这项研究是由美国国家心脏，肺和血液研究所资助HL-103542，并在散麻醉研究基金会的杰出开拓者奖CAP支持。

准备<ol><li>制备的1M K-HEPES溶液（pH调节至7.2，用KOH）。 </li><li>制备0.5M的K-EGTA溶液（调节pH值至8.0，用KOH）。 </li><li>制备的1M KH 2 PO 4的储备溶液。 </li><li>准备1米氯化镁储备溶液。 </li><li>制备均质缓冲液（pH 7.2）中300mM的蔗糖，10mM的K-HEPES，和1mM K-EGTA。保存在4℃。 </li><li>制备呼吸缓冲液250mM的蔗糖，2mM的KH 2 PO 4，10mM的MgCl 2的，20mM的K-HEPES缓冲液（pH值7.2）和0.5mM的K-EGTA（pH 8.0）中。保存在4℃。 </li><li>制备10倍的PBS原液溶解80克的NaCl，2克的KCl，14.4称量Na 2 HPO 4，和2.4克KH 2 PO 4加入到1 L双蒸H 2 O的 （pH 7.4）中。 </li><li>吹打百毫升10X PBS为1升双蒸H 2 O准备1X PBS </li><li>准备枯草杆菌蛋白酶A股称重在4毫克枯草杆菌蛋白酶A的以1.5 ml离心管中。储存在-20℃直到使用。 </li><li>准备BSA股票称取20毫克的BSA为1.5 ml离心管中。储存在-20℃直到使用。 </li></ol>线粒体的分离（图1） <ol><li>紧接隔离，溶解枯草杆菌蛋白酶A在1毫升均质缓冲液。 </li><li>紧接隔离，BSA溶解在1毫升均质缓冲液。 </li><li>使用6毫米活检样本冲和存储在1X PBS在冰50毫升离心管中收集两个新鲜组织标本。 </li><li>转移组织两个6毫米拳含有5ml冰冷的均质缓冲液的分离Ç管。 </li><li>由嵌合在组织离解的离解Ç管均质化的组织，并选择预先设定的线粒体隔离周期（60秒均化）。 </li><li>拆下分离ç管冰桶。 </li><li>加入250μl的枯草杆菌蛋白酶原液的匀浆，通过倒置混合，并培育在冰上匀浆10分钟。 </li><li>放置一个40微米的筛网过滤器上的冰和预湿的过滤器以均质缓冲液50ml锥形离心管中，并过滤匀浆成冰的50ml锥形离心管中。 </li><li>加入250微升新鲜制备的BSA储备液，向滤液并通过倒置混合。 注:省略这一步，如果需要线粒体蛋白的决心。 </li><li>放置一个40微米的过滤器上的冰和预湿的过滤器以均质缓冲液50ml锥形离心管中，并过滤匀浆成冰的50ml锥形离心管中。 </li><li>放置一个10微米的过滤器上的冰和预湿的过滤器以均质缓冲液50ml锥形离心管中，并过滤匀浆成冰的50ml锥形离心管中。 </li><li>将滤液转移到2预冷1.5毫升离心管和离心9000 xg离心10分钟4ºC。 </li><li>除去上清液并重新悬浮和结合粒料在1ml冰冷的呼吸缓冲液。 </li></ol> ATP含量注意:要确定分离线粒体的ATP荧光检测可以用一个ATP检测试剂盒进行的代谢活动。的协议，试剂和标准的测定试剂盒提供。的流程的概要进行说明。 <ol><li>平衡试剂盒至室温。 </li><li>准备10毫摩尔ATP原液在1170微升双蒸水溶解冻干ATP的颗粒。储存的ATP冰上标准储备液，准备线粒体样本。 </li><li>加入5毫升底物缓冲溶液，以冻干底物液的小瓶中。轻轻摇匀后放置在黑暗中。 </li><li>加入100μl呼吸缓冲液至黑色，不透明的底部，96孔板的所有孔中。 </li><li>从所制备的样品中添加10微升线粒体向每个孔ø发黑色，不透明的底部，96孔板中。注:样品被镀以一式三份。包括行的标准，三口井为阴性对照组（呼吸缓冲器）。 </li><li>加入50微升的哺乳动物细胞溶解液到所有孔中，包括标准和控制。 </li><li>孵育板在37℃5分钟，在定轨摇床上以125转每分钟。 </li><li>在孵育制备的0.1毫米，0.05毫米，0.01毫米，0.005毫米，0.001毫米，和0.0001毫摩尔ATP从10毫摩尔ATP储备溶液浓度的ATP标准。商店的标准在冰上。 </li><li>温育后，加入10微升的三磷酸腺苷标准，以相应的孔中的板图上标明。注意:标准进行双份。 </li><li>加入50微升重组的底物溶液的各孔中。 </li><li>孵育板在37℃在定轨振荡器进行5分钟，在125转每分钟。 </li><li>打开GEN5 1.11软件与分光光度计相连的计算机上。 </li><li>在"创建新项目"，点击"实验"。 </li><li>点击"默认协议"。单击"确定"。 </li><li>在左侧列中，选择"协议"，然后选择"程序"。 </li><li>选择"延迟"。设置为00:10:00。单击"确定"。 </li><li>选择"读取"。从下拉菜单中选择"发光"。调整其他设置如下:读取类型的端点，积分时间0:01.00 MM:SS.SS，过滤器设置1，发射孔，光学部位顶部，感光度100，顶探头垂直偏移1.00毫米。 </li><li>当板已准备好进行分析，点击最上面一排的"阅读板块"图标。点击"读取"。当分光光度计打开后，将板与A1孔在左上角的托盘。随着温度的对话框将打开。点击"读取"。注:值越高，关联与增加ATP水平和较高的代谢活动。 </li></ol>

