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 border="0" cellpadding="0" cellspacing="0" width="1000">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:369px;">Surgical Forceps</td>
			<td style="width:369px;">Kent Scientific</td>
			<td style="width:108px;">INS650916</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elastase</td>
			<td>Abcam</td>
			<td>ab95133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>A14430-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium-Chloride-Potassium (ACK) lysing buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD31</td>
			<td>BioLegend</td>
			<td>102421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD45</td>
			<td>BioLegend</td>
			<td>103125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tie2</td>
			<td>BioLegend</td>
			<td>124005</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ter-119</td>
			<td>BioLegend</td>
			<td>116233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EpCAM (CD326)</td>
			<td>eBioscience</td>
			<td>48-5791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

20.8K 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

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

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

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 lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

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 ...

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 ...

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

方法在成纤维细胞迁移的规律研究大分子复合物含MRP4，

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

分离和新型表面标记组合来标识肝祖亚群富集

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

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 ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

使用紫罗兰兴奋细胞渗透DNA结合染料分离穆林精子生成细胞

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.