Name: using caco-2 cells to study lipid transport by the intestine
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Description: Watch this Scientific Journal Video about Using Caco-2 Cells to Study Lipid Transport by the Intestine at JoVE.com

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><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&amp;nbsp;</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>&lt;a href=&quot;https://www.addgene.org/11795/&quot;&gt;https://www.addgene.org/11795/&lt;/a&gt;</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&amp;nbsp;</td><td>Fisher</td><td>IC10214625</td><td>Egg lecithin&amp;nbsp;</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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.7K 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

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

胆固醇外流检测

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

科研

医学

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

模型和方法来评价药物输送系统在整个细胞屏障的运输

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

生物工程

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

探索通过脂肪吞噬对脂滴分解代谢的调节

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This ...

癌症研究

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

X 射线辐照 Caco-2 细胞与 PBMC 间串扰的共培养方法研究

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

免疫与感染

Lipid Exchange Assay in Living Cells

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane&#39;s propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at&#160;the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

活细胞中的脂质交换测定

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the ...

生物化学

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

人类肠道器官脂滴形成特征化和定量的荧光测定

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup&gt; -</sup&gt;通道和转运

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

生物学

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

内皮细胞转吞测定作为 体外 模型评估内部血-视网膜屏障通透性

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

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 ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

采用Caco-2细胞研究脂质运输由肠

摘要

探索更多视频

此视频中的章节

胆固醇外流检测

模型和方法来评价药物输送系统在整个细胞屏障的运输

探索通过脂肪吞噬对脂滴分解代谢的调节

X 射线辐照 Caco-2 细胞与 PBMC 间串扰的共培养方法研究

活细胞中的脂质交换测定

人类肠道器官脂滴形成特征化和定量的荧光测定

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质

内皮细胞转吞测定作为体外模型评估内部血-视网膜屏障通透性

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

肠上皮细胞通透性的体外及体内测定方法

采用Caco-2细胞研究脂质运输由肠

摘要

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此视频中的章节

胆固醇外流检测

模型和方法来评价药物输送系统在整个细胞屏障的运输

探索通过脂肪吞噬对脂滴分解代谢的调节

X 射线辐照 Caco-2 细胞与 PBMC 间串扰的共培养方法研究

活细胞中的脂质交换测定

人类肠道器官脂滴形成特征化和定量的荧光测定

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质

内皮细胞转吞测定作为 体外 模型评估内部血-视网膜屏障通透性

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

肠上皮细胞通透性的体外及体内测定方法

内皮细胞转吞测定作为体外模型评估内部血-视网膜屏障通透性