We appreciate Feifei Zhang for the helpful discussions. We are grateful to Jing Gao and Yixuan Sun for the technical assistance and to Zhengshuai Liu and Fengguang Ma for the animal studies.

所有的动物实验方案通过在研究院营养科学学院，上海生命科学研究院，中国院士（中国上海）的机构动物护理和使用委员会批准。 1. DIO小鼠模型<ol><li> HFHS喂养 <ol><li>饲料八周龄雄性C57BL / 6小鼠用含有40千卡％的脂肪和40千卡％蔗糖的HFHS。房子在他们12小时暗光循环条件。 </li><li>保持在HFHS饮食喂养条件的小鼠4周至16周。检查每周体重。 </li></ol></li><li> 身体成分分析和伪彩色图像分析 <ol><li>若使小鼠身体成分分析仪分析。 <ol><li>在塑料管安全意识，不麻醉的小鼠。测量身体脂肪量，肌肉量，并使用核磁共振系统的流体，如前面2中所述。 </li></ol></li><li>使用扫描MRI系统每只小鼠的全身。 <ol><li>麻醉通过腹腔注射（25μL/ g）的1％阿佛丁的小鼠和专门的玻璃管进行成像分析。 注:成像的方法，持续每只小鼠近0.5小时。当完成测量时，返回的小鼠其笼子。 </li></ol></li><li>扫描背侧，中间，和腹侧层小鼠的整个机构;扫描在垂直方向上的肝脏。使用0.55磁铁t和下面的仪器参数:K空间= 192×256微米，截面厚度= 3毫米，TE = 14.8毫秒，TR = 400毫秒，FOVRead = 100毫米，FOVPhase = 100毫米，扫描次数= 8.扫描使用相同的磁场条件小鼠的所有组。 </li><li>根据信号强度9灰度图像映射到的伪彩色图象。 </li></ol></li><li> 鼠标安乐死 <ol><li>消毒dissectiNG工具，如镊子和剪刀。 </li><li>倒在纸巾上足够的异氟烷，并让异氟烷在玻璃罐挥发。确保将小鼠通过异氟烷安乐死。 </li><li>固定在手术台上的小鼠。喷每个腹部用75％乙醇，使在用剪刀剑突的真皮的切口，并通过与手中线纵向撕裂真皮暴露腹膜。 </li><li>夹紧钳剑突切腹膜纵横通过肋的底部，以暴露内脏。 </li><li>通过心脏穿刺收集在EDTA涂层管血。 </li><li>沿切肝隔膜，小心地取出使用细剪刀enterocoelia肝脏。切三块肝组织为以下脂质测量:一）6×3×3毫米的H＆E染色，B）6×3×3毫米油红O染色，和c）对氯仿20-40毫克肝脏/甲醇提取。 </li></ol></li><li> H＆E染色 <ol><li>固定在4％磷酸盐缓冲的福尔马林乙酸肝脏在4℃下过夜，然后在石蜡中嵌入它。 </li><li>切石蜡切片（5微米），并安装它们在载玻片上为H＆E染色，如前所述3,10。 </li></ol></li><li> 油红O染色 <ol><li>丽晶准备<ol><li>制备具有10％的福尔马林，60％异丙醇，苏木和甘油胶冻油红O工作溶液。溶解0.5油状物红O粉末用100mL异丙醇和其加热在60℃的浴中，使0.5％的储备溶液。 </li><li> 2比（毫升DDH 2 O的的储备溶液3毫升和2）:在3用双蒸水（DDH 2 O）稀释。过滤工作溶液，静置在室温下至少10分钟。 </li></ol></li><li>嵌入在AP最佳切削温度（OCT）化合物中的肝拉斯蒂克嵌入框。在-20℃30分钟将其冻结。把它切成8微米的部分在冷冻切片机，装入组织上滑动，并将其放置在室温下30分钟。 </li><li>放置纸巾在染色槽的底部，并倒10％福尔马林的少量润湿纸巾。放置在染色槽内的滑动和固定在甲醛蒸气冰冻切片在4℃5分钟。 </li><li>浸在3秒染色桶60％的异丙醇幻灯片;重复一次。 </li><li>添加的油红O工作溶液几滴到滑动覆盖所有组织孵育15分钟。在室温下避光组织。 </li><li>冲洗在3秒染色桶新的60％的异丙醇幻灯片;重复两次。 </li><li>轻轻漂洗通过蒸馏水滑动两次并取出组织周围的水。一定不要干组织。 </li><li>放在苏木幻灯片1分钟，用自来水轻轻漂洗5-10分钟。轻轻冲洗蒸馏水中滑两次，并保持它的蒸馏水，直到准备把盖玻片上。 </li><li>烧热在微波甘油果冻，并把几滴在组织之上。轻轻地在上面放置盖玻片并注意不要形成气泡。 </li><li>观察染色在显微镜下，并使用20X和40X的目标捕捉特征的照片。 </li></ol></li><li> 肝脏脂类的测量 <ol><li>放置解剖肝组织中新的2毫升的塑料离心管中。均质在1毫升的PBS用均化和裂解物转移到一个新的15毫升玻璃管中。 </li><li>加入5毫升氯仿/甲醇（2:1，体积/体积）溶液，涡旋混合物剧烈1分钟，并静置在冰上2小时。 </li><li>离心在4℃1650 XG 10分钟;该混合物应分离成3层:上层甲醇层，中间蛋白光盘，和底氯仿和脂质。 </li><li>陈德良<html> sfer底部相入用玻璃巴斯德吸管新玻璃管没有必要获取底部馏分的每一滴。抛开管冰。 </li><li>添加氯仿4mM的氯化镁和1.5毫升的600μL到留在以前的管和涡大力上清液部分。在4℃下在冰上孵育30分钟，并离心于1650×g离心10分钟。 </li><li>结合的底部相用巴斯德吸移管的第一底部相管。丢弃上层和中层。 </li><li>蒸发在氮气下的氯仿中37℃浴中。以1％的Triton X-100 /氯仿200μL的彻底清洗的玻璃试管的侧面（体积/体积）。在氮气下蒸发。溶解与DDH 2 O的200μL，涡旋脂质很好地做最后的乳液。 </li><li>根据制造商的方案通过使用甘油三酯或胆固醇试剂盒测量总甘油三酯（TG）和胆固醇（TC）水平外部参照"&gt; 3。 注:脂质值归一化到那些在初始匀浆中的蛋白质浓度的，如通过蛋白质定量3确定。 </li></ol></li></ol> 2.原发性肝细胞鼠标型号<ol><li> 小鼠原代肝细胞的分离 <ol><li>麻醉每只小鼠用6％水合氯醛（10微升/ g的腹膜内注射）。 </li><li>隔离原代肝细胞，如前所述10。生长细胞对胶原涂覆的玻璃盖玻片在油红O染色的6孔板中。用于脂质提取，种子细胞到10厘米的细胞培养皿10。 </li></ol></li><li> 细胞治疗和油红O染色 <ol><li>替换用2mL饥饿培养基的培养基，并在37℃孵育24小时。 30毫米的葡萄糖和100nM胰岛素治疗。它孵育在24小时37℃培养箱内培养。 </li><li>吸出网上平台。加入PBS毫升2和漩涡轻轻冲洗介质;重复一次。 </li><li>修复盖玻片用橡皮筋的幻灯片。暴露附着于外表面的细胞。上面（步骤1.5）中所述进行油红O染色。 注:脂质蓄积的基础水平被刺激血清饥饿后的高葡萄糖加胰岛素治疗。脂质液滴通过油红O染色可视化。 </li></ol></li><li> 血脂测定 <ol><li>治疗与高糖加胰岛素在相同的条件下（步骤2.2.1）的肝细胞。添加1毫升的PBS每10厘米细胞培养皿。刮去细胞和收获它们变成新的15毫升的玻璃管，从该100微升的等分试样转移至新的1.5毫升管中为蛋白质定量。 </li><li>添加氯仿的4毫升/甲醇（2:1，体积/体积），以细胞的剩余900微升。超声处理约20哔声，然后涡大力释放脂在细胞中。在冰上孵育混合物2小时。执行脂质提取（步骤1.6）。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。 资金来源:这项工作是由中国国家自然科学基金（81270930号，31471129，和31671224）和百人计划中国的科学院计划（2013OHTP04），以YL资助项目

