亚历山德拉·达佐（Alessandra d'Azzo）是儿童珠宝商协会（JFC）遗传学和基因治疗的主席。这项工作得到了美国国立卫生研究院（NIH）拨款R01GM104981、RO1DK095169和CA021764、孟菲斯阿西西基金会和美国黎巴嫩叙利亚联合慈善机构的部分支持。

小鼠的所有程序均根据圣裘德儿童研究医院机构动物护理和使用委员会和美国国立卫生研究院指南批准的动物方案进行。
1. 溶液和培养基的制备
<ol><li>通过将 15.4 mL PBS 与 2.5 mL 20 mg/mL 胶原酶 P（5 mg/mL 终浓度）、2 mL 11 U/mL 分散酶 II（1.2 U/mL 终浓度）和 100 μL 1.0 M CaCl2 （5 mM 终浓度）混合来制备消化溶液。</li>
	<li>通过将 440 mL DMEM 与 50 mL FBS （10%）、5.0 mL pen/链球菌（分别为 100 U/mL 和 100 μg/mL）和 5.0 mL 谷氨酰胺补充剂 （2 mM） 混合，制备 500 mL 原代成纤维细胞培养基（DMEM 完全）。用 0.22 μm 孔径真空过滤器对培养基进行过滤灭菌。</li>
	<li>通过在4°C下以100,000× g 在38.5mL聚丙烯管中过夜超速离心FBS来制备无外泌体血清，并将上清液转移到新管中。</li>
	<li>通过将 440 mL DMEM 与 50 mL 无外泌体血清 （10%）、5 mL 笔/链球菌（100 U/mL 和 100 μg/mL）和 5 mL 谷氨酰胺补充剂 （2 mM） 混合，制备 500 mL 无外泌体培养基。用 0.22 μm 孔径真空过滤器对培养基进行过滤灭菌。</li>
	<li>通过将 6.057 g Tris 碱溶解在 90 mL dH 2 O 中并用 HCl 将 pH 调节至 7.4 来制备 0.5 M Tris-HCl （pH 7.4）。 用 dH2O 将体积调节至 100 mL。</li>
	<li>通过将 97.9 mL dH 2 O 与 2 mL 0.5 M Tris-HCl （pH = 7.4） 和 100 μL 1 M Mg（Ac）2混合，制备 10 mM Tris-HCl （pH 7.4）/1 mM Mg（Ac）2 溶液（蔗糖工作溶液）。使用前将蛋白酶抑制剂添加到溶液中，并将溶液保持在冰上。</li>
	<li>根据 表1在冰上制备蔗糖密度梯度溶液。这些溶液足以制备两个蔗糖梯度管。</li>
	<li>将 40 mL dH2O 加入 100 g TCA 粉末中，制备 100% TCA，并混合直至完全溶解。用dH2O调节最终体积至100mL。</li>
	<li>通过将 20 mL dH2O 与 80 mL 100% 乙醇混合来制备 80% 乙醇。</li>
	<li>通过将 1.8 L dH2O 与 200 mL 10x 电泳缓冲液混合来制备蛋白质印迹电泳缓冲液。</li>
	<li>通过将 1.4 L dH2O 与 200 mL 10x 转印缓冲液和 400 mL 甲醇混合来制备蛋白质印迹转印缓冲液。将转移缓冲液预冷至4°C。</li>
	<li>通过将 122 g Tris 碱和 180 g 氯化钠溶解在 850 mL dH2O （pH 8.0） 中来制备 20x Tris 缓冲盐水 （TBS）。将音量调节至 1 L，dH为 2O。</li>
	<li>通过将 5 g 脱脂奶粉与 1 mL 10% 吐温-20、5 mL 20x TBS 和 94 mL dH2O 溶解来制备封闭缓冲液。</li>
	<li>通过将 3 g BSA 与 1 mL 的 10% Tween-20、5 mL 的 20x TBS 和 94 mL 的 dH2O 溶解来制备抗体缓冲液。</li>
	<li>通过将 100 mL 的 20x TBS 和 20 mL 的 10% Tween-20 （10x） 与 1.88 L dH2O 混合来制备洗涤缓冲液。</li>
	<li>通过将 1 体积的鲁米诺/增强剂与 1 体积的稳定过氧化物缓冲液混合来制备显影液。</li>
