这项工作得到了中国国家自然科学基金（81573992）的支持。我们感谢Emily K. Lo和Kathleen DiNapoli的语言编辑协助。

该协议遵循上海中医药大学动物护理方针，经动物实验上海动物伦理委员会批准。 议定书I:准备EXD <ol><li> 计算 <ol><li>将EXD给予10名6个月大的雌性印记控制区（ICR）小鼠（0.02kg /小鼠）的治疗组，每周5天，持续12周。 注意:需要总共1.702个EXD。因为EXD是以离散的增量来衡量的，在实际中，管理2个EXD。 </li><li>计算每只小鼠施用的EXD的体积（ 即 V =（0.1418EXD / kg·天）*（0.02kg /小鼠）* 50mL / EXD = 0.14mL /小鼠·天）。 </li></ol></li><li> 浸水 <ol><li>将两种EXD（ 即，Curculigo orchioides Gaertn （18 g）， Herbaa Epimedii （18 g））， 甘草 （18 g）， R将当归（18g）， 皮质黄柏 （12g）和黑曲霉 （12g），总共96g）放入具有盖子的容器中。 注意:建议使用陶瓷容器。 </li><li>用500毫升蒸馏水将原始草药切片1小时;水应该覆盖草药约一英寸。让所有的草药彻底浸泡。 </li></ol></li><li> 第一水煎剂 <ol><li>用大功率的燃气灶或电磁炉加热草药，直到水沸腾（约5-10分钟;时间间隔取决于加热功率，容器和草药和水量）。将电源降低到低炖煮2小时。 </li></ol></li><li> 先过滤 <ol><li>用常规滤纸或纱布和棉布覆盖500 mL玻璃烧杯。通过过滤器小心倒入烧杯中。把草药放在容器中。 </li><li>返还剩余的草药残留物o把滤纸放到容器上。 </li></ol></li><li> 第二汤水 <ol><li>用草药将蒸馏水加入容器中;让它覆盖草药约一英寸。 </li><li>重复步骤1.3。 </li></ol></li><li> 二次过滤 <ol><li>重复步骤1.4.1和1.4.2。 </li><li>将第二汤匙倒入同一个烧杯中。将第一和第二汤匙混合在一起。 </li></ol></li><li> 浓度 <ol><li>将烧杯放在燃气灶之间，用无石棉丝网将它们之间加热，用低温炖煮，并用玻璃棒慢慢地连续搅拌。 </li><li>当煎剂减少到200 mL时，将其转移到500 mL烧杯中。 </li><li>重复步骤1.7.1，直到煎剂减至100 mL。 </li></ol></li><li> 储存和管理 <ol><li>将浓缩汤汁转移到无菌玻璃瓶中。让它凉快到室温re（RT）。如果在一周内或-70°C使用长期储存，请在4°C储存。 </li><li>使用管饲针，向卵巢切除（OVX）小鼠施用0.14mL /小鼠煎剂一天，每周5天，持续12周。 </li></ol></li></ol> 2.议定书二:含EXD的血清的制备<ol><li> 计算 <ol><li>计算每只大鼠（1个月大）体重0.15 kg的大鼠当量（RED）:dB =（1/60）*（90/100）*（60 / 0.15） 1/3 = 0.111 EXD / kg /天。 注意:需要总共10ml的含EXD血清以制备100mL培养基（含10％含EXD的血清）。预计每只大鼠提供2mL的血清。因此，每天一次，需要6只大鼠（20％的损失被考虑在内）3天。 EXD的数量是:（0.111EXD / kg•天）*（0.15kg /大鼠）*（6只大鼠）*（3天）= 0.300EXD。再次，因为EXD以离散增量测量，使用1 EXD。 </li><li>计算每只大鼠施用的EXD体积（ 即 V =（0.111EXD / kg·天）*（0.15kg /大鼠）*（50mL / EXD）= 0.83mL /大鼠·天）。 </li></ol></li><li> 浸水 <ol><li>将1 EXD的原料放入带盖的容器中。将原料在蒸馏水中浸泡1小时;水应该覆盖草药约一英寸。让所有的草药彻底浸泡。 </li></ol></li><li> 第一水煎剂 <ol><li>用高功率的电磁炉加热草药，直到水沸腾（约5 - 10分钟;时间间隔取决于热量，容器和草药和水量）。将电源切换到低炖煮2小时。 </li></ol></li><li> 先过滤 <ol><li>用常规滤纸或纱布和棉布覆盖500 mL玻璃烧杯。通过过滤器小心倒入烧杯中。把草药放在容器中。 </li><li&gt;将滤纸上的草药残渣送回容器。 </li></ol></li><li> 第二汤水 <ol><li>用草药将蒸馏水加入容器中;让它覆盖草药约一英寸。 </li><li>重复步骤2.4。 </li></ol></li><li> 二次过滤 <ol><li>重复步骤2.5.1和2.5.2。 </li><li>将第二汤匙倒入与步骤2.4.1中相同的烧杯中，并将第一和第二汤匙混合在一起。 </li></ol></li><li> 浓度 <ol><li>将烧杯放在燃气灶之间，用无石棉丝网将它们之间加热，并用低温炖煮，并用玻璃棒慢慢连续搅拌。 </li><li>当煎剂降至100 mL时，将其转移到100 mL烧杯中。 </li><li>重复步骤2.7.1，直到煎煮至50 mL。 </li></ol></li><li> 储存和管理 <ol><li>将浓缩汤汁转移至灭菌剂中ile玻璃瓶。让它凉爽到RT。如果在一周内或-70°C使用长期储存，请在4°C储存。 </li><li>腹腔内给予0.83mL /天，每天一次，持续3天。 </li></ol></li><li> 含EXD的血清制剂 <ol><li>在最后一次给药后1小时腹腔注射300 mL / 100g 80 mg / kg氯胺酮和10mg / kg甲苯噻嗪麻醉麻醉大鼠。通过脚趾捏捏确认适当的麻醉。 </li><li>使用手术刀（或直接操作的剪刀）将大鼠的皮肤和腹膜从腹部切除到胸部的底部。创建长度和深度分别为5厘米和0.5厘米的切口。使用薄纸将腹部内脏移至左侧。 </li><li>使用薄纸，去除腹主动脉的结缔组织，以清楚地暴露血管。 </li><li>使用10 mL，22号注射器从腹主动脉缓慢吸取血液。转移到取出针后取出15 mL无菌管;一只大鼠可以产生8-10毫升血液。 注意:开始抽血时，大鼠应该活着（ 即腹部主动脉脉动），完成后，死亡。 </li><li>在室温下将血液凝固在直立位置30-60分钟。以500-600 xg离心20分钟。将所有上清液（血清）小心地放入一个50mL无菌管中，并将所有血清（来自不同动物）混合在一起。 </li><li>通过在56℃水浴中孵育血清30分钟来进行热灭活。使用具有0.22μm孔径的亲水性聚醚砜膜的注射器过滤器过滤血清。使用新鲜血清或将其储存在-20°C。 </li></ol></li><li> 对照药物血清制剂（用于对照组） <ol><li>用相同体积的盐水（0.83mL）每天施用每只大鼠一次，持续3天。其他步骤与步骤2.9相同。</li></ol></li><li> 应用 <ol><li>向每100mL培养基加上1％的青霉素 - 链霉素（PS） 23加入含有EXD的血清或含药血清。 </li></ol></li></ol>