NAFLD是一系列与代谢综合征，肥胖，胰岛素抵抗或II型糖尿病（T2DM）11相关联的进行性肝脏疾病。 NAFLD的特点是脂肪变性，脂质在肝细胞中的积累。这里，提出来表征表型和使用的DIO小鼠和小鼠原代肝细胞脂肪肝的参数的方法的光谱。此过程可帮助阐明NAFLD和其他相关的代谢性疾病的分子机制。 在体内和体外实验相结合，这些方法提供了全面的...

肥胖是发达国家和发展中国家一个新兴的健康问题。据报道是经常与非酒精性脂肪肝病（NAFLD）相关联的共存条件之一，具有患病30％和100％的NAFLD患者1之间的范围内。由于脂肪肝和肥胖症之间的强相关性，膳食诱导的肥胖（DIO）小鼠模型被广泛用于研究与NAFLD 2，3，4，5，6的发展相关联的复杂的分子机制。肝脂肪变性是NAFLD的最早阶段，它可以前进到非酒精性脂肪性肝炎（NASH），肝硬化，最终，肝癌7。因此，这种方法的总体目标是产生的肝脂肪变性条件动物和细胞模型和PR奥维德的高效，准确的测量血脂详细的协议。这些模型和测量也可用于其他代谢疾病，如胰岛素抵抗和2型糖尿病的调查是有用的。 由于肥胖被确定成为NAFLD，高脂肪的主要危险因素之一，高糖饮食（HFHS）模仿西式高脂饮食是用于诱导的肥胖小鼠。接着，肝脂肪变性的程度可以用不同的方法来评估。首先，体重和核磁共振（NMR）身体成分的分析表明喂养时间内脂质积累。的脂肪量和瘦体重可以以非侵入性的和实时的方式进行量化。 此外，磁共振成像（MRI）是用来同时显示全身和脂肪肝的分布。在MRI分析的灰度级信号可以被转换成清晰的伪彩色图象，和强度在灰度和彩色是半定量的。该技术提供了独特的优势，为在活的动物脂肪积累的测量。第二，肝脏的组织学分析是最常用的方法来确定肝脂肪变性。苏木精和曙红（H＆E）染色的组织学提供的信息，如肝细胞形态和巨噬细胞浸润，而油红O染色显示肝细胞中脂质小滴的大小和位置。第三，用氯仿/甲醇萃取脂质含量分析是肝脂质的精确和定量的测量。总甘油三酯和胆固醇水平可与生化方法来测量。重要的是，脂质提取分析和油红O染色，也可在基因操作或药学治疗肝细胞使用。 本方法的优点是多种优化方法及其利用，以产生肝脂肪变性模型和全面表征表型在体内和体外 。该DIO小鼠模型可以概括人类脂肪肝疾病的病理和代谢表型。在人类其他代谢参数可以在这个模型以及8被复制。响应于高糖加胰岛素的脂肪肝的肝细胞模型的生成是有效的，有用的，并且克服了昂贵和耗时鼠标工作的限制。两者合计，这些方法是足够和必不可少的肝脏脂质功能障碍和胰岛素抵抗养分超载在研究。

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			<td height="20" style="height:20px;width:216px;">10cm cell culture dish</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">420167</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">6-well-plate</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">3516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">BCA assay</td>
			<td style="width:183px;">Beyotime</td>
			<td style="width:163px;">P0010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Nuclear Magnetic Resonance</td>
			<td style="width:183px;">Niumag technology</td>
			<td style="width:163px;">MiniQMR23-060H-I</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">High fat high surcose diet(HFHS)</td>
			<td style="width:183px;">Research Diets</td>
			<td style="width:163px;">D12327</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

如在图1A中所示，小鼠体重为16周HFHS馈送，这大约是1.5倍食物饮食喂养组更高的后增加至45±1.2克核磁共振身体组成分析示出的脂肪质量和小鼠的瘦体质量指示（ 图1B）。全身和肝脏的脂肪分布是通过MRI测定，并在活，有意识的小鼠代表伪彩色图像在图1C-D中所示。体重和身体组成进行测定，在HFHS饮食喂养过程可视化。 <p class="jove_con...

Watch this Scientific Journal Video about Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis at JoVE.com

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 ...

optimized-analysis-of-in-vivo-and-in-vitro-hepatic-steatosis

Research

JoVE Journal

Medicine

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

15.8K 次观看.  Chinese Academy of Sciences. 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. 

视频: Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

可视化和肝脏微循环血流量和耗氧量分析:急性肝炎模型中的应用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

科研

医学

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. ...

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 ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

全身及肝脏血流动力学参数的监测小鼠

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

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 ...

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 ...

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 ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

划痕迁移分析和背皮折叠室的体外和在Vivo分析伤口愈合

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...