</ol>2. 腓肠肌 （GA） 小鼠肌肉解剖15,16
<ol><li>在冰上制备含有 10 mL PBS 的 50 mL 试管。</li>
	<li>在CO2 室中对小鼠实施安乐死，然后进行颈椎脱位。</li>
	<li>从双腿上取下腓肠肌，并将它们转移到冰上装有PBS的管子中。 注意：在将 GA 肌肉转移到 PBS 之前，从 GA 肌肉中取出比目鱼肌。</li>
</ol>3. 小鼠原代成纤维细胞分离和培养
<ol><li>称量肌肉并将它们转移到生物安全罩下的 10 厘米培养皿中。用手术刀将肌肉切碎，直到它变成细糊状。</li>
	<li>将切碎的肌肉糊状物转移到50mL管中，并向管中加入3.5x体积/ mg的消化溶液组织，并在37°C下孵育45分钟。 每 10 分钟用 5 mL 移液管将悬浮液彻底混合（这将有助于完全解离）。 注意：或者，组织解离器可用于此步骤。</li>
	<li>向细胞悬液中加入 20 mL DMEM 以灭活消化溶液。转移到放置在 50 mL 试管上的 70 μm 尼龙细胞过滤器中。收集流出物并用另外 5 mL 的 DMEM 完成洗涤细胞过滤器。</li>
	<li>在室温（RT）下以300× g 离心细胞悬浮液10分钟，并小心地除去上清液。</li>
	<li>将沉淀重悬于 10 mL DMEM 中，并将细胞接种到 10 cm 培养皿中。</li>
	<li>在37°C，5%CO2,3%O2 （传代0或P0）下培养细胞。 注：1） 这些培养物需要保持在低氧水平，以确保生理条件（骨骼肌中的 O 2 水平约为2.5%）17。2）细胞培养物的传代数（例如，P0）是培养物传代培养次数的记录。3） P0 处的原代成纤维细胞常规培养至 100% 汇合加 1 天（并且仅适用于 P0）。这是为了清除其他类型的细胞，并确保培养物中存在的细胞只是成纤维细胞，根据显微镜检查耗尽了肌原细胞。2 个月大的成年动物的骨骼肌含有约 2% 的肌源性祖细胞4。此外，肌源性祖细胞通常不会附着在未涂层的培养皿上;因此，它们在传代培养过程中丢失 18,19,20。成肌细胞需要补充有 20% FBS 和碱性成纤维细胞生长因子 （bFGF） 18 的不同培养基 （F10）。在DMEM完全中，中等成纤维细胞的增殖率高于原肌细胞，这导致这些污染细胞在第一次传代之前被消除。</li>
	<li>当 100% 汇合加 1 天时，用 PBS 冲洗 P0 细胞。加入1mL胰蛋白酶消化溶液，在37°C，5%CO2,3%O2 下孵育以分离细胞。使用 10 mL DMEM 完全停止酶活性。</li>
	<li>在室温下以 300 x g 离心细胞 10 分钟，并将沉淀重悬于 20 mL DMEM 完全中，并接种到 15 cm 培养皿中（传代 1 或 P1）。细胞生长至80%-90%汇合，并可扩增至第3代。 注：根据下游实验，可以使用传代 1、传代 2 （P2） 或传代 3 （P3）。</li>
</ol>4. 细胞接种和条件培养基的收集
<ol><li>用10mL PBS洗涤成纤维细胞（P1，P2或P3），向细胞中加入2.5mL胰蛋白酶消化溶液，并在37°C，5%CO 2,3%O2下孵育。使用 10 mL DMEM 完全停止酶活性。 注意：在第 4 次传代后，原代细胞被丢弃，不应用于进一步的实验，因为它们会改变特性。</li>
	<li>收集细胞并在室温下以300× g 离心10分钟。</li>
	<li>将细胞沉淀重悬于 10 mL 无外泌体培养基中并计数细胞。</li>
	<li>在1.5-2.0×10 每培养皿（15cm）下接种6个细胞，并在37°C，5%CO 2,3%O2下孵育。</li>
	<li>在冰上的 50 mL 试管中收集 16-24 小时的调节培养基。 注意：如果细胞不是 80% 汇合，可以再次添加新鲜的游离外泌体培养基，并在额外的 16-24 小时后收集。这两个集合可以合并以进行进一步处理。</li>
</ol>5. 使用差速和超速离心纯化外泌体
注意：所有步骤均在4°C或冰上进行。需要时用装满水的管子平衡管子。
