Name: isolation and differentiation of primary myoblasts from mouse skeletal muscle explants
Uploaded: 2019-10-15T14:45:00
Description: Watch this Scientific Journal Video about Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants at JoVE.com

作者感谢墨尔本大学的马修·瓦特博士和约翰霍普金斯大学的阿纳斯塔西娅·克拉利博士，他们根据莫克贝尔等人6号的工作，协助采纳这一协议。我们还感谢萨宾·乔丹博士协助在我们的实验室中制定和通过这一议定书。这项工作由美国国家卫生研究院R01s DK097164和DK112927资助。

该协议遵循斯克里普斯研究的动物护理指南。
1. 肌肉组织外泄的收集和处理
<ol><li>解剖前一天，对所有解剖设备（钳子、剃须刀和剪刀）进行消毒，并准备所有必需的介质：磷酸盐缓冲盐水（PBS）、甲基溴电镀介质（DMEM 12.5 mL、HAMS F12 12 12.12 mL、20 mL 热灭活胎牛血清 （FBS）， 5 mL 羊水介质补充剂） 和涂层溶液 （24 mL DMEM， 24 mL HAMS F12， 1.7 mL 胶原蛋白， 1 mL 母体凝胶）.</li>
...

<ol>
	<li>Srikanthan, P., Karlamangla, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+mass+index+as+a+predictor+of+longevity+in+older+adults.">Muscle mass index as a predictor of longevity in older adults.</a> American Journal of Medicine. 127 (6), 547-553 (2014).</li><li>Lee-Young, R. S., Kang, L., Ayala, J. E., Wasserman, D. H., Fueger, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiological+regulation+of+glucose+flux+into+muscle+in+vivo.">The physiological regulation of glucose flux into muscle in vivo.</a> Journal of Experimental Biology. 214 (2), 254-262 (2010).</li><li>Sj&#248;berg, 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=Exercise+increases+human+skeletal+muscle+insulin+sensitivity+via+coordinated+increases+in+microvascular+perfusion+and+molecular+signaling.">Exercise increases human skeletal muscle insulin sensitivity via coordinated increases in microvascular perfusion and molecular signaling.</a> Diabetes. 66 (6), 1501-1510 (2017).</li><li>Girgis, C. M., Clifton-Bligh, R. J., Mokbel, N., Cheng, K., Gunton, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+D+signaling+regulates+proliferation,+differentiation,+and+myotube+size+in+C2C12+skeletal+muscle+cells.">Vitamin D signaling regulates proliferation, differentiation, and myotube size in C2C12 skeletal muscle cells.</a> Endocrinology. 155 (2), 347-357 (2014).</li><li>Smith, J., Merrick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+skeletal+muscle+microexplant+culture+and+isolation+of+skeletal+muscle+stem+cells.">Embryonic skeletal muscle microexplant culture and isolation of skeletal muscle stem cells.</a> Methods in Molecular Biology. 633, 29-56 (2010).</li><li>Mokbel, 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=K7del+is+a+common+TPM2+gene+mutation+associated+with+nemaline+myopathy+and+raised+myofibre+calcium+sensitivity.">K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity.</a> Brain. 136 (2), 494-507 (2013).</li><li>Yaffe, D., Saxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+passaging+and+differentiation+of+myogenic+cells+isolated+from+dystrophic+mouse+muscle.">Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.</a> Nature. 270, 725-727 (1977).</li><li>Rando, T. A., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+mouse+myoblast+purification,+characterization,+and+transplantation+for+cell-mediated+gene+therapy.">Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.</a> Journal of Cell Biology. 125 (6), 1275-1287 (1994).</li><li>Musar&#242;, A., Carosio, S. <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+Culture+of+Satellite+Cells+from+Mouse+Skeletal+Muscle.">Isolation and Culture of Satellite Cells from Mouse Skeletal Muscle.