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B., Jacob, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+practice+guide+for+dose+conversion+between+animals+and+human.">A simple practice guide for dose conversion between animals and human.</a> J Basic Clin Pharm. 7 (2), 27-31 (2016).</li><li>Xu, X., 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=Protective+effect+of+the+traditional+Chinese+medicine+xuesaitong+on+intestinal+ischemia-reperfusion+injury+in+rats.">Protective effect of the traditional Chinese medicine xuesaitong on intestinal ischemia-reperfusion injury in rats.</a> Int J Clin Exp Med. 8 (2), 1768-1779 (2015).</li><li>Li, Z., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+methods+for+Chinese+herbs+serum+pharmcology.">On the methods for Chinese herbs serum pharmcology.</a> Zhong Guo Zhong Yi Yao Xing Xi Za Zhi. 9 (2), 5-6 (2002).</li><li>Jiang, Y. R., 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=Effect+of+chinese+herbal+drug-containing+serum+for+activating-blood+and+dispelling-toxin+on+ox-LDL-induced+inflammatory+factors'+expression+in+endothelial+cells.">Effect of chinese herbal drug-containing serum for activating-blood and dispelling-toxin on ox-LDL-induced inflammatory factors' expression in endothelial cells.</a> Chin J Integr Med. 18 (1), 30-33 (2012).</li><li>Guo, C. Y., Ma, X. J., Liu, Q., Yin, H. J., Shi, D. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Effect+of+Chinese+herbal+drug-containing+serum+for+activating+blood,+activating+blood+and+dispelling+toxin+on+TNF-alpha-induced+adherence+between+endothelial+cells+and+neutrophils+and+the+expression+of+MAPK+pathway].">[Effect of Chinese herbal drug-containing serum for activating blood, activating blood and dispelling toxin on TNF-alpha-induced adherence between endothelial cells and neutrophils and the expression of MAPK pathway].</a> Zhongguo Zhong Xi Yi Jie He Za Zhi. 35 (2), 204-209 (2015).</li><li>Li, Y., Xia, J. Y., Chen, W., Deng, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ling+Qi+Juan+Gan+capsule+drug-containing+serum+on+PDGF-induced+proliferation+and+JAK/STAT+signaling+of+HSC-T6+cells].">Effects of Ling Qi Juan Gan capsule drug-containing serum on PDGF-induced proliferation and JAK/STAT signaling of HSC-T6 cells].</a> Zhonghua Gan Zang Bing Za Zhi. 21 (9), 663-667 (2013).</li><li>Li, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Some+issues+in+methology+of+Chinese+herbs+serum+pharmcology].">[Some issues in methology of Chinese herbs serum pharmcology].</a> Zhong Yao Xin Yao Yu Lin Chuang Yao Li. 10 (5), 263 (1999).</li><li>Zhang, 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=[A+review+of+Chinese+herbs+serum+pharmcology+methodological+study].">[A review of Chinese herbs serum pharmcology methodological study].</a> Nan Jing Zhong Yi Yao Da Xue Xue Bao. 18 (4), 254 (2002).</li><li>Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Issues+and+strategies+for+study+of+serum+pharmcology+in+oncology].">[Issues and strategies for study of serum pharmcology in oncology].</a> Zhong Yi Yan Jiu. 17 (5), 13-14 (2004).</li></ol>

