这项工作得到了NIAMS AR064873，Epigen Project PB的支持。 P01.001.019 / Progetto Bandiera Epigenomica IFT to LL

根据标准动物设施程序培育和维持小鼠，所有实验方案均经过动物福利保证和意大利卫生部内部动物研究伦理委员会批准，并遵守"NIH护理和使用指南"实验动物。 肌肉损伤和自噬通量的体内阻滞<ol><li> 肌肉受伤。 <ol><li>为了诱导急性骨骼肌损伤，将20μL的10μM心脏毒素（CTX）直接注射到体重约为20g的2个月大的C57BL / 6J小鼠的左胫骨前部（TA）肌肉中。使用相等数量的男性和女性。使用不受干扰的对侧肢体作为对照。 注意:在0.9％氯化钠溶液中的10μMCTX应过滤（0.22μmPVDF过滤器）并储存在-20°C。避免反复冻融循环。 </li><li>使用我具有30G针头的nsulin注射器，在TA的中间注射CTX。为了在TA中引起精确的伤害，确保注射器的针进入从肌肉的底部到顶部5mm深的远端肌腱附近（ 图1A ）。 </li></ol></li><li> 在不受干扰和受伤的小鼠中阻断自噬通量。 <ol><li>为了评估自噬通量，24小时后损伤（pi）通过腹膜内注射（IP）每24小时给予50mg / kg氯喹（CLQ）4天（ 图1B ）。 <ol><li>将CLQ（10mg / mL）溶于PBS 1X中并过滤0.2μm膜。称量每只老鼠，计算注射量的CLQ。 注意:在同一天准备和使用CLQ解决方案。 CLQ库存可在-20°C下储存长达一个月。由于自噬是一种与代谢有关的过程，因此总是在同一时间执行CLQ治疗（ 即早晨befo）10 AM）。 </li></ol></li></ol></li></ol>肌肉组织切片<ol><li> 老鼠牺牲 <ol><li>损伤后5天对小鼠进行安乐死。 </li><li>通过颈椎脱位或CO 2进行安乐死（随后进行颈椎脱位确认）。由于自噬过程与小鼠睡眠/饮食习惯有关，因此始终同时牺牲小鼠，优选在早晨（ 即早上10点之前）。 </li></ol></li><li> TA隔离和包容。 <ol><li>解剖前，用70％乙醇喷雾小鼠。 </li><li>使用剪刀在臀部水平的小鼠的背部皮肤上做一个小的垂直切口（3毫米长）。切割尾巴和脚部以简化皮肤清除。将皮肤拉向尾部，并将其取出以暴露下面的肌肉; TA肌肉容易定位， 如图2A所示 </STRONG&gt;。 </li><li>在两个镊子的帮助下，抓住远端肌腱。 </li><li>切远端肌腱; TA和伸肌腱（EDL）肌腱通常被共同剪切并分开（见步骤2.2.6）。 </li><li>使用镊子，用肌腱保持TA肌肉，并小心地将肌肉拉近近端（靠近膝盖; 图2B ）。 注意:此时，EDL和TA肌肉易于识别，TA比EDL更大，更肤浅。 </li><li>通过沿相反方向拉拽两根远端肌腱将TA与EDL肌肉分开（ 图2C ）。 </li><li>切开近端肌腱。 </li><li>使用镊子，去除覆盖肌肉的薄筋膜，而不会损坏组织。 </li><li>借助纸巾去除样品中的过多水分。确保肌肉既不潮湿也不会过度干燥，因为过多的水分会产生信号对肌肉造成重大损害，危及下一次染色。 </li><li>将最佳切割温度（OCT）化合物放置在模具底部，并将肌肉放入其中，方向如图2D所示。向烧杯中加入100％异戊烷，直至达到约3-4cm的深度。将放置在聚苯乙烯箱中的烧杯与液氮接触。 </li><li>不要让液氮进入烧杯，因为它会产生一个泡沫泡沫，这可能会影响夹杂物并损伤肌肉部分。 </li><li>观察异戊烷，并等到达到适当的温度（-140°C和-150°C之间）;在适当的温度下，冷却​​的异戊烷的固体白色层将在烧杯的底部结晶。 </li><li>如果异戊烷完全冻结固体，熔化并冷却至冷冻温度，然后再进行下一步骤。 </li><li>使用预制镊子，精细地将模具降低异戊烷约20-30秒。将冷冻的样品放入带有干冰的容器中，直至转移到-80°C的冷冻箱中。 注意:协议可以在这里暂停。 </li></ol></li><li> 肌肉组织冷冻切片。 <ol><li>在-80°C至少一夜之后，进行冷冻切片。 </li><li>将低温恒温刀和防滚板从-20°C冷冻箱中取出，并将它们放在相应的支架上进入低温恒温柜。 </li><li>将OCT上的一滴OCT放在样品柱上，并将其放置在垂直NS方向的冷冻样品上， 如图2D所示 。 </li><li>将样品桩放在卡盘上。 </li><li>不要将抗卷板向下拉在刀上，在40微米处做一些切割，以摆脱不包括肌肉的OCT。当肌肉变得可见时，将切片厚度减小到7-8μm。 </li><li>拉下防卷板直到与其对齐刀的边缘或一点点以上。 </li><li>切割7-8μm厚的横切面，每个组织学载玻片安装3-4片。 注意:建议的低温恒温器温度在-15°C和-23°C之间。样本的横截面需要随后的分析来确定卫星位置的肌肉干细胞。 </li><li>为了使切片的粘附最大化，将载玻片在室温下保持10-15分钟，以防止其在抗体孵育过程中脱落。 注意:空气干燥步骤可能会影响免疫染色，提供不明确的结果。幻灯片可以在-80°C下保存几个月。协议可以在这里暂停。 </li></ol></li><li> 通过进行苏木精曙红（H＆E）染色检查TA肌肉冷冻切片中的肌肉损伤。 <ol><li>评估肌肉的质量（ 即损伤的有效性，肌肉的分离和包容离子和冷冻切片），进行H＆E染色。对于每个实验时间点，用4％PFA固定一个幻灯片10分钟。固定后，在1x PBS的几个变化中清洗载玻片。 注意:注意。 PFA是致癌物质，必须小心处理。 </li><li>为每个实验点取一张幻灯片，并将其放入染色罐中。 </li><li>用9克/升苏木精填充罐，直到切片被覆盖并孵育8分钟。 </li><li>回收苏木精。 </li><li>将瓶子放在流水下10分钟，以除去过量的苏木精。 注意:确保不要将水直接流入各部分。 </li><li>用无菌水冲洗。 </li><li>在酸化的90％乙醇中孵育0.5％（w / v）曙红1分钟。 </li><li>回收曙红。 </li><li>用无菌水洗涤两次，洗涤3分钟。 注意:在化学防护罩上执行以下步骤。 </li><li>用70％乙醇洗涤切片。 </li><li>洗具有90％乙醇的切片。 </li><li>用100％乙醇洗涤切片。 </li><li>使用0.879 g / mL邻二甲苯孵育切片。 注意:注意。邻二甲苯是易燃和中等毒性的。在化学防护罩下操作，戴防护装备，包括手套，实验室外套和面罩。 </li><li>回收o-二甲苯。 </li><li>将幻灯片放在一张纸上干燥。 </li><li>使用快速硬化的基于二甲苯的安装介质封闭载玻片。 </li><li>以10倍放大倍率在光学显微镜下检查切片的质量（ 见图3 ） 注意:协议可以在这里暂停。 </li></ol></li></ol> 3.在损伤的肌肉组织切片中LC3和MyoD的免疫染色<ol><li> 肌肉组织切片的固定。 <ol><li>通过润湿一张纸并将其放置在可关闭的塑料盒的底部以保持较高的温度来准备孵育室室内湿度很大。将组织切片用1％PBS中的4％多聚甲醛（PFA）固定10分钟。 注意:使用湿纸张以避免样品干燥。准备新鲜的PFA或使用储存在-20°C的PFA。警告。 PFA有毒;制造原料溶液时，必须小心处理粉末。该操作必须在化学防护罩上进行，并带有防护装备，包括手套，实验室外套和面罩。此外，在解决方案时，应仔细操作PFA。 </li><li>取出PFA，用1x PBS洗涤5〜7分钟;重复这一步3次。 </li></ol></li><li> 肌肉组织切片的渗透。 <ol><li>使用pap笔在组织切片周围绘制疏水屏障。 注意:此步骤定义了组织切片周围的表面，并使步骤3.4,3.6,3.7和3.9中描述的抗体体积最小化。 </li><li>用200μL冷（-20°C）覆盖切片，甲醇，并将孵育室水平置于-20℃的冷冻箱中5分钟。 </li><li>从冷冻箱中取出孵育室，抽出甲醇，并在室温下用1X PBS洗涤切片5-7分钟;重复此步骤3x。 </li></ol></li><li> 闭塞 <ol><li>准备4％牛血清白蛋白（BSA）在PBS 1X中的新鲜块溶液。 注意:BSA溶液可以在4℃下储存1周。 </li><li>在室温下用块溶液孵育至少60分钟。 </li><li>移除阻塞解决方案。 </li><li>通过稀释1×PBS中的20μg/ mL抗Fab制备100μL抗Fab混合物。使用非偶联亲和纯化的抗小鼠IgG的F（ab）片段在室温孵育切片1小时。 </li></ol></li><li> 初级抗体孵育。 <ol><li>通过将LC3和MyoD抗体溶解在1％PBS中的4％BSA中，为每个载玻片制备100μL的一抗混合物。