Name: facs-isolation and culture of fibro-adipogenic progenitors and muscle stem cells from unperturbed and injured mouse skeletal muscle
Uploaded: 2022-06-08T14:30:00
Description: Watch this Scientific Journal Video about FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle at JoVE.com

我们要感谢Tom Cheung（香港科技大学）关于MuSC隔离的建议。这项工作由NIAMS-IRP通过NIH拨款AR041126和AR041164资助。

进行的所有动物实验均按照国家关节炎，肌肉骨骼和皮肤病研究所（NIAMS）动物护理和使用委员会（ACUC）批准的机构指南进行。执行此协议的调查人员必须遵守当地的动物伦理准则。
注意：该协议详细描述了如何从成年雄性和雌性小鼠（3-6个月）的后肢和受伤的胫骨前（TA）肌肉中分离FAP和MuSC，并提供共培养FAP和MuSC的指南。实验程序的概述如图 1所示?...

<ol>
	<li>Baskin, K. K., Winders, B. R., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+as+a+&amp;#34;mediator&amp;#34;+of+systemic+metabolism.">Muscle as a &amp;#34;mediator&amp;#34; of systemic metabolism.</a> Cell Metabolism. 21 (2), 237-248 (2015).</li><li>Dumont, N. A., Bentzinger, C. F., Sincennes, M. C., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells+and+skeletal+muscle+regeneration.">Satellite cells and skeletal muscle regeneration.</a> Comprehensive Physiology. 5 (3), 1027-1059 (2015).</li><li>Relaix, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+skeletal+muscle+stem+cells.">Perspectives on skeletal muscle stem cells.</a> Nature Communications. 12 (1), 692 (2021).</li><li>Pawlikowski, B., Pulliam, C. J., Betta, N. D., Kardon, G., Olwin, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+satellite+cell+contribution+to+uninjured+adult+muscle+fibers.">Pervasive satellite cell contribution to uninjured adult muscle fibers.</a> Skeletal Muscle. 5, 42 (2015).</li><li>Joe, A. W. 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=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nature Cell Biology. 12 (2), 153-163 (2010).</li><li>Wosczyna, M. 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=Mesenchymal+stromal+cells+are+required+for+regeneration+and+homeostatic+maintenance+of+skeletal+muscle.">Mesenchymal stromal cells are required for regeneration and homeostatic maintenance of skeletal muscle.</a> Cell Reports. 27 (7), 2029-2035 (2019).</li><li>Theret, M., Rossi, F. M. V., Contreras, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+roles+of+muscle-resident+fibro-adipogenic+progenitors+in+health,+regeneration,+neuromuscular+disorders,+and+aging.">Evolving roles of muscle-resident fibro-adipogenic progenitors in health, regeneration, neuromuscular disorders, and aging.</a> Frontiers in Physiology. 12, 673404 (2021).</li><li>Uezumi, A., Fukada, S. I., Yamamoto, N., Takeda, S., Tsuchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+progenitors+distinct+from+satellite+cells+contribute+to+ectopic+fat+cell+formation+in+skeletal+muscle.">Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle.</a> Nature Cell Biology. 12 (2), 143-152 (2010).</li><li>Wosczyna, M. N., Biswas, A. A., Cogswell, C. A., Goldhamer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+progenitors+resident+in+the+skeletal+muscle+interstitium+exhibit+robust+BMP-dependent+osteogenic+activity+and+mediate+heterotopic+ossification.">Multipotent progenitors resident in the skeletal muscle interstitium exhibit robust BMP-dependent osteogenic activity and mediate heterotopic ossification.</a> Journal of Bone and Mineral Research. 27 (5), 1004-1017 (2012).</li><li>Eisner, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+tissue-resident+PDGFR&#945;++fibro-adipogenic+progenitors+spontaneously+acquire+osteogenic+phenotype+in+an+altered+inflammatory+environment.">Murine tissue-resident PDGFR&#945;+ fibro-adipogenic progenitors spontaneously acquire osteogenic phenotype in an altered inflammatory environment.</a> Journal of Bone and Mineral Research. 35 (8), 1525-1534 (2020).</li><li>Biferali, B., Proietti, D., Mozzetta, C., Madaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro-adipogenic+progenitors+cross-talk+in+skeletal+muscle:+The+social+network.">Fibro-adipogenic progenitors cross-talk in skeletal muscle: The social network.</a> Frontiers in Physiology. 10, 1074 (2019).</li><li>Lemos, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nilotinib+reduces+muscle+fibrosis+in+chronic+muscle+injury+by+promoting+TNF-mediated+apoptosis+of+fibro/adipogenic+progenitors.">Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors.</a> Nature Medicine. 21 (7), 786-794 (2015).