<ol><li>将条件培养基以 300 x g 离心 10 分钟以除去活细胞，并将上清液转移到新的 50 mL 管中。</li>
	<li>通过以2,000× g 离心10分钟除去死细胞，并将上清液转移到38.5mL聚丙烯管中。</li>
	<li>将上清液以10,000× g 离心30分钟，以除去细胞器，凋亡小体和膜碎片。将上清液转移到 38.5 mL 超透明管中。</li>
	<li>将上清液以100,000× g 超速离心1.5小时以沉淀外泌体。</li>
	<li>小心地弃去上清液，留下约 1 mL 条件培养基，并在总体积为 30 mL 的冰冷 PBS 中洗涤外泌体沉淀。</li>
	<li>以100,000× g 离心1.5小时，并通过移液小心地弃去上清液，注意不要干扰外泌体沉淀。</li>
	<li>将沉淀重悬于冰冷的PBS（每15cm培养皿~25μL）中，并使用BCA蛋白质测定试剂盒和酶标仪在562nm处测量蛋白质浓度。 注：蛋白质浓度通过在dH 2 O中用0.1%Triton-X100以1：2稀释度测量。汇合度为 80% 的 15 cm 培养皿（20 mL 条件培养基）原代成纤维细胞的外泌体产量为 ~3–4 μg。</li>
</ol>6. 蔗糖密度梯度表征外泌体
<ol><li>通过移液（自下而上）将蔗糖梯度加载到13mL超透明管中：1.5 mL 的2.0 M，2.5 mL 的1.3 M，2.5 mL 的1.16 M，2.0 mL 的0.8 M和2.0 mL 的0.5 M蔗糖溶液。</li>
	<li>将 30–100 μg 外泌体与 0.25 M 蔗糖溶液混合，并将体积调节至 1 mL。</li>
	<li>小心地将外泌体加载到梯度顶部，并在100,000× g 和4°C下将样品超速离心2.5小时。</li>
	<li>从超速离心机中取出试管，将其置于冰上，并从梯度顶部开始收集1.0mL馏分。将它们转移到冰上的 1.7 mL 试管中。</li>
	<li>向每个试管中加入 110 μL 100% TCA，充分混合，并在室温下孵育 10 分钟。</li>
	<li>以10,000× g 和室温离心10分钟，小心弃去上清液。</li>
	<li>将沉淀重悬于500μL预冷的80%乙醇中，并让样品在-20°C下洗涤10分钟。</li>
	<li>在10,000× g 和4°C下离心10分钟，弃去上清液。</li>
	<li>在室温下干燥沉淀10-15分钟，并将沉淀重悬于PBS中用于体外实验。使用BCA蛋白测定试剂盒测量蛋白质浓度，或将沉淀直接重悬于14.5μLdH2O，5μL的4x Laemmli缓冲液和0.5μL的1M DTT中，用于蛋白质印迹分析。</li>
</ol>7. 通过蛋白质印迹分析检测外泌体
<ol><li>将步骤5.7中的5-10μg外泌体与5μL的4x Laemmli缓冲液和0.5μL的1M DTT混合，然后用dH 2O将最终体积调节至20μL。 将所有样品（包括步骤6.9中的样品）在98°C下加热5分钟。</li>
	<li>将样品加载到10%TGX无染色凝胶中，并根据制造商的建议加载蛋白质分子量标准。 注意：根据目标蛋白质的分子量选择凝胶的百分比。</li>
	<li>在电泳缓冲液中以100V电泳凝胶运行~1小时，直到上样染料位于凝胶底部。 注意：运行时间可能因所使用的设备或凝胶的类型和百分比而异。</li>
	<li>对凝胶进行成像。无染色凝胶的图像用作免疫印迹的上样对照。</li>
	<li>使用PVDF膜在80V下转移凝胶1.5小时或在4°C下在30V下过夜。</li>
	<li>在室温下封闭封闭缓冲液中的膜1小时。</li>
	<li>将特异性抗体的膜（表2）在抗体缓冲液中在4°C下搅拌孵育过夜。</li>
	<li>在洗涤缓冲液中洗涤膜，并在室温下搅拌将膜与适当的二抗（表2）孵育1小时。</li>
	<li>在洗涤缓冲液中洗涤膜，并使用显影液和基于相机的成像仪对膜进行成像。或者，使用 X 射线胶片来显影膜。</li>
</ol>