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所有的动物都按照现行体制的指导方针进行处理。我们没有竞争的经济利益披露。

成功地分离出使用该协议，必须保持所有的解决方案和组织样品在冰上保持线粒体活力线粒体。在冰上保持甚至当，分 ​​离线粒体将表现出随着时间的推移19的功能活性的降低。我们建议所有的解决方案和补充预先做好准备。我们预先称重并存储枯草杆菌蛋白酶A在4毫克等分试样在1.5ml微量离心管中，并将其存储在-20℃。同样的BSA被预先称重并存储在在1.5ml微量离心管20毫克等分，可以储存在-20℃。使用管之前，从-20℃下除去，​​枯草杆菌蛋白酶A和牛血清白蛋白溶解于1ml的均质缓冲液中的线粒体分离过程中使用。 从骨骼肌组织活检两个拳使用6毫米的活检冲床提供足够的线粒体在临床和手术过程中使用的治疗性干预17获得-18。来估计，我们使用两种方法，血细胞计数和粒径数量线粒体的数量。建议使用的粒径数量。粒度计数器使用电阻抗测量粒子穿过限定大小的孔的体积。粒度计数器是昂贵，但提供准确和可靠的估计，可由用户独立。通过血细胞计数估计线粒体数目是更经济的。我们的研究已经表明，这种方法提供了可变的估计是大小小于使用粒度计数器获得的大约一个数量级。我们也注意到，通过血细胞计数器获得的计数是高度依赖于用户。我们建议，以确保一致的估计使用血球计数都应该由一个人来完成。 我们发现ATP检测试剂盒是用于确定线粒体功能非常有用。该套件提供了所有必须的Reagents并提供了用于确定分离的线粒体代谢活性的简单和快速的方法。 ATP的测定提供了类似的结果如那些用Clark型电极得到的，因此，与以前的数据分析20-21兼容。 我们的线粒体分离方案的主要优点是，它允许一个高产可行，呼吸主管无污染，在不到30分钟的线粒体的隔离。代替差速离心的差过滤显著减少手术时间。其他协议包含若干个离心步骤，整个隔离时间为60分钟至100分钟13-17。这个协议的另一个优点是，组织均质化是标准化的。商业组织离解器提供了一个标准化的循环，并产生一致且可重复的结果。这是相对于手工匀化，是受用户可变性和不一致。我们对线粒体利用组织分离和鉴别过滤的快速分离方法，为临床和手术治疗干预17,18兼容的隔离时间框架。

A correction was made to: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/51682">Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.</a> An optional step was added to step 7 of the &#39;Mitochondrial Isolation&#39; section of the protocol. The step is:
1. Optional in the case that the tissue is fibrous: Centrifuge the solution at 750 x g for 4 min.

A correction was made to: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.