使用5μg/mL抗LC3（兔多克隆抗体）和10mg / L抗MyoD（小鼠单克隆抗体）。在4℃孵育一次抗体混合物过夜。 </li></ol></li><li> 洗。 <ol><li>取出一级抗体混合物。 </li><li>在1X PBS中用1％BSA洗涤切片10分钟;重复此步骤3x。 </li></ol></li><li> 二次抗体孵育。 <ol><li>为每个载玻片准备100μL的二抗混合物。在1％PBS中的4％BSA中溶解山羊抗兔488（5μg/ mL）和山羊抗小鼠594（5μg/ mL）。 注意:避免过度的光照，以防止荧光染色。在黑暗中执行以下步骤。 </li><li>在室温下使用二级抗体混合物孵育切片45分钟。 </li><li>取出第二抗体混合物，用1x PBS洗涤切片5-7分钟;重复此步骤3x。 </li></ol></li><li> 初级抗体孵育。 <ol><li>稀释层压板在1％PBS中的4％BSA中的n-2（α-2-链）单克隆抗体（0.33μg/ mL），每个载玻片使用100μL混合物。在一次抗体混合物中孵育1-2小时。 </li></ol></li><li> 洗。 <ol><li>取出一级抗体混合物。 </li><li>在1X PBS中用1％BSA洗涤切片10分钟;重复此步骤3x。 </li></ol></li><li> 二次抗体孵育。 <ol><li>通过在1％PBS中的4％BSA中稀释山羊抗大鼠647（5μg/ mL），为每个载玻片制备100μL的二抗混合物。在二级抗体混合物中孵育45分钟。 </li><li>取出第二抗体混合物，用1x PBS洗涤切片5-7分钟;重复此步骤3x。 </li></ol></li><li> 4&#39;，6-二脒基-2-苯基吲哚（DAPI）孵育。 <ol><li>在每个载玻片中加入200μL300 nM DAPI溶液，在1×PBS中。在室温下孵育5分钟。将DAPI溶液储存在4℃黑暗。 </li><li>用1x PBS洗涤切片5-7分钟;重复此步骤3x。 </li></ol></li><li> 安装染色肌肉部分。 <ol><li>从部分去除多余的洗涤溶液。 </li><li>将10μL甘油在1×PBS（3:1）中放置在载玻片中间的染色部分，并加入盖玻片，避免形成气泡。 </li></ol></li></ol>共聚焦显微镜获取<ol><li>使用与图像采集系统和分析软件集成的四激光共焦显微镜获取图像。 注意:MyoD与594染料（一种亮红色荧光染料）共轭，其激发理想地适用于594nm激光（激发:590nm，发射:617nm）。 LC3与488染料（其是亮绿色荧光染料）共轭，理想地适用于488nm激光线（激发:495nm，发射:519nm）。层粘连蛋白与647染料缀合一种明亮的远红荧光染料，具有理想适用于647nm激光线（激发:650nm，发射:668nm）的激发。 </li><li>将幻灯片放置在显微镜托盘中并使用放大倍数63倍以检测核信号。通过使活动再生的领域居中，依靠肌肉形态，手动聚焦在再生区域（参见图4 ）。 注意:在无刺激的肌肉中，肌纤维的大小几乎是恒定的（ 图4A ），损伤介导的肌肉再生可以通过更新的肌纤维的外观来识别，这是再生后新形成的纤维。此外，主动再生中的肌肉的特征在于由巨噬细胞，纤维成脂前体以及定位于受损肌肉以编排肌肉再生的细胞形成的广泛浸润（ 图4B ）。 </li><li>评估再生中的免疫荧光染色重点是位于肌纤维基底层下的MuSCs。 </li><li>专注于对MyoD染色阳性的MuSCs，并位于由层粘连蛋白染色标记的肌纤维内（参见图4B ，白色箭头）。 </li><li>使用至少3只小鼠/实验组，并使用显微镜获得每个实验组至少7个场的63倍放大图像。 </li><li> 一旦识别再生区域，使用DAPI染色进行聚焦，然后调整其他单个通道。 <ol><li>使用以下显微镜设置:通道A594针孔1.2通风单元，增益791;通道A488针孔1.6通风单位，增益659;通道A647针孔1.4通风单位，增益719。 </li></ol></li><li>调整单个通道的焦点后，继续采集1,024 x 1,024帧大小和12.61μs像素停留的图像。 </li><li>计算MyoD阳性细胞的百分比（对照t他正确地位于层下）LC3阴性和显示LC3信号的MyoD阳性细胞的百分比。 注意:显示LC3染色的MyoD阳性细胞的百分比是该方案的读数。 </li></ol>

