</li><li>Li, X., 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=Targeting+the+cancer-stroma+interaction:+a+potential+approach+for+pancreatic+cancer+treatment.">Targeting the cancer-stroma interaction: a potential approach for pancreatic cancer treatment.</a> Current pharmaceutical design. 18, 2404-2415 (2012).</li><li>Sorrell, J. M., Caplan, A. I. <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:+more+than+skin+deep.">Fibroblast heterogeneity: more than skin deep.</a> Journal of cell science. 117, 667-675 (2004).</li><li>Cormack, D. 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> Ham's Histology. , 450-474 (1987).</li><li>Jahoda, C. A., Reynolds, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermal-epidermal+interactions.+Adult+follicle-derived+cell+populations+and+hair+growth.">Dermal-epidermal interactions. Adult follicle-derived cell populations and hair growth.</a> Dermatologic clinics. 14, 573-583 (1996).</li><li>Jahoda, C. A., Reynolds, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+dermal+sheath+cells:+unsung+participants+in+wound+healing.">Hair follicle dermal sheath cells: unsung participants in wound healing.</a> Lancet. 358, 1445-1448 (2001).</li><li>Sorrell, J. M., Baber, M. A., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+bilayered+dermal+equivalent+containing+human+papillary+and+reticular+dermal+fibroblasts:+use+of+fluorescent+vital+dyes.">Construction of a bilayered dermal equivalent containing human papillary and reticular dermal fibroblasts: use of fluorescent vital dyes.</a> Tissue engineering. 2, 39-49 (1996).</li><li>Harper, R. A., Grove, G. <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+fibroblasts+derived+from+papillary+and+reticular+dermis:+differences+in+growth+potential+in+vitro.">Human skin fibroblasts derived from papillary and reticular dermis: differences in growth potential in vitro.</a> Science. 204, 526-527 (1979).</li><li>Schafer, I. A., Pandy, M., Ferguson, R., Davis, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+observation+of+fibroblasts+derived+from+the+papillary+and+reticular+dermis+of+infants+and+adults:+growth+kinetics,+packing+density+at+confluence+and+surface+morphology.">Comparative observation of fibroblasts derived from the papillary and reticular dermis of infants and adults: growth kinetics, packing density at confluence and surface morphology.</a> Mechanisms of ageing and development. 31, 275-293 (1985).</li><li>Sorrell, J. M., Baber, M. A., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-matched+papillary+and+reticular+human+dermal+fibroblasts+differ+in+their+release+of+specific+growth+factors/cytokines+and+in+their+interaction+with+keratinocytes.">Site-matched papillary and reticular human dermal fibroblasts differ in their release of specific growth factors/cytokines and in their interaction with keratinocytes.</a> Journal of cellular physiology. 200, 134-145 (2004).</li><li>Sharon, Y., Alon, L., Glanz, S., Servais, C., Erez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+normal+and+cancer-associated+fibroblasts+from+fresh+tissues+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Isolation of normal and cancer-associated fibroblasts from fresh tissues by Fluorescence Activated Cell Sorting (FACS).</a> Journal of visualized experiments : JoVE. , e4425 (2013).</li><li>Walmsley, G. G., 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=Live+Fibroblast+Harvest+Reveals+Surface+Marker+Shift+in+vitro.">Live Fibroblast Harvest Reveals Surface Marker Shift in vitro.</a> Tissue engineering. Part C, Methods. , (2014).</li><li>Rinkevich, Y., Walmsley, G. G., Hu, M. S., Maan, Z. N., Newman, A. M., Drukker, M., Januszyk, M., Krampitz, G. W., Gurtner, G. C., Lorenz, H. P., Weissman, I. L., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Isolation+of+a+Dermal+Lineage+with+Intrinsic+Fibrogenic+Potential.">Identification and Isolation of a Dermal Lineage with Intrinsic Fibrogenic Potential.</a> Science. , (2015).</li><li>Seluanov, A., Vaidya, A., Gorbunova, V. <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. , (2010).</li><li>Lichti, U., Anders, J., Yuspa, 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=Isolation+and+short-term+culture+of+primary+keratinocytes,+hair+follicle+populations+and+dermal+cells+from+newborn+mice+and+keratinocytes+from+adult+mice+for+in+vitro+analysis+and+for+grafting+to+immunodeficient+mice.">Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice.</a> Nature protocols. 3, 799-810 (2008).</li><li>Klein, M., Fitzgerald, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+separation+of+intact+epidermal+sheets+from+mouse+skin.">Enzymatic separation of intact epidermal sheets from mouse skin.</a> The Journal of investigative dermatology. 39, 111-114 (1962).</li><li>Biburger, M., Trenkwald, I., Nimmerjahn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=yThree+blocks+are+not+enough+-+Blocking+of+the+murine+IgG+receptor+FcgammaRIV+is+crucial+for+proper+characterization+of+cells+by+FACS+analysis.">yThree blocks are not enough - Blocking of the murine IgG receptor FcgammaRIV is crucial for proper characterization of cells by FACS analysis.</a> European journal of immunology. , (2015).</li></ol>

The authors have nothing to disclose.