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

存在的每一个细胞在体内除了红血细胞中的线粒体，并参与了大量重要的细胞代谢过程1-4。由于这些诸多功能，线粒体损伤可能具有不利的影响3。为了研究线粒体功能和功能障碍的若干线粒体的分离方法进行了描述。线粒体隔离日至1940年5-8最早公布的帐目。第一次记录尝试通过研磨肝组织在研钵中，然后离心以盐溶液在低速5,8证实线粒体隔离。后来，其他群体扩大后，原来的程序，证明组织分离的基础上差速离心6-8。这些早期方法形成的电流的技术，往往掺入均质化，和/或差速离心9-15的基础。 homogenizati数并离心步骤不同协议之间。这些重复的步骤，增加了时间线粒体隔离，并最终降低的可行性。此外，手动均化可引起线粒体损伤和不一致的结果，如果没有适当的控制，10,16。 最近，我们使用的同质化和差速离心分离线粒体移植到心肌组织17,18。这个冗长分离过程需要约90分钟，该方法的临床适用性因此受到限制。要允许在临床和手术治疗急性治疗用途，我们已经开发出可以在不到30分钟内完成快速的线粒体分离过程。 该协议的主要好处是标准化的组织分解允许统一和组织的一致同质化是不容易用手工同质化来实现的。在乳房清洁度离子，代替差速离心的使用微分过滤消除了耗时的和重复离心步骤，允许对高度纯化的，可行的和呼吸能力的线粒体的更快速分离。 隔离可行和呼吸主管线粒体在不到30分钟的能力允许临床应用。这种隔离协议有潜力的冠状动脉旁路移植术（CABG）和其他治疗程序中使用。

<table border="0" cellpadding="0" cellspacing="0" width="933">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:377px;">Sucrose</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:147px;">84100</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Substilsin A</td>
			<td>Sigma Aldrich</td>
			<td>P5380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KH2PO4</td>
			<td>Sigma Aldrich</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S6191</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Fisher Scientific</td>
			<td>P2173</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:377px;">ATPlite Luminescence Assay System, 
			1000 Assay Kit</td>
			<td>Perkin Elmer</td>
			<td>6016941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mL Conical Tubes</td>
			<td>BD</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 &#956;m Nylon Filters</td>
			<td>BD&#160;</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GentleMACS C tube</td>
			<td>Miltenyl Biotech</td>
			<td>120-005-331</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mL Eppendorf tube</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 mm biopsy punch</td>
			<td>Miltex</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 &#956;m Pluristrainer</td>
			<td>Pluriselect</td>
			<td>43-500-10-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf Centrifuge 5415C</td>
			<td>Marshall Scientific</td>
			<td>EP-5415C&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GentleMACS Dissociator&#160;</td>
			<td>Miltenyl Biotech</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well plates, tissue culture treated</td>
			<td>VWR</td>
			<td>82050-732</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotomax 120 orbital shaker</td>
			<td>Heidolph</td>
			<td>544-41200-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Synergy H4 Hybrid Multi-Mode 
			&#160;Microplate Reader</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multisizer 4 Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>A63076</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oxytherm System</td>
			<td>Hansatech Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration

使用差速离心和/或聚蔗糖梯度离心之前描述的线粒体分离方法需要60至100分钟内完成。我们描述了线粒体利用商业组织离解和鉴别过滤哺乳动物的活组织切片检查的快速提取方法。在这个协议中，手动均化置换为组织离解的标准化的均匀化周期。这使得均匀和组织的一致均质化是不容易用手工匀化来实现的。以下组织离解，在匀浆通过尼龙网过滤器，它消除重复离心步骤过滤。其结果是，线粒体隔离可以在不到30分钟来进行。此隔离协议产生约2×10 10活和呼吸从0.18±0.04克（湿重）的组织样品主管线粒体。