</li><li>Malecova, 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=Dynamics+of+cellular+states+of+fibro-adipogenic+progenitors+during+myogenesis+and+muscular+dystrophy.">Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy.</a> Nature Communications. 9, 3670 (2018).</li><li>Liu, L., Cheung, T. H., Charville, G. W., Rando, 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=Isolation+of+skeletal+muscle+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.</a> Nature Protocols. 10 (10), 1612-1624 (2015).</li><li>Maesner, C. C., Almada, A. E., Wagers, 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=Established+cell+surface+markers+efficiently+isolate+highly+overlapping+populations+of+skeletal+muscle+satellite+cells+by+fluorescence-activated+cell+sorting.">Established cell surface markers efficiently isolate highly overlapping populations of skeletal muscle satellite cells by fluorescence-activated cell sorting.</a> Skeletal Muscle. 8, 6-35 (2016).</li><li>Low, M., Eisner, C., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors+(FAPs):+Isolation+by+FACS+and+culture.">Fibro/adipogenic progenitors (FAPs): Isolation by FACS and culture.</a> Methods and Protocols. 1556, 179-189 (2017).</li><li>Judson, R. N., Low, M., Eisner, C., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+differentiation+of+fibro/adipogenic+progenitors+(FAPs)+from+skeletal+muscle.">Isolation, culture, and differentiation of fibro/adipogenic progenitors (FAPs) from skeletal muscle.</a> Methods in Molecular Biology. 1668, 93-103 (2017).</li><li>Hamilton, T. G., Klinghoffer, R. A., Corrin, P. D., Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+divergence+of+platelet-derived+growth+factor+alpha+receptor+signaling+mechanisms.">Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.</a> Molecular and Cellular Biology. 23 (11), 4013-4025 (2003).</li><li>Contreras, O., 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=Cross-talk+between+TGF-&#946;+and+PDGFR&#945;+signaling+pathways+regulates+the+fate+of+stromal+fibro-adipogenic+progenitors.">Cross-talk between TGF-&#946; and PDGFR&#945; signaling pathways regulates the fate of stromal fibro-adipogenic progenitors.</a> Journal of Cell Science. 132 (19), (2019).</li><li>Holmes, C., Stanford, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+Stem+cell+antigen-1:+Expression,+function,+and+enigma.">Concise review: Stem cell antigen-1: Expression, function, and enigma.</a> Stem Cells. 25 (6), 1339-1347 (2007).</li><li>Rodgers, J. T., Schroeder, M. D., Chanthia, M., Rando, 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=HGFA+Is+an+injury-regulated+systemic+factor+that+induces+the+transition+of+stem+cells+into+GAlert.">HGFA Is an injury-regulated systemic factor that induces the transition of stem cells into GAlert.</a> Cell Reports. 19 (3), 479-486 (2017).</li><li>Garc&#237;a-Prat, L., 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=FoxO+maintains+a+genuine+muscle+stem-cell+quiescent+state+until+geriatric+age.">FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age.</a> Nature Cell Biology. 22 (11), 1307-1318 (2020).</li><li>Yamamoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deterioration+and+variability+of+highly+purified+collagenase+blends+used+in+clinical+islet+isolation.">Deterioration and variability of highly purified collagenase blends used in clinical islet isolation.</a> Transplantation. 84 (8), 997-1002 (2007).</li><li>Walls, P. L. L., 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=Quantifying+the+potential+for+bursting+bubbles+to+damage+suspended+cells.">Quantifying the potential for bursting bubbles to damage suspended cells.</a> Scientific Reports. 7 (1), 1-9 (2017).</li><li>Kafadar, K. A., 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=Sca-1+expression+is+required+for+efficient+remodeling+of+the+extracellular+matrix+during+skeletal+muscle+regeneration.">Sca-1 expression is required for efficient remodeling of the extracellular matrix during skeletal muscle regeneration.</a> Developmental Biology. 326 (1), 47-59 (2009).</li></ol>

建立高效且可重复的方案来鉴定和分离纯成体干细胞群是了解其功能的第一步，也是最关键的一步。分离的FAP和MuSC可用于在移植实验中进行多组学分析，作为肌肉疾病的潜在治疗方法，或者可以进行基因改造，用于干细胞治疗中的疾病建模。 
此处描述的方案为从成年小鼠后肢肌肉获得的FAP和MuSC的鉴定，分离和培养提供了标准化指南。使用基于FACS的技术分离FAP和MuSC的纯群体...

