这项工作得到了国家健康与医学研究委员会（NHMRC）澳大利亚（526615和545906）和部分赠款，由维多利亚州政府的OIS计划。 DS是NHMRC研究员。

1。 [3H]胆固醇的制备<ol><li>在通风柜，免除到1.5 ml离心管（0.5微居里（19 kBq）每口井是一个典型的检测要求）[3H]胆固醇所需的金额。 </li><li>如果[3H]胆固醇在甲苯暂停，完全干燥氮气和重悬与100％乙醇的终浓度为1微居里（37 kBq /μL。涡拌匀。 </li></ol> 2。的电镀细胞和标签细胞内胆固醇该协议已通过测试，使用以下类型的细胞:人单核细胞4,5，THP-1人单核细胞-巨噬细胞6,7,8的RAW 264.7巨噬细胞5,9,10,11，12 HeLa细胞，人脐静脉内皮细胞（HUVEC），BHK-21细胞9，人类和小鼠成纤维细胞13,14，肝癌人肝癌细胞15，在修改后的形式，从血小板&lt;SUP&gt; 16。 <ol><li>重悬细胞，并计算它们。到12孔板板细胞最终密度0.2x10 6细胞，每孔0.9毫升完全培养基。细胞会继续生长和外排实验时将达到0.8x10 6细胞，每孔。对于不分裂的细胞分化的THP-1细胞，如播种密度应为0.8x10 6细胞每口井。如果分别用6 - 或24孔板，双或每孔细胞数的一半。井的数量应该足够一式四份决定为每个实验条件。 </li><li>如果需要的THP-1细胞的分化，加入PMA（终浓度为0.1微克/毫升）到48-72Ĥ媒体。 </li><li>含[3H]胆固醇的离心管中加入了媒体的小等份。 </li><li>与媒体之前加入[3H]胆固醇单独成一个完整的媒体等份（100μL/孔），洗管的两侧。 </li> &lt;李&gt;添加媒体细胞（每孔终体积为1毫升）含[3H]胆固醇的水井。如果没有标签之前需要细胞治疗，细胞可镀在[H]含胆固醇的媒介。 </li><li>孵育细胞在细胞培养孵化器48小时（37℃，5％CO 2）。 </li></ol> 3。平衡育成<ol><li>经过48小时的潜伏期，在显微镜下检查细胞。确保它们是健康的，并在约80％汇合。 </li><li>取出介质含有[3H]胆固醇。轻轻地用PBS洗涤细胞。重复3次洗涤。 </li><li>准备无血清培养基。如果需要，加入LXR激动剂（如在1-4毫摩尔/升-901317）或cAMP（0.3μmol/ L的;只小鼠起源细胞）激活细胞无血清媒体。 </li><li>每孔加入500μL无血清媒体。 </li><li>在细胞培养孵化器18小时的孵育（37℃，5％CO 2）。 </li></ol> &lt;类p ="jove_title"&gt; 4。胆固醇流出孵化<ol><li>经过18小时无血清培养基中培养，在显微镜下检查细胞。确保他们健康和融合（最低80％汇合）。 </li><li>准备在无血清培养基中的胆固醇流出受体的解决方案。受体的例子包括: 载脂蛋白AI（载脂蛋白A-I），（终浓度为10微克/毫升） 高密度脂蛋白（HDL）（终浓度为20微克/毫升） 环糊精（终浓度为200微克/毫升） 等离子（终浓度为1-2％） </li><li>轻轻地用PBS洗涤细胞。 </li><li>添加受体，每孔250μL无血清培养基。 </li><li>离开一个集水井，以确定背景流出（流出受体与NO无血清媒体） </li><li>孵育细胞，在细胞培养孵化器为2小时（37℃，5％CO 2）。