<ol>
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所有作者均无任何利益冲突需要声明。

如本协议所述，从培养基中成功分离外泌体的关键步骤是从成人骨骼肌中正确建立和维持原代小鼠成纤维细胞培养物。这些培养物需要保持在低氧水平，以确保生理条件（骨骼肌中的 O2 水平为 ~2.5%）15。原代成纤维细胞在培养中传承过多时会改变特征。因此，低传代数对于理想的外泌体产量是必不可少的。纯化的外泌体应立即用于实验目的，或在-80°C下以等分试样冷冻?...

外泌体是异质细胞外囊泡，大小范围为 30-150 nm。鉴于它们在组织和器官中无处不在的分布，它们是生理和病理过程中的关键参与者 1,2。外泌体携带蛋白质、脂质、DNA 类型和 RNA 类型的复杂货物，这些货物根据它们来源的细胞类型而变化 1,2,3。外泌体富含具有不同功能的蛋白质（即四跨膜蛋白，包括 CD9 和 CD63）负责融合事件。例如，热休克蛋白 HSP70 和 HSP90 参与抗原结合和呈递。此外，Alix、Tsg101 和 flotillin 参与外泌体生物发生和释放，并被广泛用作这些纳米囊泡的标志物 2,3,4。
外泌体还含有多种 RNA（即 microRNA、长链非编码 RNA、核糖体 RNA），它们可以转移到受体细胞中，从而影响下游信号转导3。外泌体被单个单元膜包围，其生物活性不仅取决于蛋白质和核酸的货物，还取决于限制膜1的脂质成分。外泌体膜富含磷脂酰丝氨酸、磷脂酸、胆固醇、鞘磷脂、花生四烯酸和其他脂肪酸，所有这些都会影响外泌体稳定性和膜拓扑结构 2,3。由于货物和脂质排列，外泌体在接收细胞中启动信号通路并参与维持正常组织生理学 1,2,4,5。在某些病理条件下（即神经退行性变、纤维化和癌症），它们已被证明会触发和传播病理刺激 4,6,7,8,9,10,11。
由于外泌体能够将信号传播到邻近或遥远的部位，因此已成为诊断或预后疾病状况的有价值的生物标志物。此外，外泌体已被实验用作治疗化合物的载体 2,12。这些纳米囊泡在临床上的潜在应用使得分离方法变得越来越重要，以实现最大的产量、纯度和可重复性。已经开发和实施了用于分离外泌体的不同技术。通常，可以通过差速离心、体积排阻色谱和免疫捕获（使用市售试剂盒）从条件的细胞培养基或体液中分离外泌体。每种方法都有独特的优点和缺点，前面已经讨论过1、2、13、14。
概述的方案侧重于1）从成年小鼠腓肠肌中分离和培养原代成纤维细胞，以及2）这些细胞释放到培养基中的外泌体的纯化和表征。目前缺乏从原代成纤维细胞中分离外泌体以进行功能研究的成熟方案。原代成纤维细胞不分泌大量外泌体，使分离和纯化过程具有挑战性。该方案描述了从大培养体积中纯化大量纯外泌体，同时保持其形态完整性和功能活性。从条件培养基中获得的纯化外泌体已成功用于体外摄取实验，以诱导受体细胞中的特异性信号通路。它们还被用于对来自多个生物样品的外泌体货物进行比较蛋白质组学分析4。