骨骼肌是人体最大的组织，占成人体重的~40%，负责维持姿势、产生运动、调节基础能量代谢和体温1。骨骼肌是一种高度动态的组织，具有适应各种刺激的非凡能力，例如机械应力、代谢变化和日常环境因素。此外，骨骼肌响应急性损伤而再生，导致其形态和功能完全恢复2。骨骼肌可塑性主要依赖于一组常驻肌肉干细胞（MuSC），也称为卫星细胞，它们位于肌纤维质膜和基底层之间2，3。在正常情况下，MuSCs以静止状态存在于肌肉壁龛中，只有少数分裂来补偿细胞更新并补充干细胞库4。作为对损伤的反应，MuSCs进入细胞周期，增殖，并有助于新肌肉纤维的形成或在自我更新过程中返回生态位2，3。除MuSCs外，骨骼肌的稳态维持和再生依赖于称为纤维脂肪祖细胞（FAPs）的肌肉驻留细胞群的支持5，6，7。FAP是嵌入肌肉结缔组织中的间充质基质细胞，能够沿着纤维化，脂肪源性，成骨性或软骨细胞系分化5，8，9，10。FAP为MuSCs提供结构支持，因为它们是肌肉干细胞生态位中细胞外基质蛋白的来源。FAP还通过分泌细胞因子和生长因子来促进骨骼肌的长期维持，这些细胞因子和生长因子为肌生成和肌肉生长提供营养支持6，11。急性肌肉损伤后，FAPs迅速增殖以产生短暂的生态位，支持再生肌肉的结构完整性，并为维持MuSCs以旁分泌方式增殖和分化提供有利的环境5。随着再生的进行，FAP通过细胞凋亡从再生肌中清除，其数量逐渐恢复到基础水平12。然而，在有利于慢性肌肉损伤的情况下，FAPs会覆盖促凋亡信号传导并积聚在肌肉壁龛中，在那里它们分化成产生胶原蛋白的成纤维细胞和脂肪细胞，导致异位脂肪浸润和纤维化瘢痕形成12，13。
由于其多能性和再生能力，FAP和MuSCs已被确定为再生医学治疗骨骼肌疾病的前瞻性靶标。因此，为了研究它们的功能和治疗潜力，建立有效且可重复的FAP和MuSC分离和培养方案非常重要。
荧光激活细胞分选（FACS）可以根据形态特征（如大小和粒度）识别不同的细胞群，并允许基于使用针对细胞表面标志物的抗体进行细胞特异性分离。在成年小鼠中，MuSCs表达血管细胞粘附分子1（VCAM-1，也称为CD106）14，15和α7-整合素15，而FAP表达血小板衍生的生长因子受体α（PDGFRα）和干细胞抗原1（Sca1或Ly6A / E）5，6，9，12，16，17.在此描述的方案中，MuSCs被鉴定为CD31-/ CD45-/Sca1-/VCAM-1 + / α7-Integrin+，而FAP被鉴定为CD31-/CD45-/Sca1+/VCAM-1-/α7-Integrin-。或者，使用PDGFRαEGFP小鼠分离FAP作为CD31-/ CD45-/PDGFRα+/VCAM-1-/α7-整合素-事件18，19。此外，我们比较了PDGFRα-GFP+细胞的荧光信号与表面标记物Sca1鉴定的细胞之间的重叠。我们的分析表明，所有表达GFP的细胞对Sca1也呈阳性，这表明任何一种方法都可以用于FAP的鉴定和分离。最后，用特异性标记抗体染色证实了每个细胞群的纯度。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL Polypropylene Round-Bottom Tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G BD Needle 1 in. single use, sterile</td>
			<td>BD Biosciences&#160;</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Alpha 7 Integrin PE (clone:R2F2) (RatIgG2b)</td>
			<td>The University of British Columbia</td>
			<td>53-0010-01</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD31 Antibody</td>
			<td>BioLegend</td>
			<td>102510</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD45 Antibody</td>
			<td>BioLegend</td>
			<td>103112</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSMelody Cell Sorter</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Luer-Lok tip control syringe, 10-mL</td>
			<td>BD Biosciences&#160;</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse CD106 Antibody</td>
			<td>BioLegend</td>
			<td>105703</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J&#160; mouse (Female and Male)</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.129S4-Pdgfratm11(EGFP)Sor/J mouse</td>
			<td>The Jackson Laboratory</td>
			<td>007669</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning BioCoat Collagen I 6-well Clear Flat Bottom TC-treated Multiwell Plate</td>
			<td>Corning</td>
			<td>356400</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning BioCoat Collagen I 12-well Clear Flat Bottom TC-treated Multiwell Plate</td>
			<td>Corning</td>
			<td>356500</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning BioCoat Collagen I 24-well Clear Flat Bottom TC-treated Multiwell Plate</td>
			<td>Corning</td>
			<td>356408</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Solution (1 mg/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>62248</td>
		</tr>
		<tr>
			<td>Disposable Aspirating Pipets, Polystyrene, Sterile</td>
			<td>VWR</td>
			<td>414004-265</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 40 &#181;m Cell Strainer, Blue, Sterile</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 60 mm TC-treated Cell Culture Dish, Sterile</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning, 15-mL</td>
			<td>VWR</td>
			<td>352196</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning, 50-mL</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Tubes, Polypropylene, Corning</td>
			<td>VWR</td>
			<td>60819-728</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Tubes, Polystyrene, with 35um Cell Strainer Cap Corning</td>
			<td>VWR</td>
			<td>21008-948</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor, Basic, Human, Recombinant (rhFGF, Basic)</td>
			<td>Promega</td>
			<td>G5071</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo 10.8.1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Collagenase, Type II, powder</td>
			<td>ThermoFisher Scientific</td>
			<td>17101015</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Dispase, powder</td>
			<td>ThermoFisher Scientific</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco DMEM, high glucose, HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>12430054</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Fetal Bovine Serum, certified, United States</td>
			<td>ThermoFisher Scientific</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Ham&#39;s F-10 Nutrient Mix</td>
			<td>ThermoFisher Scientific</td>
			<td>11550043</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Horse Serum, New Zealand origin</td>
			<td>ThermoFisher Scientific</td>
			<td>16050122</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco PBS, pH 7.4</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco PBS (10x), pH 7.4</td>
			<td>ThermoFisher Scientific</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicillin-Streptomycin-Glutamine (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG1 cross-absorbed secondary antibody, Alexa Fluor 555</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21127</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened Fine Scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen 7-AAD (7-Aminoactinomycin D)</td>
			<td>ThermoFisher Scientific</td>
			<td>A1310</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse PDGF&#160;R alpha Antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF1062</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Fisher Scientific</td>
			<td>NC9624464</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>31872</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue anti-mouse Ly-6A/E (Sca 1) Antibody</td>
			<td>BioLegend</td>
			<td>108120</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16%</td>
			<td>Fisher Scientific</td>
			<td>NCC0528893</td>
			<td></td>
		</tr>
		<tr>
			<td>Pax7 mono-clonal mouse antibody (IgG1) (supernatant)</td>
			<td>Developmental Study Hybridoma Bank</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cyanine7 Streptavidin</td>
			<td>BioLegend</td>
			<td>405206</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Vannas Spring Scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>91500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Dumont #5 Forceps</td>
			<td>Fine Science Tools Inc</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