流出潜伏期的持续时间可能会有所不同，从30分钟到8小时，如果需要的话。 </li></ol> 5。加工萨mples <ol><li> 2小时后孵化检查，显微镜下的细胞。 </li><li>收集到1.5 ml离心管媒体。在室温1-10分钟，14,000 rpm的旋转，以去除细胞碎片。 </li><li> 7毫升闪烁瓶中转入100μL媒体。新增的Insta-凝胶加5毫升（珀金埃尔默）和涡混合物。其余样品储存在4°C </li><li>放置在冰箱板为30分钟。每孔加入500μLDH 2。细胞在显微镜下检查，以确保所有的细胞都从井底解除。如果仍然附着，要么离开DH 2 O的 4°C过夜或刮井板块。 </li><li>一旦升空，所有的细胞，吸来打破细胞团块。 7毫升闪烁瓶转移到100微升。新增的Insta-凝胶加5毫升（珀金埃尔默）和涡混合物。储存在4°C其余板块</li><li>闪烁计数器放置在闪烁瓶。配置计数器计数3 H]DPM单位。 </li></ol> 6。分析结果<ol><li>胆固醇流出率通常表示为胆固醇的比例，从细胞受体。使用下列公式: </li></ol><img alt="式（1）" src="/files/ftp_upload/3810/3810eq1.jpg" /><ol start="2"><li>具体计算差异的存在或缺乏受体（空）之间的流出流出。 </li></ol><img alt="公式（2）" src="/files/ftp_upload/3810/3810eq2.jpg" /> 7。时间轴<img alt="图1" src="/files/ftp_upload/3810/3810timeline.jpg" /> 8。代表结果如图胆固醇流出实验结果的一个例子。 1。在这个实验中THP-1人单核细胞分化成巨噬细胞和胆固醇流出到不同的受体测试。没有受体（"空白"）介质的胆固醇流出为0.79％，此值被视为一个非特定的外流，并从其他值中减去。胆固醇外流的人载脂蛋白A-Ⅰ（终浓度为30微克/毫升）为4.75％。本实验参考的血浆样品被纳入监测实验间变异。参考样本，可用于跨越大量的实验数据正常化，但我们发现它审慎的重复检测，如果是高变异。测试之前和之后的病人药物治疗的病人的血浆（终浓度为2％）。得出的结论是，在这个病人服药有一个对血浆容量支持胆固醇外流的负面影响。 外排实验结果的另一个例子是在图所示。 2。在这个实验中，RAW 264.7巨噬细胞被激活或不激活LXR激动剂TO-901317过夜培养（终浓度加入1μmol/ L）和胆固醇流出的人类血浆相同的样本（2％）进行了测试。得出的结论是LXR激动剂激活细胞表达ABC转运增加胆固醇释放到细胞外受体细胞的能力。 <img alt="图1" src="/files/ftp_upload/3810/3810fig1.jpg" /> 图1。从THP-1细胞的各种受体的胆固醇流出胆固醇流出的百分比（即标记胆固醇的比例从指定的受体细胞）。后显示空白值的加减。平均值±SD一式四份决定，* P &lt;0.05。 <img alt="图2" src="/files/ftp_upload/3810/3810fig2.jpg" /> 图2。从细胞胆固醇流出的RAW 264.7激活或不激活LXR激动剂对人体血浆胆固醇流出的百分比（即标记胆固醇的比例。指定受体）后显示空白值的加减。平均值±SD一式四份决定，* P &lt;0.001。

