这个作品是通过从加拿大自然科学和工程研究委员会（RGPIN 238382-2011）和健康研究（MOP115076）的加拿大学院资助。希瑟·保罗是由加拿大自然科学和工程研究理事会研究生奖学金和艾伯塔创新的健康解决方案的奖学金支持。梅根·哈勒姆是由自然科学与工程研究理事会研究生奖学金，一个弗雷德里克·班廷和查尔斯最佳加拿大研究生奖学金，和亚省儿童医院研究所培训奖遗传学，儿童发展，健康的支持。

该协议被批准卡尔加里动物保健委员会的大学和符合该指南实验动物的护理和使用 。 从后代1.独立坝<ol><li>挤奶12之前至少5分钟分开她的后代的坝。 注:大坝可以挤奶可达5-6小时分离1,6,13后，但是分离的时间超过4小时，可能会改变乳成分14。而分离时间似乎没有影响奶源容积 12，它是表示，维持整个研究一致分离时间。乳成分在整个哺乳期15可能发生变化，因此，应努力保持奶源一致的一天。最大的牛奶生产建议将发生在哺乳期第14天12。 </li><li>使用变暖室，确保幼崽能够保持适当的身体temperatu重新没有他们的母亲的存在的挤奶过程的持续时间。 注:在研究下面列出，挤奶在断奶，当水坝约22周龄的后代21日龄进行，因此也没有变暖室使用。 </li></ol> 2.设置和准备<ol><li>收集需要的挤奶过程的所有材料。 注:所有材料可在材料和设备表中找到。 </li><li>放置一个加热垫板凳挤奶的地方将发生并覆盖垫在垫吸收板凳上。 </li><li>设置麻醉系统。确保系统有足够的氧气，并开始前异氟醚。附加麻醉剂掩模将用于初始麻醉诱导到机器。放置，将用于对附近如果比初始麻醉剂掩模不同麻醉维持掩模。 </li><li>附加将25g针1毫升注射器催产素注射用无菌技术。 </li><li>打开加热垫使母体体温挤奶过程期间保持。注:监视所述加热垫的温度，以确保垫不会变得太热并且造成烫伤。或者，使用热源，诸如外科加热表，可以设置到特定的温度。 </li></ol> 3.麻醉大坝使用异氟醚<ol><li>打开氧气罐，并打开流量1升（1000毫升）每分钟。打开异氟醚的流动，并设定为5％。注意:避免直接吸入麻醉剂，防止麻醉剂蒸气积聚。 </li><li>麻醉大坝。 </li><li>如果需要切换到维修面具，把大坝平卧于吸收板凳垫。缺乏踏板反射确认麻醉。 </li><li>降低的异氟烷至2-3％的流量为维持麻醉。持续监测大坝整个程序，以确保德不发生呼吸PRESSION。注意:一旦在麻醉状态下，坝的眼睛应该使用无菌的眼润滑剂，以防止眼睛变干或者成为划伤保护。 </li></ol> 4.注射催产素<ol><li>确保催产素（20 USP单位/毫升）还未到达其到期日期。催产素消毒的小瓶消毒酒精擦拭/酒精清洁垫。 </li><li>催产素入注射器的使用无菌技术，制定2 IU（0.1毫升）。使用一个新的针头和注射器将挤奶每个坝。 注:催产素剂量范围通常为单个注射1至5 IU 1,6,12,16。或者，4国际单位/公斤体重的剂量可以使用12。 2 IU单剂量可如果挤奶困难遇到重复一次。 </li><li>腹腔注射催产素。针插入与朝向头部的针指向腹部的右下象限，以一定的角度15-30°，约0.5厘米深。 </li><li>向后拉柱塞，以确保在注射前的负压。如果任何流体（血液，尿液，肠内容物， 等）被吸入注射器，取出针，并尝试在注射用新的针头和注射器。如果没有流体被吸入注射催产素和立即丢弃针头和注射器成生物危害容器。 </li><li>等待大约5-15分钟的催产素刺激放乳。 </li></ol> 5.准备挤奶遗址<ol><li>您可以选择其中的牛奶将被收集网站/奶嘴。乳可从任何奶嘴12被收集。 </li><li>轻轻取出周围与剪挤奶奶头的毛皮，毛皮可能造成困难样品收集由于牛奶芯吸。温柔 - 围绕乳头的皮肤非常敏感，可能是干的，因此很容易受到划痕和泪水。 </li><li>的奶嘴我灭菌不是必要的，但可选，清洁用温水将奶嘴的皮毛被删除后。因为多个站点可能需要的奶源至少准备两个站点。 