<ol>
	<li>Bentzinger, C. 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=Differential+response+of+skeletal+muscles+to+mTORC1+signaling+during+atrophy+and+hypertrophy.">Differential response of skeletal muscles to mTORC1 signaling during atrophy and hypertrophy.</a> Skelet Muscle. 3 (1), (2013).</li><li>Chakkalakal, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aged+niche+disrupts+muscle+stem+cell+quiescence.">The aged niche disrupts muscle stem cell quiescence.</a> Nature. 490 (7420), 355-360 (2012).</li><li>Jang, Y. 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=Skeletal+muscle+stem+cells:+effects+of+aging+and+metabolism+on+muscle+regenerative+function.">Skeletal muscle stem cells: effects of aging and metabolism on muscle regenerative function.</a> Cold Spring Harb Symp Quant Biol. 76, 101-111 (2011).</li><li>Cheung, T. H., 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=Molecular+regulation+of+stem+cell+quiescence.">Molecular regulation of stem cell quiescence.</a> Nat Rev Mol Cell Biol. 14 (6), 329-340 (2013).</li><li>Collins, C. A., Partridge, 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=Self-renewal+of+the+adult+skeletal+muscle+satellite+cell.">Self-renewal of the adult skeletal muscle satellite cell.</a> Cell Cycle. 4 (10), 1338-1341 (2005).</li><li>Bentzinger, C. 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=Cellular+dynamics+in+the+muscle+satellite+cell+niche.">Cellular dynamics in the muscle satellite cell niche.</a> EMBO Rep. 14 (12), 1062-1072 (2013).</li><li>Bernet, J. D., 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=p38+MAPK+signaling+underlies+a+cell-autonomous+loss+of+stem+cell+self-renewal+in+skeletal+muscle+of+aged+mice.">p38 MAPK signaling underlies a cell-autonomous loss of stem cell self-renewal in skeletal muscle of aged mice.</a> Nat Med. 20 (3), 265-271 (2014).</li><li>Cosgrove, B. D., 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=Rejuvenation+of+the+muscle+stem+cell+population+restores+strength+to+injured+aged+muscles.">Rejuvenation of the muscle stem cell population restores strength to injured aged muscles.</a> Nat Med. 20 (3), 255-264 (2014).</li><li>Sousa-Victor, P., 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=Geriatric+muscle+stem+cells+switch+reversible+quiescence+into+senescence.">Geriatric muscle stem cells switch reversible quiescence into senescence.</a> Nature. 506 (7488), 316-321 (2014).</li><li>Madaro, L., Latella, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forever+young:+rejuvenating+muscle+satellite+cells.">Forever young: rejuvenating muscle satellite cells.</a> Front Aging Neurosci. 7, 37 (2015).</li><li>Price, F. D., 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=Inhibition+of+JAK-STAT+signaling+stimulates+adult+satellite+cell+function.">Inhibition of JAK-STAT signaling stimulates adult satellite cell function.</a> Nat Med. 20 (10), 1174-1181 (2014).</li><li>Tierney, M. 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=STAT3+signaling+controls+satellite+cell+expansion+and+skeletal+muscle+repair.">STAT3 signaling controls satellite cell expansion and skeletal muscle repair.</a> Nat Med. 20 (10), 1182-1186 (2014).</li><li>Judson, R. N., Zhang, R. H., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+mesenchymal+stem/progenitor+cells+in+skeletal+muscle:+collaborators+or+saboteurs?.">Tissue-resident mesenchymal stem/progenitor cells in skeletal muscle: collaborators or saboteurs?.</a> FEBS J. 280 (17), 4100-4108 (2013).</li><li>Kroemer, G., Marino, G., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+the+integrated+stress+response.">Autophagy and the integrated stress response.</a> Mol Cell. 40 (2), 280-293 (2010).</li><li>Marino, G., Madeo, F., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+for+tissue+homeostasis+and+neuroprotection.">Autophagy for tissue homeostasis and neuroprotection.