</a> Methods in Molecular Biology. 1553, 155-167 (2017).</li><li>Smolina, N., Bruton, J., Kostareva, A., Sejersen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+mitochondrial+respiration+as+an+indicator+of+cellular+metabolism+and+fitness.">Assaying mitochondrial respiration as an indicator of cellular metabolism and fitness.</a> Methods in Molecular Biology. 1601, 79-87 (2017).</li><li>Elliott, B., Renshaw, D., Getting, S., Mackenzie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+central+role+of+myostatin+in+skeletal+muscle+and+whole+body+homeostasis.">The central role of myostatin in skeletal muscle and whole body homeostasis.</a> Acta Physiologica. 205 (3), 324-340 (2012).</li><li>Smolina, N., Kostareva, A., Bruton, J., Karpushev, A., Sjoberg, G., Sejersen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+murine+myotubes+as+a+model+for+investigating+muscular+dystrophy.">Primary murine myotubes as a model for investigating muscular dystrophy.</a> BioMed Research International. , (2015).</li><li>Nedachi, T., Fujita, H., Kanzaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+C+2+C+12+myotube+model+for+studying+exercise-inducible+responses+in+skeletal+muscle.">Contractile C 2 C 12 myotube model for studying exercise-inducible responses in skeletal muscle.</a> American Journal of Physiology-Endocrinology and Metabolism. 295 (5), E1191-E1204 (2008).</li><li>Chen, M. 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=Impaired+activation+of+AMP-kinase+and+fatty+acid+oxidation+by+globular+adiponectin+in+cultured+human+skeletal+muscle+of+obese+type+2+diabetics.">Impaired activation of AMP-kinase and fatty acid oxidation by globular adiponectin in cultured human skeletal muscle of obese type 2 diabetics.</a> Journal of Clinical Endocrinology and Metabolism. 90 (6), 3665-3672 (2005).</li><li>Douillard-Guilloux, G., Mouly, V., Caillaud, C., Richard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+murine+muscle+cells+from+lysosomal+&#945;-glucosidase+deficient+mice:+A+new+tool+to+study+pathophysiology+and+assess+therapeutic+strategies+for+Pompe+disease.">Immortalization of murine muscle cells from lysosomal &#945;-glucosidase deficient mice: A new tool to study pathophysiology and assess therapeutic strategies for Pompe disease.</a> Biochemical and Biophysical Research Communications. 388 (2), 333-338 (2009).</li><li>Varga, 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=Myotube+elasticity+of+an+amyotrophic+lateral+sclerosis+mouse+model.">Myotube elasticity of an amyotrophic lateral sclerosis mouse model.</a> Scientific Reports. 8 (1), 5917 (2018).</li><li>Kriebs, 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=Circadian+repressors+CRY1+and+CRY2+broadly+interact+with+nuclear+receptors+and+modulate+transcriptional+activity.">Circadian repressors CRY1 and CRY2 broadly interact with nuclear receptors and modulate transcriptional activity.</a> Proceedings of the National Academy of Sciences. 114 (33), 8776-8781 (2017).</li><li>Cho, Y., 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=Perm1+enhances+mitochondrial+biogenesis,+oxidative+capacity,+and+fatigue+resistance+in+adult+skeletal+muscle.">Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.</a> FASEB Journal. 30 (2), 674-687 (2016).</li></ol>