facs-isolation and culture of fibro-adipogenic progenitors and muscle stem cells from unperturbed and injured mouse skeletal muscle

纤维脂肪祖细胞 （FAP） 是一组骨骼肌驻留间充质基质细胞 （MSC），能够沿纤维化、成脂、成骨或成软骨谱系分化。FAP与肌肉干细胞（MuSC）一起在肌肉稳态，修复和再生中起着关键作用，同时积极维持和重塑细胞外基质（ECM）。在病理条件下，例如慢性损伤和肌肉萎缩症，FAPs经历异常激活并分化为产生胶原蛋白的成纤维细胞和脂肪细胞，导致纤维化和肌内脂肪浸润。因此，FAP在肌肉再生中起着双重作用，通过维持MuSC更新和促进组织修复或促进纤维化瘢痕形成和异位脂肪浸润，从而损害骨骼肌组织的完整性和功能。FAP和MuSCs的适当纯化是了解这些细胞在生理和病理条件下的生物学作用的先决条件。在这里，我们描述了一种使用荧光激活细胞分选（FACS）从成年小鼠的肢体肌肉中同时分离FAP和MuSC的标准化方法。该协议详细描述了来自整个肢体肌肉和受伤的胫骨前（TA）肌肉的单核细胞的机械和酶解离。随后使用半自动细胞分选仪分离FAP和MuSC，以获得纯细胞群。我们还描述了一种单独或在共培养条件下培养静态和活化FAP和MuSC的优化方法。