</a> Curr Opin Cell Biol. 23 (2), 198-206 (2011).</li><li>Jiang, P., Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+human+diseases.">Autophagy and human diseases.</a> Cell Res. 24 (1), 69-79 (2014).</li><li>Eskelinen, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doctor+Jekyll+and+Mister+Hyde:+autophagy+can+promote+both+cell+survival+and+cell+death.">Doctor Jekyll and Mister Hyde: autophagy can promote both cell survival and cell death.</a> Cell Death Differ. 12, 1468-1472 (2005).</li><li>Basile, V., 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=bis-Dehydroxy-Curcumin+triggers+mitochondrial-associated+cell+death+in+human+colon+cancer+cells+through+ER-stress+induced+autophagy.">bis-Dehydroxy-Curcumin triggers mitochondrial-associated cell death in human colon cancer cells through ER-stress induced autophagy.</a> PLoS One. 8 (1), e53664 (2013).</li><li>Neel, B. A., Lin, Y., Pessin, 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=Skeletal+muscle+autophagy:+a+new+metabolic+regulator.">Skeletal muscle autophagy: a new metabolic regulator.</a> Trends Endocrinol Metab. 24 (12), 635-643 (2013).</li><li>Sandri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+skeletal+muscle.">Autophagy in skeletal muscle.</a> FEBS Lett. 584 (7), 1411-1416 (2010).</li><li>Grumati, P., 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=Autophagy+is+defective+in+collagen+VI+muscular+dystrophies,+and+its+reactivation+rescues+myofiber+degeneration.">Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration.</a> Nat Med. 16 (11), 1313-1320 (2010).</li><li>Chrisam, M., 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=Reactivation+of+autophagy+by+spermidine+ameliorates+the+myopathic+defects+of+collagen+VI-null+mice.">Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice.</a> Autophagy. 11 (12), 2142-2152 (2015).</li><li>Grumati, P., 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=Autophagy+induction+rescues+muscular+dystrophy.">Autophagy induction rescues muscular dystrophy.</a> Autophagy. 7 (4), 426-428 (2011).</li><li>De Palma, 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=Autophagy+as+a+new+therapeutic+target+in+Duchenne+muscular+dystrophy.">Autophagy as a new therapeutic target in Duchenne muscular dystrophy.</a> Cell Death Dis. 3, e418 (2012).</li><li>Hindi, S. M., 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=Distinct+roles+of+TRAF6+at+early+and+late+stages+of+muscle+pathology+in+the+mdx+model+of+Duchenne+muscular+dystrophy.">Distinct roles of TRAF6 at early and late stages of muscle pathology in the mdx model of Duchenne muscular dystrophy.</a> Hum Mol Genet. 23 (6), 1492-1505 (2014).</li><li>Pauly, M., 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=AMPK+activation+stimulates+autophagy+and+ameliorates+muscular+dystrophy+in+the+mdx+mouse+diaphragm.">AMPK activation stimulates autophagy and ameliorates muscular dystrophy in the mdx mouse diaphragm.</a> Am J Pathol. 181 (2), 583-592 (2012).</li><li>Spitali, P., 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=Autophagy+is+Impaired+in+the+Tibialis+Anterior+of+Dystrophin+Null+Mice.">Autophagy is Impaired in the Tibialis Anterior of Dystrophin Null Mice.</a> PLoS Curr. 5, (2013).</li><li>Whitehead, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+autophagy+as+a+potential+mechanism+for+the+improved+physiological+function+by+simvastatin+in+muscular+dystrophy.">Enhanced autophagy as a potential mechanism for the improved physiological function by simvastatin in muscular dystrophy.</a> Autophagy. 12 (4), 705-706 (2016).</li><li>Whitehead, N. P., 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=A+new+therapeutic+effect+of+simvastatin+revealed+by+functional+improvement+in+muscular+dystrophy.">A new therapeutic effect of simvastatin revealed by functional improvement in muscular dystrophy.