This work was supported by the Seed Grant Award from California Northstate University College of Pharmacy (to AMN). The authors would like to thank California Northstate University College of Pharmacy for covering the publication cost of this article, and George Talbott for his help in editing this manuscript.

在Caco-2细胞的1.维持<ol><li>使用常规组织培养皿<ol><li>解冻的Caco-2细胞从冷冻的小瓶通过将小瓶在37℃水浴中，并立即将其添加到含有10预温热的生长培养基（15％胎牛血清毫升10cm的组织培养皿（FBS ）在Dulbecco氏改良Eagle培养基（DMEM））。 <ol><li>当Caco-2细胞达到50-70％汇合时，将它们分割1：直到它们被分离的6通过在37℃下孵育细胞用3ml的0.05％胰蛋白酶/ 0.53毫乙二胺四乙酸（EDTA）（15分钟） 。为了避免细胞聚集，轻柔吹打混合细胞几次。加入0.5毫升胰蛋白酶细胞对含有10ml预热的生长培养基的10cm的组织培养皿。 </li></ol></li><li>轻轻摇动菜几次在向前和向后方向接着是向左和向右的方向。避免纷飞的菜因为这可能导致不平等的细胞分散。 </li><li>放置在平坦表面上的菜在5％的CO 2供给的37℃培养箱中。倾斜表面也可能导致在一个不平等细胞分散。 </li><li>每天在开始培养后改变生长培养基。 </li><li>监测细胞并记录它们达到汇合的那一天。这将需要约1周的细胞，从一天他们被分开达到汇合。 </li><li>每周两次更换生长培养基之前，细胞达到汇合。一旦细胞达到汇合，每隔一天更换培养基。后的细胞是7-日后汇合，每天更换生长培养基。 注：细胞准备用于实验时，他们的13天后汇合（ 图1）。因为在生产脂蛋白细胞将最终变得不那么有效，使用细胞，13至17天后汇合8之间。转到如何进行的实验步骤3。</li></ol></li><li>利用渗透膜系统<ol><li>如在步骤1.1.1.1中描述的种子的Caco-2细胞中的渗透膜的插入。上面。避免使用细胞冷冻瓶，因为他们的细胞生长较慢正常（恢复期）。简言之，加入0.5毫升胰蛋白酶细胞对心尖室（上室），它包含10毫升预温热的生长培养基。此外，加入10 mL预温热的生长培养基到基底外侧腔室（下室）。 注意：对于最佳操作，第一和随后的生长培养基添加到顶部室向基底外侧腔室。当拆除旧媒体，心尖媒体面前取出基底媒体。避免戳是聚碳酸酯膜，因为它可能会破裂的膜。 </li><li>由于通过聚碳酸酯膜差细胞能见度，种子的Caco-2细胞在常规组织培养皿具有类似的密度，并使用这个菜来判断细胞汇合。 </li><li>按照sTEPS 1.1.2-1.1.6以上。 </li></ol></li></ol> 2.基因表达<ol><li>使用常规方法转染<ol><li>如以上1.1.1.1.-1.1.4描述种子Caco-2细胞上的组织培养皿。 注意：当细胞约有40-50％汇合时，它们已准备好被转染。 </li><li>添加67微升转染试剂以472微升减少血清培养基的微量离心管中。避免未稀释的转染试剂与所述管壁接触。 </li><li>加23微克pLL3.7的增强型绿色荧光蛋白（EGFP）9成转染试剂/减少血清的培养基混合物中。 </li><li>为20分钟，在RT孵育混合物。 </li><li>用8ml 10％胎牛血清的DMEM中取代的Caco-2细胞的生长培养基。 </li><li>添加该DNA /转染试剂/低血清培养基混合物逐滴的菜。 </li><li>轻轻摇动的菜好几次向前和Backward方向接着是向左和向右的方向。 </li><li>放置在37℃培养箱菜。 </li><li>在开始培养后替换媒体用了一天10毫升生长培养基的菜。 </li><li>监测基因表达。 <ol><li>监测基因表达而不4&#39;，6-二脒基-2-苯基吲哚（DAPI）染色。使用荧光显微镜，确定细胞是绿色的百分比。百分比表示的转染效率。 </li><li>监视器DAPI染色基因表达。 <ol><li>用磷酸盐缓冲盐水（PBS）洗涤三次洗细胞。 </li><li>通过在PBS（10分钟温育在室温下）加入10ml 4％的甲醛固定细胞。 </li><li>用PBS三次洗细胞。 </li><li>孵育细胞在黑暗中进行15分钟，在RT下用8ml PBS中含1：500的DAPI，1％BSA，0.01％毛地黄皂苷。 </li><li>用PBS三次洗细胞。 </li><li> ü唱的荧光显微镜，确定细胞，绿色/蓝色相对那些蓝色（ 图2上图）的数量。所计算的百分比表示的转染效率。 </li></ol></li></ol></li></ol></li><li>利用慢病毒表达系统10 <ol><li>种子人胚肾（HEK）293T细胞在15厘米的组织培养皿（10％FBS的DMEM中为它们的生长介质）。 注意：当细胞约有60-70％汇合时，它们已准备好被转染。 </li><li>添加162微升转染试剂到1133微升减少血清的培养基的微量离心管11。避免未稀释的转染试剂与所述管壁接触。 </li><li>添加24 pLL3.7的eGFP（或另一个构建体）的微克，15.6微克启反应元件（RRE），6.0微克的REV和8.4微克的水泡性口炎病毒糖蛋白（VSVG）插入转染试剂/ R得出血清培养基混合。 </li><li>为20分钟，在RT孵育混合物。 </li><li>以16毫升10％的FBS的DMEM更换生长培养基。 </li><li>添加该DNA /转染试剂/低血清培养基混合物逐滴的菜。 </li><li>轻轻摇动菜几次在向前和向后方向接着是向左和向右的方向。这是正常的看到一些在这一点超脱HEK293T细胞。 </li><li>放置在孵化器的菜。 </li><li>孵化后24小时，在用25毫升生长培养基的盘更换介质。 </li><li>第二天，收集含有慢病毒（第一集）的生长介质。 </li><li>重复步骤2.2.10收集更多的慢病毒（第二个集合）。 </li><li>池的第一和第二集合，并通过离心除去细胞碎片（2,500×g离心5分钟）。 </li><li>过滤收集，用一次性瓶顶滤器（0.45μm孔径）和通过在RT（JA-20转子）离心收集在31400 XG 2小时浓缩病毒。标记的离心管，其中团粒是位于底部。滗析出上清液，和离开倒在RT管约15分钟。 </li><li>重悬在含病毒沉淀用100μl1X PBS中。避免气泡。 </li><li>存储集中病毒在-80℃。避免反复冻融。 </li><li>确定病毒滴定转导的Caco-2细胞所需要的最佳量。为了节省成本，使用24孔板（0.3毫升生长培养基/孔的），而不是10cm的组织培养皿滴定。 <ol><li>添加增加量的浓缩病毒的（0，1，2，5，10，25，和50微升/孔），补充用聚凝胺（最终浓度= 5微克/毫升），以40-70％汇合Caco-2细胞。轻轻摇动24孔板如在1.1.2中描述。 </li><li>监测其基因表达（步骤2.1.10。）（ 图2底部4板）。 </li></ol></li><li>由于转基因的表达一般是持续，维持转导的细胞用于以后的实验和持续监测其基因表达（步骤2.1.10）。 </li></ol></li></ol> 3.刺激分泌脂蛋白<ol><li>使用常规的组织培养皿<ol><li>通过混合50毫克油酸，40毫克卵磷脂，和48毫克的牛磺胆酸钠制得100X脂质混合物。使体积达到900微升用PBS（足够8菜）。大力旋涡脂质混合物。 </li><li>添加900微升100X脂质混合物的成89.1毫升生长培养基。混合，并过滤该含脂介质。油酸，卵磷脂和牛磺胆酸钠的最终浓度（1X）是2.0毫摩尔，1.36毫和1.0毫米。 </li><li>加10毫升含脂质介质的细胞中。 </li><li>在37孵育将细胞与含脂介质4小时°Ç孵化器。 </li><li>用PBS三次洗细胞。 </li><li>加入10毫升生长培养基向细胞收集脂蛋白分泌。 </li><li>孵育2小时在37℃的培养箱中。 </li><li>收集含有脂蛋白媒体。 </li></ol></li><li>利用渗透膜系统<ol><li>按照步骤3.1.1-3.1.2准备脂质混合物。 </li><li>加10毫升含脂质介质到心尖室和10ml生长培养基的至基底外侧腔室。 </li><li>孵育在37℃培养箱中4小时。收集含有脂蛋白媒体在基底室。 </li></ol></li></ol> 4.脂蛋白的分离（图3） <ol><li>通过离心（2,000×g离心5分钟）从含脂蛋白介质除去细胞碎片。 </li><li>滗媒体到50毫升管中。 </li><li>添加5.95克氯化钠给媒体。 </li><li>如果SAmple将利用TEM进行分析，加入1蛋白酶抑制剂混合物片剂保持脂蛋白的完整性。 </li><li>使体积至23毫升水（1.2微克/毫升的NaCl浓度溶液）。 </li><li>完全溶解的溶质。 </li><li>滗所有溶液的成聚碳酸酯超速离心管。 </li><li>轻轻覆盖用0.5ml水（1.0微克/毫升的密度）1.2克/毫升的密度溶液。 </li><li>平衡管（重量），而不是由体积。 </li><li>轻轻装入试管中的T865转子。 </li><li>旋在超速离心机样品在429460×g离心24小时，在4℃下。 </li><li>立即隔离前0.5 ml的溶液轻柔吹打。保持管，还是越好。 </li></ol> 5. TEM分析<ol><li>准备加入5ml 2％磷钨酸（重量/体积）并调节pH至6.0。 </li><li>过滤2％磷钨酸，用注射器过滤器（孔径：0.2微米）。 </li><li>一滴无菌培养皿20微升脂蛋白的样品。 </li><li>滴20微升过滤2％磷钨酸的旁边在同一盘的样品。 </li><li>轻轻地落了EM电网与样品上的沉闷边休息。 </li><li>在室温下孵育1分钟。 </li><li>轻轻拍打在滤纸上的EM格一侧去除从电网样品。 </li><li>轻轻在2％磷钨酸的暗面搁滴EM网格。 </li><li>在室温下孵育1分钟。 </li><li>轻轻拍打在滤纸上的EM网格的一侧，以从电网除去磷钨酸。 </li><li>用TEM，捕捉脂蛋白（ 图4）的图像。 </li></ol>