该协议允许从野生型成年小鼠（3-6个月）的未受伤后肢中分离约100万个FAP和多达350，000个MuSC，对应于FAP的产量为8%，MuSC的产量为3%的总事件。当从受伤后 7 天受损的 TA 中分选细胞时，将两到三块 TA 肌肉汇集以获得多达 300，000 个 FAP 和 120，000 个 MuSC，分别对应于 11% 和 4% 的产量。FAP和MuSC的分选后纯度值通常高于95%。
用于分离FAP和MuSC的门控策略如图 2所...

Watch this Scientific Journal Video about FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle at JoVE.com

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

纤维脂肪祖细胞（FAPs）和肌肉干细胞（MuSCs）的精确鉴定对于研究它们在生理和病理条件下的生物学功能至关重要。该协议为从成年小鼠肌肉中分离，纯化和培养FAP和MuSC提供了指南。

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 ...

该协议描述了一种使用分选荧光激活剂同时分离纤维脂肪，祖细胞和肌肉干细胞的方法。这些细胞的纯群体是了解它们在生理和病理条件下的生物学作用的先决条件。使用这种技术，福布斯和肌肉干细胞被鉴定并从腋窝或受伤的肌肉中分离出来。福布斯和肌肉干细胞的高饱满有罪不罚使我们能够将这些细胞用于不同的下游技术，如糖精移植和多混合分析。首先，将安乐死的小鼠仰卧位放在解剖垫上并喷洒70%乙醇为避免污染，使用镊子捏住小鼠腹部皮肤的中心并水平切开约一厘米的开口。抓住鼠标的伤口边缘和椎间盘，向相反方向拉动开口，露出下面的肌肉，露出后肢的一侧。在收集肌肉之前，去除股四头肌近端腘绳肌之间的肌间脂肪，以改善纤维性脂肪祖细胞和肌肉干细胞的分离。接下来，在不损伤组织的情况下，使用锋利的镊子折断并剥离筋膜，以露出胫骨下前部或TA和趾伸肌或EDL肌肉。将镊子的尖头放在TA EDL肌肉下方，将肌肉从胫骨上分离。切断肌腱，将 TA EDL 连接到脚踝，同时用镊子握住它，用剪刀沿着 TA EDL 的纵线修剪肌肉。切开膝盖周围的肌腱，分离整个TA EDL肌肉。将肌肉放在一个六厘米长的盘子里，放在冰上。为了隔离腓肠肌，切断脚踝处的所有肌腱，并将肌肉与胫骨和腓骨分离。切开膝盖，将肌肉转移到盘子上。剥下股四头肌周围的筋膜，并通过在肌肉和骨骼之间运行镊子的尖锐尖端将其与股骨分开。切断膝盖周围的肌腱，并通过沿着股骨切割来修剪其余肌肉，同时用镊子在远端握住股四头肌。将肌肉放在六厘米的盘子中。分离股骨周围的腿筋和剩余肌肉，并将其转移到盘子中。将后肢肌肉收集在冰上的盘子中。从培养皿中吸出洗涤介质，加入一到两毫升肌肉解离缓冲液，以保持肌肉湿润。接下来，将肌肉彻底切碎 用手术刀撕裂和切片肌肉，然后用剪刀将肌肉不断切成小块一到两分钟，直到获得切碎组织的浆糊。然后将切碎的肌肉转移到含有肌肉解离缓冲液的锥形管中。用实验室薄膜密封试管，并在37摄氏度的水浴中搅拌孵育一小时。一小时后，从摇床中取出试管并将其填充至50毫升 用洗涤介质，轻轻倒置试管两次以确保混合。将细胞在水平桶转子中以 250 倍 G 在 4 摄氏度下离心五分钟。将42毫升上清液转移到两个新管中，并在原始管中留下8毫升。用洗涤介质填充含有21毫升上清液至50毫升的新管。然后将细胞在 350 倍 G 下在 4 摄氏度下离心 8 分钟。吸出上清液并将颗粒保持在冰上。接下来，在含有八毫升肌肉解离缓冲液的原始管中加入一毫升胶原酶二储备液和一毫升二基原液。使用5毫升血清移液管重悬沉淀10次而不会堵塞。