</a> Proc Natl Acad Sci U S A. 112 (41), 12864-12869 (2015).</li><li>Fiacco, E., 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=Autophagy+regulates+satellite+cell+ability+to+regenerate+normal+and+dystrophic+muscles.">Autophagy regulates satellite cell ability to regenerate normal and dystrophic muscles.</a> Cell Death Differ. 23 (11), 1839-1849 (2016).</li><li>Lee, I. H., 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=A+role+for+the+NAD-dependent+deacetylase+Sirt1+in+the+regulation+of+autophagy.">A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy.</a> Proc Natl Acad Sci U S A. 105 (9), 3374-3379 (2008).</li><li>Rubinsztein, D. C., Mari&#241;o, G., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+aging.">Autophagy and aging.</a> Cell. 146 (5), 682-695 (2011).</li><li>Colman, R. J., 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=Caloric+restriction+delays+disease+onset+and+mortality+in+rhesus+monkeys.">Caloric restriction delays disease onset and mortality in rhesus monkeys.</a> Science. 325 (5937), 201-204 (2009).</li><li>Levine, B., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+the+pathogenesis+of+disease.">Autophagy in the pathogenesis of disease.</a> Cell. 132 (1), 27-42 (2008).</li><li>Yang, 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=Long-Term+Calorie+Restriction+Enhances+Cellular+Quality-Control+Processes+in+Human+Skeletal+Muscle.">Long-Term Calorie Restriction Enhances Cellular Quality-Control Processes in Human Skeletal Muscle.</a> Cell Rep. 14 (3), 422-428 (2016).</li><li>Wenz, 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=Increased+muscle+PGC-1alpha+expression+protects+from+sarcopenia+and+metabolic+disease+during+aging.">Increased muscle PGC-1alpha expression protects from sarcopenia and metabolic disease during aging.</a> Proc Natl Acad Sci USA. 106 (48), 20405-20410 (2009).</li><li>Carnio, S., 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=Autophagy+Impairment+in+Muscle+Induces+Neuromuscular+Junction+Degeneration+and+Precocious+Aging.">Autophagy Impairment in Muscle Induces Neuromuscular Junction Degeneration and Precocious Aging.</a> Cell Rep. , (2014).</li><li>Sandri, M., 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=Misregulation+of+autophagy+and+protein+degradation+systems+in+myopathies+and+muscular+dystrophies.">Misregulation of autophagy and protein degradation systems in myopathies and muscular dystrophies.</a> J Cell Sci. 126 (Pt 23), 5325-5333 (2013).</li><li>Cerletti, M., 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=Short-term+calorie+restriction+enhances+skeletal+muscle+stem+cell+function.">Short-term calorie restriction enhances skeletal muscle stem cell function.</a> Cell Stem Cell. 10 (5), 515-519 (2012).</li><li>Gopinath, S. D., 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=FOXO3+promotes+quiescence+in+adult+muscle+stem+cells+during+the+process+of+self-renewal.">FOXO3 promotes quiescence in adult muscle stem cells during the process of self-renewal.</a> Stem Cell Reports. 2 (4), 414-426 (2014).</li><li>Tang, A. H., 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=Induction+of+autophagy+supports+the+bioenergetic+demands+of+quiescent+muscle+stem+cell+activation.">Induction of autophagy supports the bioenergetic demands of quiescent muscle stem cell activation.</a> EMBO J. 33 (23), 2782-2797 (2014).</li><li>Garcia-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=Autophagy+maintains+stemness+by+preventing+senescence.">Autophagy maintains stemness by preventing senescence.</a> Nature. 529 (7584), 37-42 (2016).</li><li>Klionsky, D. J., 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=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+(3rd+edition).">Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).</a> Autophagy. 12 (1), 1-222 (2016).</li></ol>