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The author has nothing to disclose.

在本文中，可以使用两个系统，以保持Caco-2细胞进行了描述，即，常规的组织培养皿和渗透膜。利用渗透膜系统的优点包括心尖的和分离基底外侧室，并孵育​​所述脂质混合物并同时收集所述脂蛋白的分泌的能力。然而，渗透膜刀片是昂贵的，并且它们的聚碳酸酯膜不允许良好的细胞可见性。一个这种体外模型的一个优点是，遗传操作的研究将是更经济的和耗时少。为了获得更好的效果?...

4 -的饮食脂肪，和脂溶性药物和维生素肠吸收的研究可以在体内通过使用淋巴瘘模型1进行。然而，所涉及的手术技术不仅具有挑战性的，而且成本很高。虽然体内方法的基础上的粪便分析可以利用，它们被用于主要由胃肠道2,5，以确定百分摄取。本文所述的体外模型更符合成本效益，以及所涉及的技术，可以说是不太具有挑战性。基因修饰的研究也更经济和耗时更少时，他们利用这种体外模型进行的。 因为这是采取由肠细胞的脂溶性物质被打包成脂蛋白6,7，该体外模型的有效性，以产生脂蛋白是至关重要的。两个主要intestina升脂蛋白是乳糜微粒和极低密​​度脂蛋白（VLDL）。乳糜微粒，其定义为具有80nm以上脂蛋白直径，严格生产由小肠时的脂质是大量存在于胃肠内腔。因为它们是最大的脂蛋白，乳糜微粒是可以想象的最有效的脂质转运。此体外模型，其能够产生乳糜微粒8，可用于研究饮食脂肪吸收，脂溶性维生素的吸收由肠道和口服脂溶性药物的生物利用度。脂溶性分子，维生素，或药物中的脂蛋白组分的存在是其吸收由小肠的指标。如先前讨论的，该模型可以用于改善口腔脂溶性药物的生物利用度6。 本文介绍Caco-2细胞如何应保持在渗透膜或定期组织培养皿，如何脂质英里夹具用于刺激脂蛋白生产应准备，如何慢病毒表达系统可以被用来实现有效的过表达，而分离出的脂蛋白应如​​何进行分析。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>VWR</td>
			<td>16750-112</td>
			<td>Pre-warm the growth media in individual tissue culture dish before adding cells</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Fisher</td>
			<td>3600511</td>
			<td>We do not heat inactivate our serum</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-660</td>
			<td>Cells can be washed with PBS prior to trypsin treatment&#160;</td>
		</tr>
		<tr>
			<td>Permeable membrane system</td>
			<td>Fisher</td>
			<td>7200173</td>
			<td>10-cm dish, 3-mm pore size, polycarbonate membrane</td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>Fisher</td>
			<td>08-772-E</td>
			<td>Tissue culture dish</td>
		</tr>
		<tr>
			<td>15 cm dish</td>
			<td>Fisher</td>
			<td>08-772-24</td>
			<td>Tissue culture dish</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Fisher</td>
			<td>12565163</td>
			<td>Tissue culture plate</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent</td>
			<td>Fisher</td>
			<td>PRE2691</td>
			<td>Can be substituted with other transfection reagent</td>
		</tr>
		<tr>
			<td>Reduced serum media</td>
			<td>Invitrogen</td>
			<td>11058021</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>pLL3.7 eGFP</td>
			<td>Addgene</td>
			<td>11795</td>
			<td><a href="https://www.addgene.org/11795/">https://www.addgene.org/11795/</a></td>
		</tr>
		<tr>
			<td>Bottle-top filter</td>
			<td>Fisher</td>
			<td>9761120</td>
			<td>0.45 mm pore</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Fisher</td>
			<td>NC9840454</td>
			<td>10 mg/mL</td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Sigma</td>
			<td>01383-5G</td>
			<td>Prevent freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>Lecithin&#160;</td>
			<td>Fisher</td>
			<td>IC10214625</td>
			<td>Egg lecithin&#160;</td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Fisher</td>
			<td>NC9620276</td>
			<td>Product discontinued; alternative catalog number: 50-121-7956</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablet (EDTA-free)</td>
			<td>Fisher</td>
			<td>5892791001</td>