注:如果牛奶分析包括微生物分析，乳头区，可能需要消毒用碘5。 </li></ol> 6.奶源<ol><li>轻轻挤压奶嘴的基础上，手动排出乳汁收集。 注:如果牛奶分析包括微生物分析，第一滴几滴牛奶应丢弃。 </li><li>收集奶液滴进入毛细管时，填充所述毛细管。可以容纳体积较大（ 例如，50微升）缓解这一进程毛细管。 注意:如果难以收集奶遇到，催产素的第二剂量可以施用。推荐剂量不超过4 IU总和。 </li><li>分配从毛细管的牛奶到由无菌的离心管动它被用来从乳头到微量离心管的侧吸取乳汁的管的端部 - 观察通过毛细作用被拉出的毛细管的牛奶。 注:"吹出"使用连接到1ml注射器18G的针头未吸入到管内的奶。 </li><li>连续监视大坝疼痛或呼吸抑制的迹象，并相应地调整异氟醚的流动。 </li><li>继续如在本节中所述，直到足够的牛奶已收集为所选奶分析收集奶。对于下面的creamatocrit和蛋白质浓度测定描述，收集0.25毫升牛奶。 注:使用不同的挤奶网站，如果放乳减慢或所选择的网站不排出乳汁充分。最多约2.5毫升每只动物乳汁可以收集17，或至多每奶头0.5毫升作者建议，根据麻醉总时间限制为约45-60分钟，邻ř挤奶的45分钟。 </li><li>收集乳成microhematocrit管用于从新鲜牛奶样品creamatocrit测量，并密封与粘土密封剂管的端部。标注microhematocrit管坝ID和直立存放牛奶样品。 </li><li>当挤奶完成后，关闭异氟烷和氧气的流量。从大坝取下面膜，继续监测大坝，直到清醒。建议如果没有充分意识到，大坝应在恢复期放在吸收板凳垫，而不是直接在笼子里的床上用品，防止被褥被吸入或恢复过程中刮伤大坝的眼睛。 注意:不要让水坝无人看管，直到它重新获得足够的意识，以保持胸骨recumbancy。 </li><li>如果没有进一步的分析是必需的，冻牛奶在-80℃。其他人已经提出，牛奶可以在-20℃18被储存长达3小时，在4℃或5个月。 </li></ol> 7. Creamatocrit测量<ol><li>通过计算乳creamatocrit（奶油的乳样品中的百分比）19估计在牛奶中的脂肪含量。 注:测量与人乳已显示，无论是新鲜或冷冻牛奶可以用于一个creamatocrit测量，然而鲜奶更高度相关（r = 0.92与R = 0.90）与脂浓度11。使用新鲜或冷冻牛奶creamatocrit测量应保持一致的整个研究中，作为解冻牛奶都与一个小的下降creamatocrit相关值11。 </li><li>对于鲜奶，收集乳汁从乳头到microhematocrit管的样品;填写至少¾满（约15-20微升）。另外，搅拌好后得出鲜牛奶从收集的样品放入毛细管。密封到底用粘土密封胶。 </li><li>将毛细管插入比容离心器，用密封端指向外，保证了离心机是平衡的。 </li><li>开始的血细胞比容旋转（120秒，13700 XG）。 注:旋转时间或速度可能因离心分离机所用的模式改变。 </li><li>从离心机中取出管后的自旋是完整的，进行测量来计算creamatocrit。仔细观察样品的分离成奶油层和透明层。 </li><li>测量和记录流体在管的总长度和的脂肪（霜）的长度使用卡尺或尺子层。 注:creamatocrit表示为奶样本19内的膏层的百分比，计算为（奶油层的长度/牛奶柱的总长度）×100（ 图1A）。计算从creamatocrit测量脂肪浓度和能量值如下:脂肪浓度（g / L）=（creamatocrit（％） - 0.59）/0.146（图1B）19;能量值（KC人/ L）= 290 +（66.8 * creamatocrit（％）（图1C）19。 </li></ol> 8.蛋白质浓度测定<ol><li>使用牛血清白蛋白作为蛋白标准，确定使用标准蛋白质测定，总乳蛋白质浓度如洛瑞蛋白质测定法6。 注:牛奶稀释可能需要的乳蛋白质测量落入测定法的标准曲线内。 </li></ol>