<table>
	<tbody>
		<tr>
			<td>10 cm dishes</td>
			<td>Midwest Scientific, TPP</td>
			<td>TP93100</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm dishes</td>
			<td>Midwest Scientific</td>
			<td>TP93150</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo Fisher Scientific, Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin Fraction V</td>
			<td>Roche</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430R with rotors FA-35-6-30/ FA-45-48-11</td>
			<td>Eppendorf</td>
			<td>022620659/5427754008</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemidoc MP imaging system</td>
			<td>BioRad</td>
			<td>12003154</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase P</td>
			<td>Sigma, Roche</td>
			<td>11 213 857 001</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>cOmplete protease inhibitor cocktail</td>
			<td>Millipore/Sigma, Roche</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion Blotter with plate electrodes</td>
			<td>BioRad</td>
			<td>1704070</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion TGX stain-free protein gel</td>
			<td>BioRad</td>
			<td>5678034</td>
			<td>10% 18-well, midi-gel</td>
		</tr>
		<tr>
			<td>Criterion vertical electrophoresis cell (midi)</td>
			<td>BioRad</td>
			<td>NC0165100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma, Roche</td>
			<td>04 942 078 001</td>
			<td>neutral protease, grade II</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma/Millipore, Roche</td>
			<td>10708984001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modification Eagles Medium</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Pharmco by Greenfield Global</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluostar Omega multi-mode microplate reader</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P Transfer membranes</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli sample buffer (4x)</td>
			<td>BioRad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate solution</td>
			<td>Sigma</td>
			<td>63052-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat dry milk</td>
			<td>LabScientific</td>
			<td>M-0842</td>
			<td></td>
		</tr>
		<tr>
			<td>O2/CO2 incubator</td>
			<td>Sanyo</td>
			<td>MC0-18M</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Premium microcentrifuge tubes</td>
			<td>Fisher Scientific, Midwest Scientific</td>
			<td>AVSC1510</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Protected disposable scalpels</td>
			<td>Fisher Scientific, Aspen Surgical Bard-Parker</td>
			<td>372610</td>
			<td></td>
		</tr>
		<tr>
			<td>Running buffer</td>
			<td>BioRad</td>
			<td>1610732</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Quick release-GP sterile vacuum filtration system</td>
			<td>Millipore</td>
			<td>S2GPU05RE</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Sterile cell strainer (70 mm)</td>
			<td>Fisher Scientific, Fisher brand</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal west Femto</td>
			<td>Thermo Fisher Scientific</td>
			<td>34096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin wall Polypropylene tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td>BioRad</td>
			<td>16110734</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic Acid</td>
			<td>Sigma</td>
			<td>91228-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>BioRad</td>
			<td>1610719</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100 solution</td>
			<td>Sigma</td>
			<td>93443-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>12604-013</td>
			<td>No phenol red</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>BioRad</td>
			<td>#1610781</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-centrifuge Optima XPM</td>
			<td>Beckman Coulter</td>
			<td>A99842</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (14x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tubes (25x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath Isotemp 220</td>
			<td>Fisher Scientific</td>
			<td>FS220</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of exosomes from skeletal muscle fibroblasts

外泌体是几乎所有细胞释放并在所有生物体液中分泌的小细胞外囊泡。已经开发了许多用于分离这些囊泡的方法，包括超速离心、超滤和体积排阻色谱法。然而，并非所有方法都适合大规模外泌体纯化和表征。这里概述了一种方案，用于建立从成年小鼠骨骼肌中分离的原代成纤维细胞的培养物，然后从这些细胞的培养基中纯化和表征外泌体。该方法基于使用连续离心步骤，然后是蔗糖密度梯度。然后使用一组经典标记物（即 Alix、CD9 和 CD81）通过蛋白质印迹分析来验证外泌体制剂的纯度。该方案描述了如何分离和浓缩生物活性外泌体，用于电子显微镜、质谱和功能研究的摄取实验。它可以很容易地放大或缩小，并适用于从不同细胞类型、组织和生物体液中分离外泌体。

该方案适用于以具有成本效益的方式从大量条件培养基中纯化外泌体。该过程具有高度的可重复性和一致性。 图1 显示了从小鼠原代成纤维细胞培养基中纯化的外泌体的透射电子显微镜（TEM）图像。 图 2 显示了典型外泌体标记物的蛋白质表达模式，以及无胞质 （LDH） 和 ER（钙联蛋白）蛋白污染物。 图 3 显示了蔗糖密度梯度后典...

Watch this Scientific Journal Video about Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts at JoVE.com

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

该方案说明了1）从成年小鼠腓肠肌中分离和培养原代成纤维细胞，以及2）使用差异超速离心法结合蔗糖密度梯度，然后进行蛋白质印迹分析的外泌体纯化和表征。

骨骼肌成纤维细胞外泌体的分离和表征

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

isolation-characterization-exosomes-from-skeletal-muscle

Research

JoVE Journal

Biology

3.2K 次观看.  St. Jude Children's Research Hospital. 该方案说明了1）从成年小鼠腓肠肌中分离和培养原代成纤维细胞，以及2）使用差异超速离心法结合蔗糖密度梯度，然后进行蛋白质印迹分析的外泌体纯化和表征。

视频: Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

科研

生物学

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

骨骼肌完整的线粒体的分离差速离心高分辨率测量呼吸运动

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

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 Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

人体心室心肌细胞与颤音-切切心肌切片分离

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Methods Article

骨骼肌成纤维细胞外泌体的分离和表征