作者宣称他们没有竞争的经济利益。

近年来，更多的关注草药，一种在东方世界临床环境中应用了数千年的替代药物。传统草药与现代医学的"长凳式"床不同，首先需要"床到凳"的模式来解释他们的机制。这可以在后台进行验证，并进行开发新优化药物的处理。到目前为止，已经报道了从整个配方或草药释放活性成分的几种提取方法。其中，水提取是最广泛使用的。 中草药的提取有四个基本步骤。第一步是浸泡?...

对传统草药研究和应用的兴趣目前正在增长。与现代药物相反，化学成分明确，草药配方有一些未知的成分，需要提取过程才能使其活性化合物交付。虽然许多研究尝试选择一种具有众所周知结构的小化合物作为全草药或草药配方的代表，但药理作用和机制均不能等同于1,2 。虽然指纹图谱能够分析复方草药配方的成分，但一些成分仍未得到清晰分析，结合所有研究提取物3时会带来挑战。许多成分/提取物的相互作用介导中草药的治疗效果。为了保留这个优势，传统的提取物形式 - 煎煮离子 - 仍然广泛应用于诊所。由于提取方法对治疗效果有很大影响，因此传统水煎剂的标准方案是必要的，特别是对于体内研究4,5 。 另一方面，当探索药理机制时，体外或体外研究中草药煎剂的施用也是一个挑战。药物血清的概念最初由Tashino于1988年提出6 。从那时起，越来越多的研究人员将其应用于草药7,8,9 。虽然含药血清的方法有一定的局限性，如血清某些成分的影响，但仍被认为是与生理条件密切相关的方法。 14 ，更年期骨质疏松症15,16,17 ，早产卵巢功能衰竭18 ，乳腺癌19 ，卵巢癌20和延迟青春期21 。在这里，我们提出了制定呃ian汤（EXD）及其含药血清的详细方案。此外，我们描述了含有EXD和EXD的血清对鼠绝经期骨质疏松模型的应用。 一个EXD由9g Curculigo orchioides Gaertn ， Herbaa Epimedii组成 + ， 甘草和当归，每个成年患者每天6g 皮质黄柏和根瘤菌 。基于以下等式22，小鼠的等效剂量为0.1418EXD / kg /天:dB = dA * RB / RA *（WA / WB） 1/3 。 dA和dB分别表示人和小鼠每体重的剂量（mg / kg）。剂量（mg / kg）由EXD / kg的数量代替。 RA和RB分别代表（体表面积（m 2 ）/体重（kg）） 2/3 （ 参见表1 ）成比例的人体因子和小鼠体内因子。 WA和WB分别表示人和小鼠的体重（kg）。