在这个手稿中描述的协议提供了通过基于FACS分选来分离成纤维细胞，在比较现有方法，这两种选择的亚群或随后的分析之前需要时间在细胞培养物的方法。从收获的皮肤所需的成纤维细胞的分选的时间是约6小时;然而，在收获用于小鼠的数目将影响这一估计。 有几个点在协议中需要特别的照顾。第一是由以下中的分离过程的消化和离心除去脂肪消化和除去上清液的上部脂质?...

成纤维细胞经常定义形态作为坚持以塑料基板梭形细胞。成纤维细胞是负责合成和重塑细胞外基质在胚胎和成年器官1中的原理的细胞类型。成纤维细胞因此至关重要哺乳动物发育和细胞外环境是影响相邻存在于各组织和器官中的细胞类型的行为有助于基本上。 成纤维细胞也落后造成巨大的临床负担的医疗条件多样化的主要细胞类型。病理的成纤维细胞活性的损害正常组织功能，并且包括组织和器官的纤维化（如肺和肝），疤痕下列皮肤伤口愈合，动脉粥样硬化，全身性硬化症，和形成血管损伤后2-5粥样硬化斑块的。特别是伤口愈合，急性和慢性，涉及ðeposition既不酷似也不功能类似正常组织周围，并导致发病率显著跨不同病理状态的疤痕组织。损伤后，存在成纤维细胞的肌成纤维细胞，以过渡，然后分泌结构ECM成分，发挥对邻近的细胞类型旁分泌效果，并且通过沉积疤痕组织6恢复机械稳定性。 在皮肤组织存在于跨越发育时间伤口修复的质量解剖部位之间和显著变化。在人生的前两三个月的胎儿愈合，不留疤痕;然而，从和整个成年期的晚期，治愈人类与疤痕。位点特异性，除了年龄特异性，在伤口愈合的差异存在。在口腔中的伤口改造以最小的疤痕形成7,8-，而皮肤伤口内的瘢痕组织沉积是显著9。争议仍然存在Çoncerning环境与对伤口愈合的结果本地的成纤维细胞的固有性质的在问候年龄和位置10,11的相对影响。定在小鼠口服与皮肤真皮和早期胚胎（E15）愈合的显著差异与购买胚胎（E18）真皮，它很可能是在某些发育年龄和各种解剖部位之间在成纤维细胞的种群特性存在差异。 1986年，哈罗德·F.德沃夏克假定肿瘤伤口不愈合12。 Dvorak的结论是，肿瘤行为类似于伤口在主体和通过激活伤口愈合的主机的响应诱导其基质。许多研究以来研究了成纤维细胞，以癌13-15的进展的贡献，但如在伤口愈合中，身份并有助于皮肤CA的基质隔室的成纤维细胞的胚胎起源的情况下rcinomas还没有得到充分的界定。在回答这个问题负有鉴于最近的研究暴露肿瘤相关成纤维细胞作为抗癌治疗16潜在有效靶点医疗相关性。 识别和前瞻性隔离赋有纤维化潜力体内成纤维细胞系是实现有效地操纵其范围广泛的急性和慢性疾病损伤反应的一个重要步骤。在1987年，科马克表明两个亚群的成纤维细胞，一种驻留在乳头和一个网状真皮层17,18内内。第三个亚群被发现在毛囊19,20的毛乳头区毛囊有关。当进行培养，成长潜力，形态和生长因子/细胞因子，这些成纤维细胞亚型表现出不同型材21-24。 迄今为止，研究探讨成纤维细胞，他terogeneity在很大程度上未能充分表征体内成纤维细胞之间的发育和功能的多样性。这部分地是对培养的成纤维细胞种群的依赖和细胞培养物或阳性选择的不是由所有的成纤维细胞25表示的自表面受体的基础上的均化效果的结果。从我们的实验室最近的一项研究证实了深刻的表面标志，并在培养与未培养成纤维细胞中分离的流式细胞仪为基础的分离方法，在此手稿26呈现转录变化。 接着，我们确定了鼠背真皮内的特定的成纤维细胞谱系并确定此谱系，通过ENGRAILED-1的胚胎表达的定义，主要负责在背部皮肤结缔组织沉积。期间急性和慢性纤维化形式，包括伤口愈合，癌症间质的形成，和谱系的功能辐射诱发的纤维化27。鲜明的成纤维细胞系的表征具有治疗旨在调节纤维化的行为至关重要的意义。 而不是使用依赖在体外操作 ，以实现细胞分离28,29的现有协议，收获协议（图1）这里详述将有助于成纤维细胞更准确地捕捉和表型的行为的体内产率信息进行分析。