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作者有没有利益冲突披露。所有的动物实验均按照批准廉政公署的协议进行的。

Investigations into maternal milk components have increased as interest in early life development research rises. As the sole source of nutrition during the neonatal period, the bioactive compounds in milk are essential for ideal growth and development, especially in the context of intestinal and immune health3. The method presented here is a simple, non-invasive method of collecting milk from the lactating rat in amounts sufficient for downstream analysis, such as oligosaccharide profiling6. The me...

牛奶是营养新生哺乳动物，对于婴儿生长发育1,2-提供能量和营养的唯一来源。而乳主要由细胞脂质和蛋白 1，它还包含了过多了调节后代生命早期发展，包括酶，碳水化合物，激素，抗体，生长因子，细胞因子，外来体，微泡，和小RNA这样的生物活性化合物的作为微小RNA 1,2。孕妇奶粉在建立后代免疫和肠道健康3中的基础性作用，再加上证据表明，母乳喂养的婴儿不易受疾病2，突出了识别与疾病过程的早期生活有关的牛奶成分和所涉及的分子机制的重要性在他们的行动。发育大鼠是一种流行调查的各种营养，生理效果模型，并在早期的化学干预-生命的发展4。因此，大鼠乳汁的分析可以提供新的洞察孕产妇和后代的健康。 目前的科学进步，现在提供了越来越多的深入调查，对健康和疾病的特定的牛奶成分的影响的机会。例如，牛奶细菌型材测序阐明在婴儿肠道5早期肠集群自己的角色，乳寡糖的质谱分析已通过母亲饮食6，和微小RNA的深度测序分泌提供的洞察的乳寡糖概况的改变母乳的脂肪球突出于基因转录，代谢和免疫功能7可能角色。 大鼠模型代表在产妇的研究8,9中最常用的模式生物之一。一个优点是其短怀孕期和哺乳期的，仅approximat伊利21天每一个;因此，从怀孕哺乳的开始的总时间表示短时间内在能够产生有价值的数据。大鼠的较大尺寸相比小鼠，在奶源的背景下，可提供相对于牛奶和易于奶源体积显著优势;牛奶产量的小鼠，例如，似乎是依赖于总体重以产生更多的奶10较重的小鼠。 在这里，提供了手动收集的牛奶哺乳大鼠的一般描述。此协议需要最少的设备，非侵入性的，廉价的，并且可以用于收集奶足够量用于进一步下游分析。简言之，大坝用异氟烷麻醉，放乳由催产素的刺激，以及牛奶通过牛奶手册表达收集到毛细管。最后，如​​牛奶两个主要组成部分是脂肪和蛋白质，简要DESCRIPTIOÑ ​​估计使用creamatocrit测量11和使用标准蛋白质测定总蛋白质浓度的定量乳脂肪含量的呈现。

<table border="0" cellpadding="0" cellspacing="0" width="829">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Milking</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ul Microdispenser Capillary Tubes</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">21-169D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (20 USP Units/ml)</td>
			<td style="width:191px;">Bimeda-MTC</td>
			<td style="width:119px;">1OXY015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PPC Vet Isoflurane Inhalation Anesthetic, 250 ml</td>
			<td style="width:191px;">Fresenius Kabi</td>
			<td style="width:119px;">M60302</td>
			<td>Used on the order of a veterinarian</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pad</td>
			<td style="width:191px;">Dukal</td>
			<td style="width:119px;">853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbent Bench Underpad</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">82020-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Maxi-Therm Hyper/Hypothermia Blanket</td>
			<td style="width:191px;">Cincinnati Sub-Zero</td>
			<td style="width:119px;">274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Rodent Anesthesia Machine with Vaporizer</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;"></td>
			<td>Subject to individual laboratory needs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">Animal Masks</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;">50100/50102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tubes</td>
			<td style="width:191px;">Axygen</td>
			<td style="width:119px;">MCT-060-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChroMini Professional Trimmer</td>
			<td style="width:191px;">Wahl</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Creamatocrit</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">22-274-914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Critoseal Capillary Tube Sealant Tray</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">470161-478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin CritSpin Microhematocrit Centrifuge</td>
			<td style="width:191px;">Beckman Coulter, Inc</td>
			<td style="width:119px;">X00-004999-001</td>
			<td></td>
		</tr>
	</tbody>
</table>

milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

截至取Wistar水坝断奶描述的牛奶收集（约22周龄，体重350至400g），其消耗的控制（AIN-93G中，n = 5），高蛋白质（40％酪蛋白重量/重量，N = 5）或高益生纤维（21.6％重量/重量，1:1的比例的寡果糖和菊粉中，n = 4）的饮食整个怀孕和哺乳期。催产素剂量为2 IU。使用毛细管奶收集，并用血细胞比容喷丝确定creamatocrit（图1A），然后将其用于估计根据脂肪浓度和能量值一个管进行纺丝:脂肪浓?...

Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

奶源在大鼠毛细管管和乳脂肪含量Creamatocrit估计

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

milk-collection-rat-using-capillary-tubes-estimation-milk-fat-content

Research

JoVE Journal

Biology

12.2K 次观看.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

视频: Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&amp;#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

使用数字免疫荧光显微镜成像在大鼠软脑膜小动脉雌激素受体α

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

大鼠门户成纤维细胞的分离<em&gt;原位</em&gt;肝脏灌注

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

牛奶收集方法小鼠和里夫斯&#39;麂鹿

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

收集和分析<em&gt;拟南芥</em&gt;韧皮部分泌物使用EDTA促进方法

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

泄殖腔按摩与精子 DNA 分离法采集禽精

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...