这项研究是由赠款RO1 CA 135561，R01 CA151494，R01 CA174432和P3013330来自美国国立卫生研究院的支持。 我们要感谢米歇尔·休斯顿，艾琳娜Dhima和安娜Velcich博士开发的地下室隔离协议提出宝贵的意见。 我们也感谢糖尿病的培训和医学用NIH P60DK20541支持的阿尔伯特爱因斯坦医学院的研究中心，以及迈克尔·布朗利博士和博士学梁笃，直接和运营海马工厂，分别是谁。

按照指南中的美国国立卫生研究院实验动物的护理和使用的建议进行研究。该方案经委员会医学爱因斯坦医学院动物实验的伦理。 1.地穴的分离与培养<ol><li>隔离小肠隐窝： <ol><li>从任何感兴趣的小鼠模型中分离出肠腺。安乐死的小鼠用CO 2，随后通过颈脱位。 </li><li>打开腹部纵向和补小肠（SI）的用冰冷的磷酸盐缓冲盐水，PBS（-Ca 2+ -Mg 2+）用2×抗生素-抗真菌剂（防反）。快速隔离小肠。 </li><li>深入剖析免费使用手术刀肠系膜脂肪。要小心，不要穿透组织 - 在任何时候都保持组织湿润冰冷的PBS。打开INTE斯坦纵向和用冰冷的PBS彻底清洗。 </li><li>切小肠分成两部分，并拉平用湿式棉尖，轻轻用预冷却的滑动刮去绒毛。在冰冷的PBS几次大力清洗。 </li><li>孵育于20ml的1×PBS每3毫乙二胺四乙酸（EDTA）/0.5毫二硫苏糖醇（DTT），3分钟为组织的非酶分解。 </li><li>切割组织成使用于预冷的滑动刀片的小块（2-4毫米）。传输块放入50ml管含有20ml冰冷的PBS中。 </li><li>移液器上下轻轻地用10倍的有10毫升无菌一次性吸管。让组织碎片沉淀在重力作用。用移液管去除上清。重复附加三次;确保上清液是明确的。 </li><li>加入20毫升的PBS每2mM EDTA的。漩涡孵育在4℃下进行30分钟，并轻轻摇动。 </li><li>让组织中沉积，弃上清。加入15ml冰冷的PBS和吸管上下5倍，用10毫升吸管。让组织碎片沉淀，收集上清液作为馏分1（F1）。重复采集F2-F5，单独维护每一个部分。 </li><li>添加15ml冰冷的PBS及此时抖动用手剧烈持续15秒。收集F6和重复F7，F8。如果组织块开始浮动，挖掘，帮助他们解决。 </li><li>检查在显微镜下每个级分的等分试样。普尔那些含有隐窝分数。 </li><li>通过70微米尼龙细胞过滤器的汇集级分，收集隐窝在50ml管中。 </li><li>离心机在100×g离心5分钟，在4℃，弃上清，重悬沉淀于10ml冰冷的PBS用2×防反和转印隐窝到15毫升管。重复洗一次。 </li><li> 2个防反 - 用10毫升冰冷的自动进稿器，先进的DMEM / F-12（贝科“改良Eagle培养基/火腿”S F-12）洗一次隐窝。 </li><li>洗一次隐窝用10mlDF - 1×防反。 </li><li>计数使用血球隐窝。调整隐窝溶液体积并转移到1.5ml试管中，使悬浮在最终隐窝浓度将每50μl胶状蛋白质混合物（基质胶）100-500隐窝。离心机在100×g离心5分钟，在4℃下重悬隐窝中的胶状蛋白质混合物。 </li><li>板50微升隐窝-每孔胶状蛋白质混合物的悬浮液在24孔板中（生长面积：2平方厘米 ），小心地放置在每一个的中心的悬浮液一滴良好。保持所述板在CO 2，37℃培养箱中培养约30分钟，直到悬浮液凝固。 </li><li>加入500微升冰冷的完全培养基（先进的DMEM / F12，带有1反 - 反，10mM的4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES），1个含有GlutaMAX，1×B27的补充液，1×N2补充液，1毫N-乙酰基-L-半胱氨酸（NAC），50纳克/毫升的表皮生长因子（EGF），100ng / ml的脑袋，500毫微克/毫升的R-脊椎）。 <ol><li>重组生长因子在0.1％牛血清白蛋白（BSA）的PBS中。 </li></ol></li></ol></li><li>地穴化培养和传代： <ol><li>通道隐窝组织体14-21天接种后（或根据需要），如下所示： <ol><li>变化的媒体，每星期五和星期一。