This study was supported by the Swiss National Science Foundation (Grant 32003B_127619).

股四头肌肌肉活检是从麻醉的猪，从线粒体通过差速离心分离出服用。猪是后来用于另一个实验。这项研究是根据健康指引全国学院为照顾和使用实验动物，并与广州瑞士伯尔尼的动物保健委员会批准执行。 1.骨骼肌同质化和线粒体隔离<ol><li> 消费税从麻醉猪5-10克股四头肌标本。在本测定中，使用猪骨骼肌。该协议也可以被用于分离来自其它物种的线粒体（ 例如 ，大鼠，小鼠，人， 等 ）。 注:该步骤是通过用在外科手术专长的医学专家进行。 <ol><li>肌注氯胺酮（20毫克/公斤）和甲苯噻嗪（2毫克/公斤），猪稳重麻醉前诱导与内静脉咪达唑仑（0.5毫克/公斤，加阿托品0.02毫克/千克）。保持与手术期间异丙酚连续静脉输注（每股H 4-8毫克/千克）和芬太尼（每小时30微克/千克）麻醉。 </li></ol></li><li>立即浸泡在含有20毫升冰冷的线粒体隔离缓冲液（ 见表1）的烧杯中的组织样品。 同质化的过程中使用的缓冲区和离心应该是冰冷的，有一个生理相关pH与细胞质兼容离子和渗透强度:注意。 </li><li>称重在分析配衡天平烧杯中的组织样品。去皮与含有20毫升分离缓冲液的烧杯中的平衡。 </li><li>放在烧杯上一个冰桶。 注:执行所有在冰桶温度均匀化过程的下一步骤，并保持所有的缓冲区上的冰桶。 </li><li>剁碎烧杯中的骨骼肌使用细sciss（保持在冰上）到1-2毫米小块3-4分钟口服补液盐。 </li><li>用冰冷的分离缓冲液（20毫升，每次洗涤步骤）漂洗烧杯中的组织糜的两倍。 </li><li>暂停在10体积每含有5mg蛋白酶/ g组织的组织重量（g）隔离缓冲器的组织。 </li><li>放置烧杯中在磁性搅拌器冰浴，并搅拌10分钟。 </li><li>稀释在烧杯中的悬浮液用额外的10体积每补充有0.2％（重量/体积）脱脂牛血清白蛋白（BSA）的组织重量（g）的分离缓冲液中。 </li><li>滗所有补充有0.2％BSA的隔离缓冲液中，用补充有0.2％BSA（20毫升，每次洗涤工序）冰冷隔离缓冲器冲洗烧杯中的组织的两倍。 </li><li>悬浮在10体积的每补充有0.2％与脱脂BSA的组织重量（g）隔离缓冲器的组织。 </li><li>用均质半自动玻璃匀浆（放在冰上）用宽松杵（10杆）的组织。 注:HomogenizE在相同的方式始终组织（相同笔画数， 例如 ，10杆）。较高的笔画数可能会损坏和脱开线粒体。优选地，使用自动均化器均化该组织。手动（主观和）的玻璃匀浆组织匀浆样品可能会产生分离线粒体的不同特质。 </li><li>离心机在4℃的均质化的组织为在10,000×g下10分钟。 注:所有离心步骤与固定角转子离心机进行。 </li><li>使用血清吸管弃上清，悬浮在BSA-补充冰冷的分离培养基颗粒（10毫升/克组织）。 </li><li>离心该悬浮液在4℃下在350×g下10分钟。 </li><li>预留使用血清吸管将上清液弃沉淀（细胞碎片）。 </li><li>过滤上清液成通过两层纱布的烧杯（置于冰上）（17线/厘米2），以除去细胞debr是。 </li><li>离心机在4℃下经过滤的悬浮液以7,000×g的10分钟。 </li><li>弃去上清，重悬在补充有0.2％（重量/体积）BSA和离心悬浮液中分离缓冲液（5毫升/克组织）粗线粒体沉淀在4℃以7,000×g的10分钟。 </li><li>弃去上清，重悬在洗涤缓冲液（见表1）的粗线粒体沉淀。离心机在4℃下再次悬浮液以7,000×g的10分钟。 </li><li>弃去上清，重悬在洗涤缓冲液的粗线粒体沉淀并以7,000×于4℃再次离心悬浮液10分钟g。 </li><li>弃上清，悬浮粗颗粒的线粒体在1.0毫升洗涤缓冲液。 </li><li>确定线粒体悬浮液的蛋白质浓度，并保持浓缩的线粒体悬浮液在冰上。 注:蛋白质浓度可以使用任何标准方法来测定。 </li><li>前夕到呼吸测量测定法，重悬在呼吸缓冲器线粒体悬浮液至0.4毫克/毫升的线粒体蛋白的终浓度。 </li></ol> 2.高分辨率呼​​吸测量<ol><li>吸移管2.1毫升呼吸缓冲液（ 表1）16到一个高分辨率oxygraph室和搅拌缓冲器在37℃下连续地使用存在于所述腔室（700转）磁性搅拌棒进行1小时，直到稳定的氧通量信号得到极谱氧传感器。 注:各oxygraph腔内极谱氧电极测量氧浓度和各室内计算耗氧量（光通量）。氧浓度和耗氧速率（流量）正在使用的数据采集和分析软件显示实时在线在计算机中。此外，为了确保准确和可靠的结果，器乐氧背景校正应根据制造商的说明进行。 注:准确2.1毫升呼吸缓冲液闭挡块后添加到腔室为2.0 mL的最终室体积校准。 </li><li>根据制造商的协议17执行极谱氧传感器的空气校准。 注:在空气校准，具有本气相，媒体氧气浓度将达到平衡的15-20分钟的量级。根据不同的温度，气压，和媒体 - 氧浓度的溶解度可以计算出来。 </li><li>空气校准后，吸从oxygraph室呼吸介质和添加2.1毫升由步骤1.24孤立线粒体悬浮液（0.4毫克/毫升线粒体蛋白）向oxygraph的腔室。 注:呼吸缓冲区的容量取决于设置仪表和制造商。对于高分辨率呼​​吸测量，2.1毫升呼吸马步FFER正在关闭塞子后添加到腔室为2.0 mL的最终室体积校准。 </li><li>由止动器的插入关闭oxygraph室中。 </li><li>搅拌线粒体悬浮液连续地使用存在于所述腔室（700转），在37℃在基线磁性搅拌棒并记录细胞呼吸3-5分钟，直到一个稳定的氧通量信号得以实现。 注意:氧浓度和氧通量信号正在使用的数据采集和分析17软件显示实时在线在计算机中。 </li><li>之后，注入底物和抑制剂用于通过使用以下标准协议的挡块的钛喷射口线粒体呼吸。 </li></ol> 3.复杂我依赖呼吸<ol><li>制备含有分离的线粒体悬浮液（0.4毫克/毫升）按照步骤2.1-2.5和描述的方法的oxygraph室等待稳定的信号。 </li><li>注入12.5微升的0.8M的苹果酸（5mM的终浓度）和2M的谷氨酸10μL的（10毫米终浓度）至含有分离的线粒体悬浮液（0.4毫克/毫升）通过腔室止动件的钛喷射口和oxygraph室直到一个稳定的氧气流量信号实现了创记录的细胞呼吸3-5分钟。 注意:在下面的步骤的所有注射通过使用注射器塞子的钛喷射口进行。 </li><li>注入的0.05M的ADP（0.25毫摩尔）进oxygraph室10微升通过室止动件的钛注射口和记录线粒体呼吸，直到氧通量信号增加，然后减小，稳定（3-5分钟）。 注意:ADP加入后呼吸高原表明ADP浓度高，饱和。取决于呼吸期间使用线粒体的量的ADP浓度应该滴定一个稳定的状态3耗氧量（最大呼吸）。 ADP到线粒体悬浮液的除将引起在氧消耗的增加和氧通量信号将增加（状态3呼吸）。 ADP的被消耗后，氧通量信号将降低（状态4呼吸）。 </li></ol> 4.复合物II依赖呼吸<ol><li>制备oxygraph室包含以下步骤2.1-2.5中描述，并等待一个稳定的信号的步骤分离的线粒体悬浮液（0.4毫克/毫升）。 </li><li>注入0.2mM的鱼藤酮（0.2μM）"警告"的2微升到通过使用注射器腔室止动件的钛喷射口oxygraph室并记录3-5分钟的细胞呼吸，直到稳定的氧通量信号得以实现。 注:鱼藤酮是一种危险的毒药，可能对人体健康有显著的影响。 </li><li>注射1M的琥珀酸酯（10毫米）的20微升至oxygraph通道琥珀3-5分钟记录线粒体呼吸，直到一个稳定的氧气流量信号来实现的。 </li><li>注入的0.05M的ADP（0.25毫摩尔）进oxygraph室10微升通过室止动件的钛注入口，并记录细胞呼吸，直到氧通量信号的增加，稳定，然后下降并稳定（3-5分钟）。 注意:ADP到线粒体悬浮液中加入将引起在氧消耗的增加和氧通量信号将增加（状态3呼吸）。 ADP的被消耗后，氧通量信号将降低（状态4呼吸）。 </li></ol> 5.复合物IV依赖呼吸<ol><li>制备含有分离的线粒体悬浮以下步骤2.1到协议2.5和等待稳定的信号中描述的方法的oxygraph室（0.4毫克/毫升）。 </li><li>再注入2.5微升0.8毫米抗坏血酸（1毫米）。此后立即注入0.2 2.5微升中号TMPD（0.25毫摩尔）和0.05摩尔的ADP（0.25毫摩尔）进oxygraph腔和记录细胞呼吸，直到氧通量信号增加10μL的并稳定（3-5分钟）。 </li><li>最后注入的1M叠氮化钠（5毫摩）&#39;小心"入oxygraph室的10微升并记录细胞呼吸，直到氧通量信号下降并稳定。 注:叠氮化钠是一种危险的毒药，可能对人体健康有显著影响。 </li></ol> 6.细胞色素C测试<ol><li>制备含有分离的线粒体悬浮以下步骤2.1到协议2.5和等待稳定的信号中描述的方法的oxygraph室（0.4毫克/毫升）。 </li><li>注入12.5微升的0.8M的苹果酸（5mM的终浓度）和2M的谷氨酸10μL的（10毫米终浓度）至含有分离的线粒体悬浮液（0.4毫克/毫升）通过腔室止动件的钛喷射口oxygraph室并记录细胞呼吸3-5分钟，直到一个稳定的氧通量信号得以实现。 </li><li>注入0.5M的ADP（2.5毫摩尔）到oxygraph室10微升通过腔室挡块和记录线粒体呼吸，直到氧通量信号增加的钛注入口，然后下降并稳定（3-5分钟）。 </li><li>最后，注入4毫色素c（10μM）插入oxygraph室的5微升和直到一个稳定的氧通量信号实现记录细胞呼吸5分钟。 </li></ol>