作者没有什么可以披露的

<html>该协议描述了如何在代偿性肌肉再生期间监测骨骼肌干细胞中的自噬。尝试了几种用于LC3和MyoD共染色的抗体，在这里列出了在小鼠组织切片中工作并产生成功结果的抗体 （参见材料表 ）。强烈推荐使用甲醇渗透（见步骤3.2.2）以进行成功染色。 该方案的局限性与小鼠的内在变异性相关，强迫使用至少3只小鼠/实验组。该方法反映了在骨骼肌再生...

骨骼肌再生是成体干细胞（肌肉卫星细胞，MuSCs）与参与再生过程的其他细胞类型之间相互作用的结果。肌肉的体内平衡和功能由肌肉利基和全身线索1,2所组合的信号保持。在整个一生中，已经报告了MuSC功能，肌肉利基和系统线索的变化，导致老年人的功能能力下降3 。 MuSCs位于基底层下方的一个利基，并且在肌肉损伤时被激活以修复损伤的肌肉4,5 。为了确保有效的再生反应，至关重要的是，MuSC协调退出静止，自我更新和增殖扩张阶段所需的不同过程通过肌原性分化6 。在老年人和肌肉慢性疾病中，所有这些功能受到损害，导致肌肉功能变化2,3,6,7,8,9,10,11,12,13。 Macroautophagy（以下称为自噬）正在成为维持组织稳态至关重要的生物过程14 。自噬过程包括贩运机制，其中细胞质，细胞器和蛋白质的部分被吞入囊泡，其最终通过溶酶体途径降解，促进有毒分子的去除和大分子的再循环cules。这提供了能量丰富的化合物，以支持在应激或其他不利条件下的细胞和组织适应15,16 。与其细胞存活活动一起，自噬也可以作为细胞死亡诱导剂，取决于细胞组织背景（ 例如，正常与癌组织）和应激刺激的类型17,18 。 最近的证据表明，自噬需要维持肌肉质量和肌纤维完整性19,20 ，据报道在不同的肌营养不良21,22,23 中受损，包括Duchenne肌营养不良症（DMD）24,25,26,27 </su同样，在失去肌肉量（称为肌肉减少症）后，31,33,34,35岁的老年人中观察到自噬过程的逐渐减少32,33,34 ， 35,36,37和肌纤维存活38 。 热带实验室的一项研究预计自噬和骨骼肌再生潜能之间有着密切的关系，这表明热量限制增强了MuSC的可用性和活性。这不行最近观察到Foxo3-Notch轴激活了自我更新期间的自噬过程40以及从静止到增殖状态的MuSC转变。这些数据符合青年时期老年人和老年人的基础性自噬的逐渐减少，以及老年期间MuSCs的数值和功能下降42 。 在最近的一篇论文中，我们展示了自噬和补偿性肌肉再生之间的密切关系，区分了DMD进展的早期阶段。因此，当肌肉再生受损和纤维化组织沉积发生时，我们观察到疾病进展后期阶段的自噬通量减少。有趣的是，我们表明，在再生条件下，自噬在MuSCs中被激活，并且调节自噬过程影响MuSC激活和fu功能30 总而言之，这些数据突显了在正常和病理状况下以及整个寿命期间，在肌肉再生过程中探索MuSCs自噬过程的紧迫性。在这里，我们提供了一个协议，通过对微管相关蛋白1A / 1B-轻链3（LC3） 进行原位免疫染色监测肌肉再生条件下的自噬过程，LC3是自噬标志物43 ，MyoD是一种标记肌原纤维谱系，在对照组和受损小鼠的肌肉组织切片中。报告的方法允许监测一个特定细胞室的自噬过程，MuSC在编排肌肉再生中起关键作用。