<table border="0" cellpadding="0" cellspacing="0" width="651">
	
	<tbody>
		<tr height="165">
			<td height="165" style="height:165px;width:216px;">Curculigo orchioides Gaertn (9&#8201;g), Herbaa Epimedii (9&#8201;g), Radix Morindae Officinalis (9&#8201;g), Radix Angelicae Sinensis (9&#8201;g), Cortex Phellodendri (6&#8201;g), and Rhizoma Anemarrhenae (6&#8201;g)</td>
			<td style="width:149px;">Kang-qiao Chinese Medicine Yinpian Co. Ltd (Shanghai, CN)</td>
			<td style="width:119px;">160922</td>
			<td style="width:167px;">EXD components</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Filter paper</td>
			<td style="width:149px;">GElifesciences</td>
			<td style="width:119px;">99-103-952</td>
			<td style="width:167px;">Filter EXD decoction before concentration</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Imprinting Control Region (ICR) mice</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">In vivo study</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sprague Dawley rats</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">EXD-containing serum preparation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Syringe filter</td>
			<td style="width:149px;">Millipore</td>
			<td style="width:119px;">SLGP033RB</td>
			<td style="width:167px;">0.22 &#181;m</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">gavage needles (10 ml)</td>
			<td style="width:149px;">Shanghai BO Ge trade sales department</td>
			<td align="right" style="width:119px;">59104274</td>
			<td style="width:167px;">Adminstration of EXD</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Ketamine (80 mg/kg)&#160;</td>
			<td style="width:149px;">Fujian Gutian Pharma Co. Ltd</td>
			<td style="width:119px;">H35020148</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Xylazine (10 mg/kg)</td>
			<td style="width:149px;">Sunway Pharma Co. Ltd</td>
			<td style="width:119px;">CB07591</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">Dulbecco&#8217;s modification of Eagle&#8217;s medium Dulbecco (DMEM) culture medium</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:119px;">12800-116</td>
			<td style="width:167px;">DMEM with 2 mM L-glutamine and without ribonucleosides and ribonucleotides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">1277</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">4687</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
	</tbody>
</table>

preparation of herbal medicine: er-xian decoction and er-xian-containing serum for in vivo and in vitro experiments

传统草药是临床环境中的替代药物，近年来受到越来越多的关注。在运送到身体之前，通常需要额外的提取方法来从原料中释放活性成分。水煎剂是一种经典的提取方法，在临床环境中仍然广泛应用。在这里，我们提出了一个详细的呃ian汤（EXD）方案，以便将草药煎药应用于实验研究。描述了动物适当剂量的计算，以及EXD的四个主要步骤:浸泡，水煎剂，过滤和浓缩。此外，将含有血清的EXD作为体外验证手段引入大鼠。在这里，大鼠经口给予EXD三天。然后收集血液样品，灭活，离心和过滤。用培养基稀释的血清可用于治疗v。中的细胞或组织itro。例如，EXD被应用于体内和体外研究，并且表明EXD增强成骨。该方案可作为草药的制备和应用参考。

EXD对OVX小鼠骨密度的影响腰椎椎体部分的苏木精和伊红（H＆E）染色显示体外EXD处理后增加的骨小梁（ 图1A ，右图）与OVX组相比（ 图1A ，左图）。 图1B显示OVX小鼠（ 图1B ，左图）和EXD处理12周的OVX小鼠的代表性μCT图像（ 图1B ，右图）。在EXD治疗的小鼠的腰椎椎弓?...

Watch this Scientific Journal Video about Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments at JoVE.com

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

在这里，我们以四个步骤 - 浸泡，煎煮，过滤和浓缩四个步骤介绍了一种呃ian汤（EXD）的制备方法，并且证明了给大鼠制备的含有EXD的血清。这些方法适用于中药等中草药的体内和体外研究。

草药的制备:洱西汤和含茜素的血清<em&gt;体内</em&gt;和<em&gt;体外</em&gt;实验

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

preparation-herbal-medicine-er-xian-decoction-er-xian-containing

Research

JoVE Journal

Medicine

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

10.9K 次观看.  Shanghai University of Traditional Chinese Medicine. 在这里，我们以四个步骤 - 浸泡，煎煮，过滤和浓缩四个步骤介绍了一种呃ian汤（EXD）的制备方法，并且证明了给大鼠制备的含有EXD的血清。这些方法适用于中药等中草药的体内和体外研究。 

视频: Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

科研

医学

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

评估癌症药物过敏的有效性<em&gt;在体外</em&gt;和<em&gt;在体内</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

纳米抗体和基于抗体的验证<em&gt;在体内</em&gt;肿瘤异种移植NIRF成像的小鼠实验使用<em&gt;离体</em&gt;流式细胞仪和显微镜

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

草药的制备:洱西汤和含茜素的血清

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颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

评估癌症药物过敏的有效性

纳米抗体和基于抗体的验证

宫内遥测来衡量鼠标收缩压力

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

优化分析

制备和

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验