骨骼肌对于建立和维护代谢平衡11至关重要。肌肉生理学的研究因个体间变异性以及获取样本的困难而变得复杂，特别是在人类研究中。培养的原发性肌管已被证明可以重述肌肉生理学的许多特征，包括钙平衡12、受损肌肉组织的再生5、对运动13的代谢改变，糖尿病等疾病引起的新陈代谢的改变。小鼠的肌细...

虽然经常被引证为整体代谢健康的指示，但多项研究表明，老年人的身体质量指数（BMI）与较高的死亡率风险并不一致。迄今为止，唯一与降低这一种群死亡率相一致的因素是增加肌肉质量1。肌肉组织是体内胰岛素敏感细胞的最大来源之一，因此对维持整体代谢平衡至关重要。通过运动激活骨骼肌组织与增加局部胰岛素敏感性和整体代谢健康3相关。虽然体内模型对于研究肌肉生理学和肌肉功能对综合代谢的影响至关重要，但肌管的主要培养物提供了一个可处理的系统，可降低动物研究的复杂性。
从产后肌肉衍生的肌末代酶可用于以高度可重复的方式研究多种治疗和生长条件的影响。这早已得到承认，几种方法的月经隔离和文化已经描述了4，5，6，7，8，9。其中一些方法使用新生儿肌肉，产生相对较少的肌体5，8，需要几种动物进行更大规模的研究。此外，大多数广泛使用的培养霉细胞的方法使用"预镀"来丰富于于其他细胞类型粘附性较差的细胞。我们发现这里描述的替代富集方法对于丰富高度增殖的美洲人群体更有效和可重复。总之，该协议允许从年轻的成人肌肉外植分离高增殖性肌体，通过生长到培养培养。月子可以反复收获，在几天内，迅速扩大，并诱导分化成近管。该协议可重复产生大量健康的肌细胞，这些细胞会强力分化成自发抽搐肌细胞。它使我们能够研究各种基因型小鼠的原发性月管的代谢和昼夜节律。最后，我们采用96孔板中耗氧率的测量方法，为氧化代谢研究制备肌管。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Coating Solution:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 24 mL</td>
		</tr>
		<tr>
			<td>HAMS F12</td>
			<td>Lonza</td>
			<td>12-615F</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 24 mL</td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Life Technologies</td>
			<td>A1064401</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Fisher</td>
			<td>CB40234A</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Plating Media:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 12.5 mL</td>
		</tr>
		<tr>
			<td>HAMS F12</td>
			<td>Lonza</td>
			<td>12-615F</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 12.5 mL</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Life Technologies</td>
			<td>16000044</td>
			<td>20 mL; can be purchased as regular FBS and heat-inactivated by placing in a 40 &#176;C water bath for 20 minutes</td>
		</tr>
		<tr>
			<td>Amniomax</td>
			<td>Life Technologies</td>
			<td>12556023</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Myoblast Media:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 17.5 mL</td>
		</tr>
		<tr>
			<td>HAMS F12</td>
			<td>Lonza</td>
			<td>12-615F</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 17.5 mL</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Life Technologies</td>
			<td>16000044</td>
			<td>10 mL; can be purchased as regular FBS and heat-inactivated by placing in a 40 &#176;C water bath for 20 minutes</td>
		</tr>
		<tr>
			<td>Amniomax</td>
			<td>Life Technologies</td>
			<td>12556023</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Differentiation Media:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 24 mL</td>
		</tr>
		<tr>
			<td>HAMS F12</td>
			<td>Lonza</td>
			<td>12-615F</td>
			<td>Always add gentamicin (1:1000 by volume) prior to use; 24 mL</td>
		</tr>
		<tr>
			<td>Heat Inactivated Horse Serum</td>
			<td>Sigma</td>
			<td>H1138</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Insulin-Selenium-Transferrin</td>
			<td>Life Technologies</td>
			<td>41400045</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>Other Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>472301</td>
			<td>Prepare as 10% DMSO in Myoblast Media for freezing cells</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman paper</td>
			<td>VWR</td>
			<td>21427-648</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm plate</td>
			<td>VWR</td>
			<td>734-2318</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm plate</td>
			<td>VWR</td>
			<td>25382-428 (CS)</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 Flasks</td>
			<td>ThermoFisher</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>ThermoFisher</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (15mL)</td>
			<td>BioPioneer</td>
			<td>CNT-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Consumption Rates:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent</td>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Instrument used to measure oxygen consumption rates read out by acidification of the extracellular media</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent</td>
			<td>102416-100</td>
			<td>96-well plates for use in XFe96 Analyzer</td>
		</tr>
		<tr>
			<td>Seahorse XF Cell Mito Stress Test Kit</td>
			<td>Agilent</td>
			<td>103015-100</td>
			<td>components may be purchased from other suppliers once assay is established; some recommendations are listed below</td>
		</tr>
		<tr>
			<td>Seahorse XF Palmitate-BSA FAO substrate</td>
			<td>Agilent</td>
			<td>102720-100</td>
			<td>components may be purchased from other suppliers once assay is established; some recommendations are listed below</td>
		</tr>
		<tr>
			<td>Palmitic acid</td>
			<td>Sigma</td>
			<td>P5585-10G</td>
			<td>for measurement of fatty acid oxidation</td>
		</tr>
		<tr>
			<td>carnitine</td>
			<td>Sigma</td>
			<td>C0283-5G</td>
			<td>for measurement of fatty acid oxidation</td>
		</tr>
		<tr>
			<td>Etomoxir</td>
			<td>Sigma</td>
			<td>E1905</td>
			<td>for measurement of fatty acid oxidation</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td>used as control or in conjugation with palmitic acid for use in measurement of fatty acid oxidation</td>
		</tr>
	</tbody>
</table>

isolation and differentiation of primary myoblasts from mouse skeletal muscle explants

原发性肌体是骨骼肌的无差别增殖前体。它们可以作为肌肉前体培养和研究，或诱导分化到肌肉发育的后期阶段。此处提供的协议描述了一种强大的方法，用于从年轻的成年小鼠骨骼肌外植体中分离和培养高度增殖的肌细胞群。这些细胞可用于研究不同小鼠模型骨骼肌的代谢特性，以及其他下游应用，如外源DNA转染或病毒表达载体转导。这些细胞的分化水平和代谢特征取决于暴露的长度，以及用于诱导月细胞分化的介质的组成。这些方法为研究小鼠肌肉细胞外体代谢提供了强有力的系统。重要的是，与体内模型不同，本文描述的方法提供了一个细胞群，可以扩展和研究具有高重复性。

按照所提供的协议第 1 节，应产生从植物外部产生的原细胞，这些细胞将在标准光显微镜下可见（图 2）。异质细胞群将生长在除外每个肌肉组织外部和周围。微球体将显示为小，圆，明亮的球体。根据协议第2节，将产生从组织外植的细胞细胞的早期收获，这将包含很少的细胞，并且将是异质的（图3）。该协议第3节描述了用PB...