<table><tbody><tr><td>Surgical Forceps</td><td>Kent Scientific</td><td>INS650916</td><td></td></tr><tr><td>Micro-scissors</td><td>Kent Scientific</td><td>INS600127</td><td></td></tr><tr><td>Povidone Iodine Prep Solution</td><td>Dynarex</td><td>1415</td><td></td></tr><tr><td>Nair (depilatory cream)</td><td>Church and Dwight Co.</td><td>22600267058</td><td></td></tr><tr><td>Collagenase IV</td><td>Gibco</td><td>17104-019</td><td></td></tr><tr><td>Elastase</td><td>Abcam</td><td>ab95133</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>A14430-01</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Ammonium-Chloride-Potassium (ACK) lysing buffer</td><td>Gibco</td><td>A10492-01</td><td></td></tr><tr><td>40 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>70 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>100 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>CD31</td><td>BioLegend</td><td>102421</td><td></td></tr><tr><td>CD45</td><td>BioLegend</td><td>103125</td><td></td></tr><tr><td>Tie2</td><td>BioLegend</td><td>124005</td><td></td></tr><tr><td>Ter-119</td><td>BioLegend</td><td>116233</td><td></td></tr><tr><td>EpCAM (CD326)</td><td>eBioscience</td><td>48-5791</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D3571</td><td></td></tr><tr><td>propidium iodide (PI viability stain)</td><td>BioLegend</td><td>421301</td><td></td></tr></tbody></table>

murine dermal fibroblast isolation by facs

成纤维细胞是负责分泌细胞外基质的原则细胞类型和许多器官和组织的重要组成部分。成纤维细胞的生理和病理背后临床实体，包括的纤维化在多个器官，增生性疤痕以下烧伤，缺血心脏功能的损失，和癌症间质的形成的频谱。然而，成纤维细胞仍然是一个不良的特征类型的细胞，主要是由于其固有的异质性。成纤维细胞的分离现有方法需要的时间在细胞培养的深刻影响细胞的表型和行为。因此，许多研究调查的成纤维细胞生物学依靠在体外操作 ，不准确捕获体内成纤维细胞的行为。为了克服这个问题，我们开发了一种基于FACS的协议，用于成纤维细胞的成年小鼠的背部皮肤，不需要细胞培养物中分离，由此preservi纳克生理转录和每个细胞的蛋白质组曲线。我们的策略允许通过一个谱系负栅极排斥非间充质细胞系（林- ），而不是一个积极的选择策略，以避免预选的成纤维细胞亚群的或富集表达特异性表面标志，并尽可能具有包容性跨越这个异类细胞类型。

这种方法（图1）的有效性已经在许多方面，它可以详细检查在我们最近的出版物27中得到验证。这些措施包括分选的细胞和细胞的大量和新鲜排序细胞的单细胞转录分析的免疫细胞化学。成纤维细胞进行排序，而不是直接更准确地依赖于文化的捕捉它们在体内的表型。使用谱系阴性耗竭的方法（图2A），而不是阳性选择方法避免预选特定亚群。这种方法的值由Rinkevich 等...

Watch this Scientific Journal Video about Murine Dermal Fibroblast Isolation by FACS at JoVE.com

Murine Dermal Fibroblast Isolation by FACS

小鼠皮肤成纤维隔离用流式细胞仪

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.

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

murine-dermal-fibroblast-isolation-by-facs

Research

JoVE Journal

Developmental Biology

21.0K 次观看.  Stanford University School of Medicine. 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.

视频: Murine Dermal Fibroblast Isolation by FACS

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

科研

发育生物学

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

初级毛质素黑色素细胞和纤维细胞培养的快速生成

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

癌症研究

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

荧光活化细胞分选分离人皮肤状和网状成纤维细胞

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

遗传学

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

从未受干扰和受伤的小鼠骨骼肌中分离和培养纤维脂肪祖细胞和肌肉干细胞的FACS-分离和培养

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工程

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

生物学

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

超声波增强原成人成纤维细胞隔离

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

医学

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

人瘢痕疙瘩组织中原代真皮成纤维细胞的分离、培养和表征

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.