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有关这项研究的作者声明没有利益冲突。

所描述的方法，测量胆固醇流出是用来衡量运动的胆固醇从细胞到细胞外的胆固醇受体。了解这种方法有几个关键因素。 标签和平衡中所述的方法被添加到标记的胆固醇血清含药血清。虽然从来没有详细调查，这是假设纳入血清脂蛋白胆固醇和他们采取由细胞组成。重要的是让脂蛋白足够的时间采取了从脂蛋白胆固醇移动到细胞膜。一般24小时的标签是足够的，但48小时的标签达到较高的细胞内胆固醇的具体活动。它需要考虑到，如果[14]胆固醇用来代替[3H]胆固醇（如双标记实验），具体行政行为前ivity是非常低，加上足够的标记的胆固醇达到目的的具体活动可能改变细胞中胆固醇含量，从而影响了结果。同样重要的是，（ 我 ）没有任何标记的胆固醇或细胞表面上的非特异性被困脂蛋白胆固醇会被迅速纳入受体通过非特异性转移;及（ii）标记的胆固醇之间的各种细胞内的平衡池。虽然平衡的无血清培养基培养24小时，在我们的经验是不足以实现这些目标，通常48 h后使用。许多类型的细胞不存活48小时，甚至24小时无血清培养基中。在这种情况下，0.1％BSA（基本上脂肪无酸）可以作为替代。脂蛋白耗尽血清或各种血清替代补充，可以作为最后的手段使用，但重要的是要认识到，脂蛋白缺乏血清和血清替代补充可Çontain载脂蛋白和可能影响的结果。不幸的是制造商往往不透露补充剂的成分。 时间流出孵化的胆固醇外流导致装载到细胞外受体和胆固醇耗竭胆固醇。在细胞和受体的胆固醇含量的变化可能会影响胆固醇流出的速度和其他特性。此外，如果承兑人含有胆固醇（如高密度脂蛋白胆固醇）可能会从受体细胞，如果承兑人胆固醇的具体活动变得显着，将复杂的解释结果。因此，最好是保持尽可能短的流出孵化，允许但足够数量的标记胆固醇从细胞受体，使一个可靠的检测时间。一般2-6小时是推荐的时间。孵化时间超过24小时，将反映的equili州brium，因此将是受体积累，而不是胆固醇胆固醇流出率容量指示。 受体不同的受体是胆固醇外流的不同途径的具体。用于测量具体的胆固醇流出，载脂蛋白A-我是用来反映ABCA1依赖的外流，而高密度脂蛋白胆固醇或ABCG1的重组HDL与SR-B1依赖途径。环糊精通常被用来评估非特异性流出。重要的是要包括在实验的一个"空白"的样品，不包含在所有的受体，非特异性分离崩解死亡细胞释放的胆固醇和胆固醇的贡献来衡量的。 受体的浓度胆固醇流出，包括（一）胆固醇由细胞释放及（ii）由承兑人吸收，这两个步骤可以限速。如果实验目的是探讨在变化细胞释​​放胆固醇的能力（如转染后或击倒），然后受体的浓度不应该是限速。对于载脂蛋白A-I和高密度脂蛋白的浓度为30-50微克/毫升通常非限速。如果容量，承兑人支持胆固醇流出是调查目标（如孤立的HDL或从病人或动物的血浆样本，以支持胆固醇流出的能力），然后受体的浓度应该限速。为载脂蛋白A-我和高密度脂蛋白通常是10微克/毫升，血浆样品，这通常是1-2％，但一个初步的剂量依赖实验，找到了正确的浓度是必不可少的选择剂量。 活化细胞负责具体的胆固醇流出的关键要素是ABC转运，ABCA1和ABCG1。 LXR和LXR激动剂细胞的预孵化调节（最流行的化合物是TO-901317可从SIGMA）浓度1-4μmol/ L的显着增加了特定的胆固醇流出的比例。如鼠标起源细胞的RAW 264.7或J774，也可以通过cAMP激活（0.3毫摩尔/升）。 载入细胞与胆固醇载入中胆固醇的细胞预孵化与乙酰化或氧化低密度脂蛋白或胆固醇环糊精复合物，也刺激胆固醇流出。加载细胞，尤其是巨噬细胞胆固醇改变其新陈代谢的许多方面，需要牢记。 （胆固醇加载与加载不胆固醇）模式的选择取决于研究的科学目标。 结果介绍通常胆固醇流出实验的结果是胆固醇从细胞释放的百分比:消除变异细胞数量在个别井和标记效率。然而，TH是唯一有效的，如果治疗不影响胆固醇的吸收，并在承兑人（空白）的情况下流出。这两个条件，需要实验证明。 从理论上讲，这种方法可以用来研究任何蜂窝选区流出到细胞外受体。如果不像胆固醇，这种化合物被代谢，然后分析纯化步骤需要包含。此外，如果在细胞中合成的化合物，合成的前体，可用于标注。例如，[14]醋酸和[3Ĥ]水被用来研究新合成的胆固醇流出和[14 C]研究磷脂流出的胆。需要通常是由薄层色谱分离前体和中间体，在这种情况下，新合成的胆固醇。

<table border="1"><tbody><tr><td> 试剂\细胞 </td><td> HeLa细胞 </td><td> THP-1 </td><td> 未处理 </td><td> 人脐静脉内皮细胞 </td><td> 的BHK-21 </td><td> 卵泡液 </td></tr><tr><td> 完整的媒体 </td><td colspan="3"> 的RPMI - 10％FCS - 1％L-谷氨酰胺 - 0.2％，笔链球菌</td><td> M199 - 5％FCS - 1％L-谷氨酰胺 - 1％钢笔链球菌 - 2％肝素钠（1M） - 7％的内皮细胞生长添加物（ECGS） </td><td colspan="2"> DMEM培养液 - 10％FCS - 1％L-谷氨酰胺 - 0.2％，笔链球菌</td></tr><tr><td> 无血清 </td><td colspan="3"> 的RPMI - 1％L-谷氨酰胺 - 0.2％，笔链球菌</td><td> M199 - 1％L-谷氨酰胺 - 1％钢笔链球菌 - 2％肝素钠（1M） - 7％的内皮细胞生长日补充（ECGS） </td><td colspan="2"> DMEM培养液 - 1％L-谷氨酰胺 - 0.2％，笔链球菌</td></tr><tr><td> 通过方法 </td><td> 1 x胰蛋白酶</td><td>悬挂</td><td>刮</td><td colspan="3"> 1 x胰蛋白酶</td></tr><tr><td> 通过比 </td><td> 3:10 </td><td> 1:3 </td><td></td><td> 1:20 </td><td> 1:3 </td><td> 1:3 </td></tr><tr><td> 细胞活化（可选） </td><td></td><td colspan="5"> TO-901317 - 的LXR激动剂 - 终浓度。 4μM的</td></tr><tr><td> 其他试剂 </td><td></td><td> 佛12豆蔻13酯（PMA） - 细胞分化 - 终浓度。 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol efflux assay