将溶液喷射到管壁，避免形成气泡。用实验室薄膜密封试管，并在37摄氏度的水浴中搅拌30分钟。30分钟后，从摇床吸出物中取出试管，并通过带有20号针头的10毫升注射器弹出肌肉悬浮液10次，用洗涤介质填充每个管最多50毫升并倒置管。然后在4摄氏度下以250倍G离心5分钟，并将上清液转移到新管中。在原管中留下八毫升。用洗涤介质填充新管至50毫升，再次以350倍G在4摄氏度下离心细胞8分钟。除去上清液并将沉淀放在冰上。将 40 微米尼龙细胞过滤器放入装有 8 毫升洗涤介质的原始 50 毫升锥形管中，并用 1 至 2 毫升洗涤介质预润湿过滤器。握住过滤器的同时，使用 10 毫升移液管轻轻重悬沉淀。通过细胞过滤器将悬浮液过滤回同一管中，以最大程度地减少细胞损失。通过向每个管中加入四到五毫升洗涤介质来将其他管颗粒过滤回原始管中，以重悬沉淀并将溶液转移到位于原始管中的自过滤器中。将所有颗粒收集到原来的50毫升管中后，用另外4至5毫升洗涤介质冲洗自过滤器。使用1000微升移液器从自过滤器底部收集所有液体。用最多 50 毫升的洗涤介质填充每个管子，然后将管子倒置。在 250 倍 G 下以 4 摄氏度离心管五分钟。立即除去上清液而不干扰沉淀，并将沉淀重悬于600微升洗涤介质中。将悬浮液转移到两毫升微量离心管中。将抗体 CD 31 APC CD 45 APC SCA 一个太平洋蓝 VCAM 一个生物素和 α 7 整合素 PE 添加到含有细胞悬液的 2 毫米管中。将细胞在 4 摄氏度的旋转摇床中轻轻混合并孵育 45 分钟，保护它们免受光照。孵育后，用洗涤介质填充所有管最多两毫升，并在 250 倍 G 下以 4 摄氏度离心五分钟。除去上清液并将沉淀重悬于300微升洗涤介质中。接下来，将链霉亲和素抗体添加到试管中，并将细胞在4摄氏度的旋转振荡器中孵育20分钟。孵育后，用洗涤介质将含有链霉亲和素抗体的试管填充至两毫升。然后，离心管并完全吸出上清液。第二次洗涤后，将实验管中的细胞重悬于500微升洗涤培养基中。用 200 微升洗涤介质预润湿 5 毫升聚苯乙烯圆底管的细胞过滤器帽。将细胞悬液从实验管转移到五毫升聚苯乙烯圆底管中，并使其通过重力过滤。用300微升洗涤介质打印含有实验样品悬浮液的两毫升管，并将其穿过相同的过滤器盖。使用200微升移液器从细胞过滤器底部收集所有液体。用盖子密封，将管子放在冰上并保护它们免受光照。此处显示了用于分离纤维成脂祖细胞和肌肉干细胞的门控策略。报告小鼠中的PD GFR α E G F P敲击表达来自内源性PD G FFR Α位点的H two B E G F P融合基因，允许对PD G GFR Α谱系进行有效和特异性标记。通过用PAC-7抗体对共培养的GFP阳性纤维成脂祖细胞和肌肉干细胞进行免疫染色，证实了分离细胞群的纯度。GFP阳性纤维成脂祖细胞不表达PAC-7标志物，而肌肉干细胞与该抗体发生反应。所有表达GFP的细胞均呈SCA 1阳性，CD 31，CD 45 VCAM一和α7整合素呈阴性。显示了从损伤后七天肌内注射无毒素损伤的小鼠中分离出的纤维成脂祖细胞和肌肉干细胞。随着肌肉干细胞被激活，细胞大小增加。调整 F SCA 和 SS CA 参数并扩展门以将这些单元包含在中心非常重要。此处显示了未受伤和受伤后 7 天 TA 肌肉中纤维脂肪生成祖细胞和肌肉干细胞的定量。尽管在第2天和第3天之间观察到峰值和增殖，但在损伤后7天仍然可以看到细胞的低增殖率。进行适当的肌肉切碎对于释放单个细胞至关重要。此外，通过将悬浮液与带有20号针头的10毫升注射器运行来实现单核苷酸细胞分离。这项技术将帮助科学家在正常状态和肌肉再生的更深阶段研究福布斯和肌肉干细胞的生物转录和表观基因组谱。

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...