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:332px;">C57BL/6J</td>
			<td style="width:251px;">The Jackson Laboratory</td>
			<td style="width:128px;">000664</td>
			<td style="width:196px;">WT mice</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cardiotoxin 1</td>
			<td>Latoxan</td>
			<td>L8102</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Millex-VV</td>
			<td>Merck Millipore</td>
			<td>SLVV033RS</td>
			<td style="width:196px;">Syringe Filter Unit, 0.1 &#181;m, PVDF, 33 mm, gamma sterilized</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Chloroquine diphosphate salt</td>
			<td>Sigma-Aldrich</td>
			<td>C6628</td>
			<td style="width:196px;">Caution: 
			Harmful if swallowed</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">BD Micro-Fine + 0,5 mL</td>
			<td>BD</td>
			<td>324825</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue-Tek O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue-Tek Cryomold Intermediate</td>
			<td>Sakura Finetek</td>
			<td>4566</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2-Methylbutane</td>
			<td>Sigma-Aldrich</td>
			<td>277258</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hematoxylin Solution, Harris Modified</td>
			<td>Sigma-Aldrich</td>
			<td>HHS32</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Eosin Y solution, alcoholic</td>
			<td>Sigma-Aldrich</td>
			<td>HT110132</td>
			<td></td>
		</tr>
		<tr height="122">
			<td height="122" style="height:122px;">o-Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>X1040</td>
			<td style="width:196px;">Caution: 
			Flammable liquid and vapour; May be fatal if swallowed and enters airways; Harmful in contact with skin; May cause respiratory irritation; Causes serious eye irritation</td>
		</tr>
		<tr height="136">
			<td height="136" style="height:136px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td style="width:196px;">Caution: 
			Flammable solid; Harmful if swallowed; Causes skin irritation; May cause an allergic skin reaction; Causes serious eye damage; May cause respiratory irritation; Suspected of causing cancer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DPBS, no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Eukitt - Quick-hardening mounting medium</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-007-003</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LC3B Antibody</td>
			<td>Cell signaling Technology</td>
			<td>2775</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:332px;">Monoclonal mouse anti-MyoD 
			(concentrated) clone 5.8A</td>
			<td>DAKO - Agilent Pathology Solutions</td>
			<td>M3512</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Laminin-2 (&#945;-2-chain) monoclonal antibody</td>
			<td>Enzo Life Sciences</td>
			<td>4H8-2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)</td>
			<td style="width:251px;">Life technologies</td>
			<td style="width:128px;">A11008</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Alexa Fluor 594 Goat Anti-Mouse IgG (H+L)</td>
			<td style="width:251px;">Life technologies</td>
			<td style="width:128px;">A11005</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Alexa Fluor Goat Anti-Rat IgM Antibody</td>
			<td style="width:251px;">Life technologies</td>
			<td style="width:128px;">A21248</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ immunofluorescent staining of autophagy in muscle stem cells

越来越多的证据表明自噬是维持组织稳态的重要调节过程。已知自噬涉及骨骼肌发育和再生，并且自噬过程已经在几种肌肉病理学和年龄相关的肌肉疾病中描述。与肌肉修复期间卫星细胞的功能衰竭相关的自噬过程的最近描述的块支持活性自噬与生产性肌肉再生相结合的概念。这些数据揭示了在正常和病理状态（如肌营养不良）的肌肉再生过程中自噬在卫星细胞活化中的关键作用。在这里，我们提供了在肌肉再生条件下监测成年肌肉干细胞（MuSC）隔室中的自噬过程的方案。该协议描述了LC3的原位免疫荧光成像的设置方法，autophagy标记和MyoD，一种肌原性谱系标记，来自对照组和受损小鼠的肌肉组织切片。报告的方法允许监测一个特定细胞室的自噬过程，MuSC隔室在调节肌肉再生中起着核心作用。

该方案描述了在肌肉再生过程中检测MuSCs中的自噬的有效的原位方法。 CTX 体内治疗: 使用CTX诱导TA肌肉的肌肉损伤，并使用不受干扰的肌肉作为对照。由于自噬是高度动态的，通过执行CLQ的IP注射来阻断自噬通量（ 图1 ）。 CLQ治疗对于评估自噬通?...

Watch this Scientific Journal Video about In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells at JoVE.com

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

活动性自噬与生产性肌肉再生有关，肌肉再生对肌肉干细胞（MuSC）的激活至关重要。在这里，我们提供了原位检测LC3的方案，这是来自对照组和受伤小鼠的肌肉组织切片的MyoD阳性MuSC中的自噬标记。

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

in-situ-immunofluorescent-staining-of-autophagy-in-muscle-stem-cells

Research

JoVE Journal

Biology

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

10.1K 次观看.  Italian National Research Council. 活动性自噬与生产性肌肉再生有关，肌肉再生对肌肉干细胞（MuSC）的激活至关重要。在这里，我们提供了原位检测LC3的方案，这是来自对照组和受伤小鼠的肌肉组织切片的MyoD阳性MuSC中的自噬标记。 

视频: In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

对大鼠的过程<em在原位</em骨骼肌收缩性能

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

科研

生物学

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

伸趾长肌的肌肉功能评价<em&gt;体外</em和胫骨前肌<em在原位</em在小鼠

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...