上周三，用新鲜培养基替代一半的媒体。 </li></ol></li><li>除去培养基。以5分钟的间隔用500μl的PBS洗两次的样品。 </li><li>除去PBS，轻轻向上突破用无菌P1000微枪头胶状蛋白质混合物。 </li><li>加入1 ml完全培养基，以单井，重悬在1ml媒体的组织体。 </li><li>轻轻地破坏通过上下吹打20-30x使用P1000的组织体（检查显微镜进行适当的组织体离解以良好产率隐窝直到一致的技术下被显影）。 </li><li>转让隐窝到1.5 ml离心管中;离心机在100×g离心在4℃，5分钟。 </li><li>用1毫升完全培养基两次洗涤沉淀。 </li><li>裂以1：3的比例，或1：6​​根据需要，再悬浮隐窝中每孔加入50μl基质胶。继续上面的（ 部分1.1.16-18）的说明。 </li></ol></li><li>地穴类器官冷冻： <ol><li>除去培养基。洗500微升PBS样品。 </li><li>分散使用的是P1000的枪头的胶状蛋白质混合物。 </li><li>加入1毫升媒体单井重悬在1ml媒体的组织体。 </li><li>轻轻打散使用P1000的组织体吹打向上和向下20-30x。 </li><li>转移的组织体到1.5ml管中。离心机在100×g离心在4℃，5分钟。 </li><li>用1毫升完全培养基两次洗涤沉淀。 </li><li>隐窝重悬在500μl冷冻介质。 </li><li>转移隐窝到冻存管，将管在冷冻容器，存放于-80℃冰箱overniGHT。 </li><li>隐窝转移到液态氮罐。 <ol><li>通过在37℃水浴中快速解冻冷冻复苏隐窝类器官。用500微升，在100 XG，5分钟，悬浮在胶状蛋白质混合物，文化如上所述洗完整的培养基一次，离心机。为更好的恢复，ROCK抑制剂（Y-27632）加入到冷冻介质。 </li></ol></li></ol></li></ol> 2.地穴类器官代谢分析<ol><li> 24孔板准备： <ol><li>隔离并根据协议（见隐窝隔离和1.2节中的地穴通道第1.1节 ）数量隐窝。 </li><li>在胶状蛋白质混合物悬浮隐窝（每20微升100-200隐窝）。 </li><li>在24孔测定板中板隐窝胶状蛋白质混合物的悬浮液（确保有至少一式三份对各样品），并允许该悬浮液在37℃下固化，在CO 2孵化器;再加入500μl完整的培养基。 </li><li>文化隐窝（如1.1节所述），并在显微镜下观察到隐窝成长为全面发展类器官。 </li></ol></li><li>外流量分析： <ol><li>水合物盒过夜在非CO 2（0％CO 2）中 ，37℃培养箱中培养。 </li><li>除去培养基，并用500μl的DMEM洗两次组织体（无：葡萄糖，L-谷氨酰胺，丙酮酸钠和碳酸氢钠;以及与：酚红）。等待5分钟。 </li><li>添加675微升每孔的测定培养基（DMEM有2mM L-谷氨酰胺和5mM D-葡萄糖）到每个孔中。 </li><li>检查隐窝组织体的微观形貌，以确保组织体和凝胶状蛋白混合物的洗涤后完好无损。孵育1小时的非CO 2，37℃培养箱中培养。 </li><li>制备10μM的可注射的化合物（寡霉素，羰基氰化物对 - 三氟甲氧基苯基酮腙（FCCP）中，rotenone）在测定培养基中。 </li><li>建立墨盒通过加载75微升10微米的化合物注射进顺序墨盒的端口：端口A - 寡; B口 - FCCP;和C口 - 鱼藤酮（在试验过程中的最终浓度为1微米）。 </li><li>培养盒30分钟-在非CO 2培养箱中1小时，在37℃。 </li><li>同时，打开XF分析仪，并创建分析方案模板。 </li><li>将墨盒和公用事业板块成为XF分析和运行“校准”墨盒。 </li><li>检查地穴组织体显微镜，以确保它们连接到胶状蛋白质混合物。 </li><li>负载细胞培养板为工具和运行试验的协议。 </li></ol></li></ol>