一个数字，概述采用组织分离和鉴别过滤线粒体的隔离程序步骤如图1。总过程时间小于30分钟。 组织样品用6毫米活检穿孔获得。组织的重量为0.18±0.04克（湿重）。线粒体分离为通过粒径数量确定的数目为2.4×10 10±0.1×10 10线粒体对骨骼肌和2.75×10 10±0.1×10 10线粒体肝制剂（ 图2A）。以允许比较线粒体数目也通过血细胞计数器确定。线粒体的数量被低估了血球的0.11厘定×10 10±0.04×10 10线粒体骨骼肌和0.34×10 10±0.09×10 10线粒体肝制剂（Figure 2A）。线粒体直径为通过基于大小的粒子计数器测定示于图2B中 。代表跟踪示出的分离的线粒体中被下一个峰为0.38±0.17微米的协议平均直径与以前的报告7本地化。 线粒体蛋白/克（湿重）起组织如通过二喹啉甲酸（BCA）测定法测定为4.8±2.9毫克/克（湿重）和7.3±3.5毫克/克（湿重），用于分别骨骼肌和肝脏样品（ 图图2C）。 线粒体纯度通过透射型电子显微镜测定，并示于图2D中 。线粒体被示出为电子致密少于0.01％的断裂或损坏。污染由非线粒体颗粒小于0.001％。 线粒体活力; MitoTracker红如前面去确定划线17,18。我们的结果表明，分离的线粒体保持膜电位（ 图3A - C）。 三磷酸腺苷是使用荧光检测试剂盒测定。 A板的地图为ATP的测定示于图4。的ATP标准铺板一式两份。线粒体样品和阴性对照铺板三份。 ATP的含量为10.67±4.38纳摩尔/毫克线粒体蛋白和14.83±4.36纳摩尔/毫克线粒体蛋白的骨骼肌和肝脏样品分别（ 图3D）。 用克拉克型电极如前所述17,18线粒体呼吸进行了评估。线粒体耗氧率分别为178±17纳米O2 /分钟/毫克线粒体蛋白的骨骼肌和176±23纳米O2 /分钟/毫克线粒体蛋白肝的准备。呼吸控制指数（RCI）值分别为2.45±0.34ð2.67±0.17对骨骼肌和肝脏样品制备分别（ 图3E）。这些结果是类似报道在我们以前使用手动均化和差速离心分离线粒体17-18研究。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51682/51682fig1highres.jpg" width="600" /> 图1模式用于使用组织离解和微分过滤线粒体的分离。 （A）将两个6毫米活检样品冲至5ml均质缓冲液中的解离Ç管以及使用该组织离解的1分钟均质化方案使样品均质化。（B）中加入250μl的枯草杆菌蛋白酶中的解离的储备溶液，以匀浆Ç管在冰上孵育10分钟（C）通过预过滤匀浆<html> -wetted 40微米的筛网过滤器中在冰上的50ml离心管中，然后加入250微升BSA储备液，向滤液（D）再过滤，通过一个新的预润湿的40微米筛网过滤，滤液在50毫升锥形离心冰上。（E）再过滤，通过在冰上的50ml离心管中一个新的预润湿的10微米的筛网过滤器的滤液（F）将滤液转移到1.5 ml离心管中并离心分离机以9000×g离心10分钟，在4℃。（G）弃上清，重新悬浮，并结合线粒体颗粒在1毫升呼吸缓冲。总过程时间小于30分钟。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51682/51682fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> D / 51682 / 51682fig2highres.jpg"WIDTH ="600"/&gt; 图2线粒体产量和纯度。分离出0.18±0.04 g组织（湿重）为骨骼肌和肝脏（A）的血细胞计数和粒径计数器线粒体数目（B）线粒体尺寸（毫克/克）分布由粒径计数器检测到的（C）线粒体蛋白毫克/克组织湿重为骨骼肌和肝脏。分离的线粒体中的（D）的透射电子显微镜图像。比例尺为100nm。箭头表示可能的污染，非线粒体颗粒和损伤的线粒体。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51682/51682fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51682/51682fig3highres.jpg" width="600" /> &lt;<html> BR /&gt; 图3线粒体活力 。根据荧光分离线粒体（一）相衬照明和（B和C）下的代表性显微照片，线粒体标记MitoTracker红CMXRos。比例尺是25微米（A，B）和5微米（C）。这些图像表明，线粒体保持膜电位。 &#160;箭头表示缺乏线粒体膜电位或碎片（四）ATP含量纳摩尔/毫克线粒体蛋白由三磷酸腺苷检测和（E）RCI的决定（国家3/4的状态），由克拉克的电极决定。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51682/51682fig3highres.jpg" target="_blank">请点击这里查看大图这个数字。</a> 负载/ 51682 / 51682fig4highres.jpg"宽度="600"/&gt; 图4标示的地图为ATP的测定该板图说明了如何设置标准（A1 - A12），线粒体样品（B1 - C 6），和阴性对照（C 7 - C 9）对ATP的测定。在测定过程中，加入100μl呼吸缓冲液中，加入50μl的哺乳动物细胞裂解液和50μl重建的底物溶液加入到所有孔中（A1 - C9）。

Watch this Scientific Journal Video about Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration at JoVE.com

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

一种用于线粒体的从哺乳动物组织活检快速分离方法进行说明。大鼠肝或骨骼肌肉制剂匀浆与商业组织离解和线粒体是通过尼龙网过滤分离差过滤。线粒体隔离时间是相对于60 &lt;30分钟 - 用替代的方法100分钟。

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 ...

rapid-isolation-purification-mitochondria-for-transplantation-tissue

Nanyang Technological University

Research

JoVE Journal

Biology

37.9K 次观看.  Beth Israel Deaconess Medical Center and Harvard Medical School. 一种用于线粒体的从哺乳动物组织活检快速分离方法进行说明。大鼠肝或骨骼肌肉制剂匀浆与商业组织离解和线粒体是通过尼龙网过滤分离差过滤。线粒体隔离时间是相对于60 

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

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

分离和造血干细胞移植（造血干细胞）

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

涡固定，局部照射，组织移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

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 ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

协议主要人肝细胞的分离和相应的非实质肝细胞的主要种群

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...