细胞中胆固醇的含量必须保持在非常紧张的限制，过多或过少的胆固醇在一个细胞在细胞膜破坏，细胞凋亡和坏死1。细胞可以从细胞内合成和血浆脂蛋白胆固醇，这两个来源是足够充分满足细胞胆固醇的要求。胆固醇的合成和吸收的过程中，严格监管和胆固醇的不足之处是罕见的2。过多的胆固醇是比较常见的问题3。细胞与肝细胞的异常和肾上腺​​皮质细胞在一定程度上，都无法降低胆固醇。细胞有两种选择，以降低他们的胆固醇含量:转换成胆固醇酯，能力有限的选项中的胆固醇，胆固醇酯细胞超载也是有毒，和胆固醇流出，潜在的无限容量的选择。胆固醇外流是一个规范的IFIC过程中，细胞内的转运，如三磷酸腺苷结合盒转运蛋白体A1（ABCA1）和G1（ABCG1的）和清道夫受体B1型监管。血浆中的胆固醇的天然受体是高密度脂蛋白（HDL）和载脂蛋白AI。 胆固醇外流试验设计定量从培养细胞的胆固醇流出率。它测量的能力，以维持细胞胆固醇流出和/或血浆受体的能力，接受从细胞释放的胆固醇。该法包括以下步骤。步骤1:标记细胞内胆固醇含血清培养基中加入标记的胆固醇，并与细胞孵育24-48小时。这一步，​​可装载细胞与胆固醇结合。第2步:在无血清培养基培养细胞之间的所有细胞内胆固醇池标记的胆固醇平衡。此阶段可结合激活细胞CHolesterol转运。第三步:培养细胞与胞外受体和定量标记的胆固醇从细胞受体的运动。如果胆固醇的前体被用来标记新合成的胆固醇，第四步，胆固醇的净化，可能需要。 该法提供了以下信息:（i）一个特定的治疗（一个突变，敲下来，过度或治疗）如何影响细胞的能力流出胆固醇和（ii）如何接受胆固醇血浆受体的能力受疾病或治疗。这种方法通常用于研究心血管，代谢和神经退行性疾病，传染病和生殖系统疾病方面。

Watch this Scientific Journal Video about Cholesterol Efflux Assay at JoVE.com

胆固醇流出测定|医学|视频

Cholesterol Efflux Assay

胆固醇检测到定量，从培养细胞的胆固醇流出率和血浆受体的能力，接受从细胞释放的胆固醇。该法由标记细胞内池和胆固醇释放到细胞外受体之间的胆固醇，胆固醇平衡。

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 ...

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29.7K 次观看.  Baker IDI Heart and Diabetes Institute. 胆固醇检测到定量，从培养细胞的胆固醇流出率和血浆受体的能力，接受从细胞释放的胆固醇。该法由标记细胞内池和胆固醇释放到细胞外受体之间的胆固醇，胆固醇平衡。

视频: Cholesterol Efflux Assay

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

哺乳动物组织和异种卵母细胞与胆固醇的丰富

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

科研

生物化学

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

点变异荧光相关光谱用于分析活细胞质膜上的分子扩散

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

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 ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

降血脂药物对胆固醇颗粒形态的差异化作用

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

医学

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

高通量硝基苯并二氮唑标记的胆固醇积液测定

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

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

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).

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 ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

基于细胞健康监测的实时细胞成像分析对低密度脂蛋白胆固醇的吸收检测

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的 体内 动力学

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

胆固醇外流检测

摘要

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此视频中的章节

哺乳动物组织和异种卵母细胞与胆固醇的丰富

点变异荧光相关光谱用于分析活细胞质膜上的分子扩散

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

活细胞中的脂质交换测定

降血脂药物对胆固醇颗粒形态的差异化作用

高通量硝基苯并二氮唑标记的胆固醇积液测定

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质

采用Caco-2细胞研究脂质运输由肠

基于细胞健康监测的实时细胞成像分析对低密度脂蛋白胆固醇的吸收检测

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的体内动力学

胆固醇外流检测

摘要

探索更多视频

此视频中的章节

哺乳动物组织和异种卵母细胞与胆固醇的丰富

点变异荧光相关光谱用于分析活细胞质膜上的分子扩散

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

活细胞中的脂质交换测定

降血脂药物对胆固醇颗粒形态的差异化作用

高通量硝基苯并二氮唑标记的胆固醇积液测定

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质

采用Caco-2细胞研究脂质运输由肠

基于细胞健康监测的实时细胞成像分析对低密度脂蛋白胆固醇的吸收检测

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的 体内 动力学

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的体内动力学