Watch this Scientific Journal Video about Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants at JoVE.com

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

肌细胞是增殖前体细胞，分化形成多核肌管，并最终骨骼肌肌纤维。在这里，我们提出了一个协议，从年轻的成年小鼠骨骼肌肉有效隔离和培养原发性肌体。该方法支持对培养中的肌肉细胞进行分子、遗传和代谢研究。

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

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

这是一个协议，用于分离原发性小鼠肌肉干细胞，用于研究代谢外体。与过去尝试过的其他协议相比，该协议提供了更多的干细胞。更重要的是，一旦我们把这些干细胞分化成 myotube，它们就表现出正常的生理，所以像昼夜节律一样。首次尝试此过程时，请做详细的笔记并保存多个图像，因为每个组织外植看起来都不同，并且肌细胞的外培养会有变化。如果没有视觉演示，很难知道是否隔离了正确的单元格。当我们在实验室中建立这种方法时，我们受益于其他人的慷慨帮助， 他们向我们展示了这种方法。根据手稿准备消毒盘后，将两到三张厚吸水纸放入塑料袋中，用移液器用无菌水润湿纸张表面，准备湿润室。将腔室在紫外线下放置五分钟进行消毒。从四到八周大的老鼠解剖所需的肌肉，并在含有每毫升根霉素40微克的PBS中轻轻冲洗以进行灭菌。使用无菌钳将肌肉转移到无菌的10厘米非涂层培养皿。在肌肉上加入0.5至1毫升电镀介质，使组织湿润，但无法浮动。使用无菌手术刀或剃刀刀片轻轻地将肌肉切成小碎片。使用钳子或移液器将肌肉碎片转移到预涂覆的六厘米板表面。非常轻轻地将额外的 0.8 毫升电镀介质覆盖在组织上。将含有湿润室肌肉碎片的六厘米培养皿放在湿润室内，并在 37 摄氏度和 5%的二氧化碳下返回孵化器 48 小时。在37摄氏度的水浴中准备和预热肌细胞介质、三辛和PBS-根霉素。要为每个肌肉组涂上一个T25烧瓶，在烧瓶表面加入两毫升的涂层溶液，轻轻摇动，在表面形成均匀的涂层，并在四摄氏度下孵育烧瓶一小时。现在用移液器，从肌肉外植中去除电镀介质，然后用两毫升PBS-根霉素轻轻冲洗肌肉外植。快速拆下 PBS，不要让板位于 PBS 中。然后轻轻加入一毫升PBS-根霉素，将盘子放在37摄氏度的培养箱中一分钟。使用 P1000 移液器将 PBS 与细胞收集到 15 毫升离心管中。接下来，在盘子中加入一毫升的三辛，然后将其返回到37摄氏度的培养箱中三分钟。轻轻敲击板，将肌细胞进行分离。用单元格收集 trypsin，并结合 PBS 集合。将八毫升肌细胞介质加入离心管，轻轻倒置混合。轻轻覆盖肌肉板上两毫升电镀溶液，然后将板返回 37 摄氏度的培养箱。在离心机中旋转含有细胞的离心管，以200倍g旋转3分钟。吸上一道液，在管中保持约一毫升。轻轻添加肌细胞介质，将细胞转移到预涂层的烧瓶中，并将烧瓶放在 37 摄氏度的培养箱中。从 P0 肌爆 T25 烧瓶中吸出介质。用两毫升温暖的PBS-根霉素短暂冲洗细胞，然后从烧瓶中吸出PBS。将两毫升温暖的PBS放入每个含有肌细胞的烧瓶中。将带 PBS 的烧瓶放入 37 摄氏度的培养箱中三分钟。然后用力敲击烧瓶的侧面，将细胞分离。在光学显微镜下检查自由漂浮的肌细胞。将烧瓶直立放在组织培养罩中，用 10 毫升肌细胞介质冲洗烧瓶底部两到三次，以确保所有细胞都脱落。在15毫升离心管中收集细胞介质混合物。在200倍g下离心3分钟。吸气介质在管中留下约一毫升，小心避免细胞颗粒。轻轻地将适当体积的肌细胞介质添加到离心管中，然后轻轻混合。将细胞混合物分配到新的T75烧瓶中，每个瓶6毫升。在每个新的T75烧瓶中加入10毫升的肌细胞介质。水平轻轻摇动烧瓶，将细胞分布，并在37摄氏度的培养箱中放置过夜。在该协议中，在标准光显微镜下观察从外植中产生原生细胞。早期收获的肌细胞出现小圆和明亮的球体。肌细胞分化需要四到六天的时间，在此期间，细胞的形态从单圆球变成拉长融合的长多核纤维。生产了完全差异化的 myotube，这些管管已准备好测量氧气消耗率。这些单元可用于许多下游应用。例如，我们经常使用它们来研究海马仪器中的基板通量。

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

从小鼠骨骼肌分离I型和II型周细胞

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

人成肌细胞的分离，造血分化的评估和存储操作的钙进入测量

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

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, ...