			<td>Used mainly for samples that need TEM analysis</td>
		</tr>
		<tr>
			<td>Polycarbonate ultracentrifuge tube</td>
			<td>Fisher</td>
			<td>NC9696153</td>
			<td>Reusing it multiple times will collapse the tube</td>
		</tr>
		<tr>
			<td>Lid for ultracentrifuge tube</td>
			<td>Fisher</td>
			<td>NC9796914</td>
			<td>A tool is required to remove the tube/lid from rotor</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Fisher</td>
			<td>50-949-261</td>
			<td>Disposable</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Fisher</td>
			<td>09-719C</td>
			<td>Pore size = 0.2 mm; nylon</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Fisher</td>
			<td>AC208310250</td>
			<td>For preparing 2% phosphotungstic acid, pH 6.0</td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Fisher</td>
			<td>50-238-62</td>
			<td>Extra fine and strong tips</td>
		</tr>
		<tr>
			<td>Formvar/carbon grid</td>
			<td>Fisher</td>
			<td>50-260-34</td>
			<td>Formvar/carbon film square grid 400 Copper</td>
		</tr>
	</tbody>
</table>

using caco-2 cells to study lipid transport by the intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

图1显示正常的13天后汇合Caco-2细胞。圆顶形结构和细胞内脂滴的外观是分化的Caco-2细胞的特征。当Caco-2细胞不播种期间同样地分散，他们会结块并长满在盘的特定区域;而且会有少数地区的菜没有任何细胞。涡流，并放置在盘上的倾斜面，应该避免。同样重要的是要注意，后汇合Caco-2细胞更易受到当新生长培养基加入到细胞中过于粗略地脱离。因此，新的媒体，应缓慢加入，以防止细...

Watch this Scientific Journal Video about Using Caco-2 Cells to Study Lipid Transport by the Intestine at JoVE.com

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

采用Caco-2细胞研究脂质运输由肠

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

using-caco-2-cells-to-study-lipid-transport-by-the-intestine

Research

JoVE Journal

Biology

18.6K 次观看.  California Northstate University. Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

视频: Using Caco-2 Cells to Study Lipid Transport by the Intestine

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

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

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

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

分离纯化果蝇</em磁性微球的排序>外周神经元

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

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

测量离子转运的Na，K，H，K-ATP酶活性的<em&gt;爪蟾</em卵母细胞的原子吸收分光光度法：另一种放射性同位素检测

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

方法来研究上皮转运蛋白功能和表达在纯肠和Caco-2细胞生长在3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

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

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

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

小鼠小肠的混合原代培养拟肠激素分泌的研究和活细胞成像的肠内分泌细胞的供

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

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

系绳双层脂膜，以监测金纳米粒子和脂膜之间的热传递

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

小鼠间质瓣细胞分离研究主动脉瓣体外钙化

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

用于测量食品铁生物利用度的Caco-2细胞生物测定法

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...