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有没有披露。

我们测试的耗氧率（OCR）和隐窝中分离出8个月大的小鼠并生长成组织体在体外的细胞外酸化速率（ECAR）的基础量的测定之后，隐窝代谢物通过增加寡霉素，羰基氰化物评价-p-trifluoromethoxyphenylhydrazone（FCCP）和鱼藤酮，按顺序。 基底OCR和基底ECAR记录从0 - 29分钟（ 图2A和2B）。在第 29 届分钟，寡霉素（来自端口A）注射到每...

结直肠癌（CRC）是癌症相关死亡在美国的第三大原因。散发性大肠癌- 即后来的生活产生（&gt; 50岁），并没有明确的遗传易感因素-占〜80％ ​​的情况下，随着发病率的长期膳食模式1,2的强烈影响。这些肿瘤中表现出对依赖性代谢移对氧化糖酵解，被称为Warburg效应，这可能部分地使较高浓度的蜂窝构件和能量可用（通过谷氨酰胺），以允许和也许驱动的肿瘤细胞的增殖3-5率高。结肠癌的研究以及其他胃肠道癌症，包括小肠肿瘤提供重要的洞察肿瘤形成的原因。调查胃肠器官系统，可协助DET正常的，有利于肿瘤发生和肿瘤发生的国家之间的代谢差异发芽对肿瘤发展的相对风险，以及早期发现肿瘤的。此外，了解生物能量代谢涉及到线粒体呼吸和糖酵解提供基本的深入了解细胞生理学，老化和疾病状态扰乱肠道内环境稳定。生物能学分析技术，细胞外流量分析，可以利用在细胞生长在培养实时6,7同时评估线粒体呼吸和糖酵解的速度。 直到最近， 在体外研究小肠被限制在由良性或恶性肿瘤8,9的细胞系，并没有代表正常的肠道上皮细胞的生理和他们所在的微环境的影响。在2009年，Sato 等人 10导入离体培养体系生长的三维（3D）的小鼠的肠上皮组织体，或epithelial“微型胆”，适用于实验，诊断和治疗的研究10,11。此外，隐窝从calorically限制小鼠中分离维持其改变的生长特性类器官在这样的文化12。相比于转化的细胞系，隐窝类器官培养物可以被用于产生生理相关数据呈现出好得多的模型，以了解在体内的状态。 我们调整生物能量学分析技术测定肠隐窝组织体的能量代谢。小鼠小肠隐窝类器官培养体外发展提出的墓穴类器官的能量代谢研究。耗氧率（OCR）和隐窝组织体的细胞外酸化速率（ECAR）测定在不存在和两种不同的代谢抑制剂（寡霉素，鱼藤酮），和离子载体（羰基氰 - 对 - trifluoromethoxyphenylhydrazone）存在下进行。地穴奥尔加这些化学物质NOID代谢反应，通过改变ECAR和OCR值成功体现。 细胞生物能的研究将阐明代谢状态和疾病的风险和表型之间的相互交流的癌症，肥胖症，糖尿病，代谢紊乱和线粒体疾病，并有助于与转化医学直接影响预先筛选方法。在这里，我们描述了一个详细的协议来隔离小肠隐窝和文化隐窝类器官。此外，我们引入了一种新的方法来使用地穴类器官培养的代谢试验。

<table border="0" cellpadding="0" cellspacing="0" width="1633">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">BD Matrigel Basement Membrane Matrix, GFR, Phenol Red-free, LDEV-free</td>
			<td style="width:229px;">BD Biosciences</td>
			<td style="width:135px;">356231</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">PBS (phosphate buffered saline), no magnesium, no calcium, pH 7.2</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">20012-027</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Advanced DMEM/F-12 (1X)</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">12634-028</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Dulbecco&#8242;s Modified Eagle&#8242;s Medium without glucose, L-glutamine, Phenol Red, sodium pyruvate and sodium bicarbonate</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">D5030</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Phenol Red sodium salt&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">P4758</td>
			<td style="width:456px;">Final Concentration 15 mg / L in DMEM (D5030) - step 2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Antibiotic-Antimycotic, 100X, 100ml</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15240-062&#160;</td>
			<td style="width:456px;">Final Concentration 1 X or 2 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Penicilin-Streptomycin, liquid</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15140-122</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; GlutaMAX&#8482; supplement</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">35050061</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 1 M</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15630-080</td>
			<td style="width:456px;">Final Concentration 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">N-Acetyl-L-Cysteine, 25g</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">A9165-25G</td>
			<td style="width:456px;">Final Concentration 1 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">100X N-2 supplement, liquid</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:135px;">17502-048</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">50X B-27&#174; supplement minus Vitamin A, liquid</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:135px;">12587-010</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Mouse R-Spondin 1, CF, 50ug</td>
			<td style="width:229px;">R&#38;D Systems</td>
			<td style="width:135px;">3474-RS-050&#160;</td>
			<td style="width:456px;">Final Concentration 500 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Murine EGF, 100ug&#160;</td>
			<td style="width:229px;">Peprotech</td>
			<td style="width:135px;">315-09&#160;</td>
			<td style="width:456px;">Final Concentration 50 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Murine Noggin, 20ug&#160;</td>
			<td style="width:229px;">Peprotech</td>
			<td style="width:135px;">250-38</td>
			<td style="width:456px;">Final Concentration 100 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; L-glutamine, 200 mM</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">25030-081</td>
			<td style="width:456px;">Final Concentration 2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; Glucose powder</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15023-021</td>
			<td style="width:456px;">Final Concentration 5 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Ambion&#174; 0.5 M EDTA (Ethylenediaminetetraacetic acid), pH 8.0</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">AM9260G</td>
			<td style="width:456px;">Final Concentration 3 mM for step 1.1.5; 2 mM for step 1.1.8&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">DTT (Dithiothreitol), 1M</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">P2325</td>
			<td style="width:456px;">Final Concentration 3 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Albumin from bovine serum (BSA)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">A2058</td>
			<td style="width:456px;">0.1 % in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">16000-044</td>
			<td style="width:456px;">1 % in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recovery&#8482; Cell Culture Freezing Medium</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">12648-010</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">ROCK inhibitor (Y-27632)&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">Y0503</td>
			<td style="width:456px;">Final Concentration 10 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Oligomycin</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">O4876</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Carbonyl cyanide-p-trifluoro-methoxy-phenyl-hydrazone (FCCP)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">C2920</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Rotenone</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">R8875</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Sodium hydroxide</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">221465</td>
			<td style="width:456px;">Final Concentration 0.1 N in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">XF24 Extracellular Flux Analyzer (XF Analyzer)</td>
			<td style="width:229px;">Seahorse Bioscience</td>
			<td style="width:135px;"></td>
			<td style="width:456px;"></td>
		</tr>
	</tbody>
</table>