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Chapter 3. 3, (2001).</li><li>Franko, 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=Efficient+isolation+of+pure+and+functional+mitochondria+from+mouse+tissues+using+automated+tissue+disruption+and+enrichment+with+anti-TOM22+magnetic+beads.">Efficient isolation of pure and functional mitochondria from mouse tissues using automated tissue disruption and enrichment with anti-TOM22 magnetic beads.</a> PLoS One. 8 (12), 382392 (2013).</li><li>Pecinov&#225;, A., Drahota, Z., N&#367;skov&#225;, H., Pecina, P., Hou&#353;t&#283;k, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+basic+mitochondrial+functions+using+rat+tissue+homogenates.">Evaluation of basic mitochondrial functions using rat tissue homogenates.</a> Mitochondrion. 11 (5), 722-728 (2011).</li><li>Lombardi, 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=Characterisation+of+oxidative+phosphorylation+in+skeletal+muscle+mitochondria+subpopulations+in+pig:+a+study+using+top-down+elasticity+analysis.">Characterisation of oxidative phosphorylation in skeletal muscle mitochondria subpopulations in pig: a study using top-down elasticity analysis.</a> FEBS Lett. 475 (2), 84-88 (2000).</li></ol>

The authors have nothing to disclose.

在本研究中，我们描述一个协议以分离高品质的，完整和差速离心紧密耦合骨骼肌线粒体可用于功能性研究，例如高分辨率呼​​吸测量。 为了分离完整和紧耦合线粒体，有要在本协议中考虑的一些关键点。收获骨骼组织后，应立即浸入冰冷线粒体隔离缓冲器。所有离心步骤应在4℃进行，并线粒体悬浮液应该总是在冰上分离过程中被保持。分离过程应尽可能快地完成。将匀?...