real time analysis of metabolic profile in ex vivo mouse intestinal crypt organoid cultures

小肠粘膜表现出组织成两个基本结构的重复架构：绒毛，伸入肠腔和成熟肠细胞，杯状细胞和肠内分泌细胞组成;和隐窝，驻留近端粘膜下层和肌层，窝藏成体干细胞和祖细胞和成熟的帕内特细胞，以及基质和隐窝微环境的免疫细胞。直到最近几年， 在小肠体外研究仅限于从良性或恶性的肿瘤衍生的细胞系，并没有表示的正常肠上皮细胞的生理机能和在它们所在的微环境的影响。在这里，我们展示了从培养C57BL / 6小鼠的原代小鼠小肠隐窝类器官改编自佐藤等人的方法。（2009）。此外，我们目前使用的crypt类器官培养法检测进行实时测量地穴新陈代谢换货基础耗氧量，糖酵解速率，ATP的产生和呼吸的能力。类器官维持他们的源代码中定义的属性，并保留其代谢改编耗氧量和胞外酸化率反映的各个方面。在这个地穴化培养系统实时代谢研究是一个强大的工具来研究隐窝类器官的能量代谢，以及如何通过营养和药物因素的调节。

地穴类器官是8月龄C57BL / 6小鼠饲喂建立纯化营养76A（AIN76A）的啮齿动物饲料美国学院。肠隐窝组织体可以生长在培养物从单个隐窝（ 图1A，单红色箭头）长时间。组织体生长出隐窝状结构在18-20天后在培养物（ 图1B，红色箭头）。隐窝传代，每3周和组织体有效回收下列各通道。 海马生物能量的仪器可以在细胞生长在培养的实时同步​​评估线粒体呼...

Watch this Scientific Journal Video about Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures at JoVE.com

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

小肠隐窝类器官培养离体提供的组织培养系统，该系统概括隐窝依赖于干细胞和它们的生态位的增长。我们建立了一个方法，以测定在实时在原代小​​鼠隐窝类器官的代谢分布图。我们发现组织体保持了其源定义的生理特性。

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

real-time-analysis-metabolic-profile-ex-vivo-mouse-intestinal-crypt

Research

JoVE Journal

Biology

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

16.1K 次观看.  Albert Einstein College of Medicine. 小肠隐窝类器官培养离体提供的组织培养系统，该系统概括隐窝依赖于干细胞和它们的生态位的增长。我们建立了一个方法，以测定在实时在原代小​​鼠隐窝类器官的代谢分布图。我们发现组织体保持了其源定义的生理特性。 

视频: Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

真核Polyribosome剖面分析

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

科研

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

逆转录病毒感染的原发性肠化培养的视频协议

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

小鼠小肠的混合原代培养拟肠激素分泌的研究和活细胞成像的肠内分泌细胞的供

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

实时在体记录了拟南芥钙信号在使用荧光传感器的昆虫饲喂时

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

眼部微血管血管生成前维沃胆球状芽分析

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

类器官来源的上皮单层：临床相关的肠道屏障功能体外模型

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...