线粒体生物能量学和呼吸能力不仅可以在透化的细胞或纤维，而且在分离线粒体进行研究。在本研究中，我们描述了一个协议，使用隔离差速离心高分辨率呼​​吸测量测量完整骨骼肌线粒体。 为了分离完整的线粒体为呼吸测量，组织是均质和线粒体通过常规差速离心法分离。的差速离心的方法是基于连续的离心（在一系列增加速度的）组织匀浆首先由Pallade引入和同事近70年前1。将组织用剪刀第一切碎并在玻璃匀浆用宽松杵机械匀化。之后将匀浆在低速和包含完整的组织中得到的沉淀，细胞离心碎片和细胞核被丢弃。然后，将上清液在高速离心几次和线粒体富集馏分被收集。的差速离心方法的优点来分离线粒体是:i）该方法是快速的和线粒体可以1-1.5小时内（呼吸实验应尽可能快地进行）来分离; ii）其是便宜;和iii）它是非常有效的，并通过差速离心获得的线粒体是呼吸测量测定足够纯的。差速离心方法的缺点以分离线粒体是ⅰ）线粒体可能损坏和均化过程中脱开; ii）与其它细胞组分线粒体（的污染可能由进一步洗涤线粒体沉淀用另外的离心步骤）来解决; iii）选择不同的线粒体亚群， 例如 ，在差分离心步骤的可能性，麻省理工学院ochondria较低致密可以排除7;和iv）线粒体细胞周围缺少和仅理论最大呼吸可以被测量。分离线粒体为呼吸测量测定法的另一种方法是在密度梯度离心2。在该技术中，组织提取物层叠在蔗糖或Percoll密度梯度（具有较高密度在离心管的底部）中的溶液，并在一定的速度离心，使线粒体根据从其他细胞组分中分离出来的密度。这种方法经常被用于脑线粒体与来自突触体非常低的污染隔离。然而，通过密度梯度离心分离大鼠肝线粒体是高度污染的其他细胞器3。这种方法的局限性是，存在于离心管中的蔗糖梯度可能破裂，以便我线粒体（渗透冲击）。 根据组织的类型;还有要考虑完整的线粒体通过差速离心分离有些重要的因素。首先需要在一个温和的方式来组织均匀。软组织，如肾，脑和肝需要均化期间施加温和的机械力。与此相反，硬组织如需要更强的机械力心肌和骨骼肌。组织糜通常与蛋白酶的同质软化组织之前处理。均化和离心分离期间使用的所有缓冲液应该是冰冷的，并有一个生理相关pH与胞浆4,5兼容离子和渗透强度。 一项研究中分离线粒体生物能量学的一个优点是，蜂窝质膜不需要是permeabilized用洗涤剂如毛地黄皂苷或皂苷4,6，这可能会危及线粒体外膜的完整性。分离的线粒体的另一个优点是不存在的其他胞质因子，这可能与线粒体功能，如氧消耗干扰分析。使用分离的线粒体的缺点是某些线粒体种群在离心步骤的可能的选择，均化过程中损坏线粒体，并且为了获得分离的线粒体7,8的良好产率的高量生物样品的要求。 分离过程后，线粒体复合物的呼吸速率I-，II-和IV依赖性（状态2，3和4）使用高分辨率呼​​吸测量确定的。对于复杂的I-驱动的呼吸，谷氨酸和苹果酸中，随后加入二磷酸腺苷（ADP）。对于复杂的Ⅱ-驱动呼吸，琥珀酸盐，然后加入由ADP。为复合物IV驱动呼吸，抗坏血酸和tetramethylphenylendiamine（TMPD）接着加入ADP 9，10，11，12。状态2是指氧消耗在单独基板的存在。状态3是指氧消耗在衬底和ADP的存在。国家4指的耗氧量ADP耗尽后。的呼吸控制率（RCR）是氧消耗ATP产生的耦合的索引，并且被计算为状态3和状态4 13，15之间的比率。 总之，我们描述了一种协议通过差速离心分离功能和完整的骨骼肌线粒体和使用这些分离mitochondria用于功能和生物能量研究，例如高分辨率呼​​吸测量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ADP</td>
			<td>Sigma</td>
			<td>A 4386</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma</td>
			<td>A 8674</td>
			<td>Chemical, dissolve in ethanol</td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>Merck</td>
			<td>1.00127</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A 7699</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A 6003</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>fluka</td>
			<td>3779</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma,</td>
			<td>G 1626</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>H 7523</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1.04936</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Merck</td>
			<td>1.04873</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>K-lactobionate</td>
			<td>Sigma</td>
			<td>L 2398</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M 9272</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Morpholinopropane sulphonic acid (MOPS)</td>
			<td>Merck</td>
			<td>1.06129</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>O2k-Core: Oxygraph-2k&#160;</td>
			<td>Oroboros Instruments</td>
			<td>10000-02</td>
			<td>High-resolution respirometry instrument</td>
		</tr>
		<tr>
			<td>Proteinase, bacterial</td>
			<td>Sigma</td>
			<td>P 8038</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma</td>
			<td>R 8875</td>
			<td>Chemical, dissolve in ethanol</td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma</td>
			<td>S 2378</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Schuett homogen-plus semiautomatic homogeniser&#160;</td>
			<td>schuett-biotec GmbH</td>
			<td>3.201 011</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T 8691</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>TMPD</td>
			<td>Sigma</td>
			<td>T 3134</td>
			<td>Chemical</td>
		</tr>
	</tbody>
</table>

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.

复杂的I-依赖呼吸 分离的线粒体复合物I依赖呼吸速率（状态2，3和4）使用高分辨率呼吸测量（ 图1，一个代表性的图表）来确定。线粒体复合物I底物，谷氨酸和苹果酸盐，接着加入加入的ADP。状态2是指氧的消耗，仅在基片的存在。状态3是指氧消耗在衬底和ADP的存在。国家4指的耗氧量ADP耗尽后。 RCR?...

Watch this Scientific Journal Video about Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements at JoVE.com

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

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-intact-mitochondria-from-skeletal-muscle-differential

Research

JoVE Journal

Biology

18.0K 次观看.  Inselspital, Bern University Hospital. 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.

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

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 ...

科研

生物学

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 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 ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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 ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

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 ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...