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。

<ol>
	<li>Tabas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+in+health+and+disease.">Cholesterol in health and disease.</a> J. Clin. Invest. 110, 583-590 (2002).</li><li>Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+feedback:+from+Schoenheimer's+bottle+to+Scap's+MELADL.">Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL.</a> J. Lipid Res. 50, 15-27 (2009).</li><li>Tabas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+cellular+cholesterol+accumulation:+basic+concepts+and+physiological+implications.">Consequences of cellular cholesterol accumulation: basic concepts and physiological implications.</a> J. Clin. Invest. 110, 905-911 (2002).</li><li>Murphy, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Density+Lipoprotein+Reduces+the+Human+Monocyte+Inflammatory+Response.">High-Density Lipoprotein Reduces the Human Monocyte Inflammatory Response.</a> Arterioscler. Thromb. Vasc. Biol. 28, 2071-2077 (2008).</li><li>Mujawar, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Immunodeficiency+Virus+Impairs+Reverse+Cholesterol+Transport+from+Macrophages.">Human Immunodeficiency Virus Impairs Reverse Cholesterol Transport from Macrophages.</a> PLoS Biology. 4, 365-36 (2006).</li><li>Patel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+High-Density+Lipoprotein+Increases+Plasma+High-Density+Lipoprotein+Anti-Inflammatory+Properties+and+Cholesterol+Efflux+Capacity+in+Patients+With+Type+2+Diabetes.">Reconstituted High-Density Lipoprotein Increases Plasma High-Density Lipoprotein Anti-Inflammatory Properties and Cholesterol Efflux Capacity in Patients With Type 2 Diabetes.</a> J. Am. Coll. Cardiol. 53, 962-971 (2009).</li><li>Shaw, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+reconstituted+high-density+lipoprotein+leads+to+acute+changes+in+human+atherosclerotic+plaque.">Infusion of reconstituted high-density lipoprotein leads to acute changes in human atherosclerotic plaque.</a> Circ. Res. 103, 1084-1091 (2008).</li><li>Kekulawala, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+freezing+on+high-density+lipoprotein+functionality.">Impact of freezing on high-density lipoprotein functionality.</a> Anal. Biochem. 379, 75-77 (2008).</li><li>D'Souza, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure/Function+Relationships+of+Apolipoprotein+A-I+Mimetic+Peptides:+Implications+for+Antiatherogenic+Activities+of+High-Density+Lipoprotein.">Structure/Function Relationships of Apolipoprotein A-I Mimetic Peptides: Implications for Antiatherogenic Activities of High-Density Lipoprotein.</a> Circ. Res. 107, 217-227 (2010).</li><li>Tchoua, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cholesteryl+ester+transfer+protein+overexpression+and+inhibition+on+reverse+cholesterol+transport.">The effect of cholesteryl ester transfer protein overexpression and inhibition on reverse cholesterol transport.</a> Cardiovasc Res. 77, 732-739 (2008).</li><li>Mukhamedova, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+apolipoprotein+A-I-dependent+cholesterol+efflux+elevates+cholesterol+export+from+macrophages+in+vivo.">Enhancing apolipoprotein A-I-dependent cholesterol efflux elevates cholesterol export from macrophages in vivo.</a> J. Lipid Res. 49, 2312-2322 (2008).</li><li>Mukhamedova, N., Fu, Y., Bukrinsky, M., Remaley, A. T., Sviridov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Different+Regions+of+ATP-Binding+Cassette+Transporter+A1+in+Cholesterol+Efflux.">The Role of Different Regions of ATP-Binding Cassette Transporter A1 in Cholesterol Efflux.</a> Biochemistry. 46, 9388-9398 (2007).</li><li>Sviridov, D., Fidge, N., Beaumier-Gallon, G., Fielding, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+A-I+stimulates+the+transport+of+intracellular+cholesterol+to+cell-surface+cholesterol-rich+domains+(caveolae.">Apolipoprotein A-I stimulates the transport of intracellular cholesterol to cell-surface cholesterol-rich domains (caveolae.</a> Biochem. J. 358, 79-86 (2001).</li><li>Smyth, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+harlequin+ichthyosis+delineates+a+key+role+for+abca12+in+lipid+homeostasis.">A mouse model of harlequin ichthyosis delineates a key role for abca12 in lipid homeostasis.</a> PLoS Genet. 4, e1000192-e1000192 (2008).</li><li>Fu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Caveolin-1+Enhances+Cholesterol+Efflux+in+Hepatic+Cells.">Expression of Caveolin-1 Enhances Cholesterol Efflux in Hepatic Cells.</a> J. Biol. Chem. 279, 14140-14146 (2004).</li><li>Calkin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+High-Density+Lipoprotein+Attenuates+Platelet+Function+in+Individuals+With+Type+2+Diabetes+Mellitus+by+Promoting+Cholesterol+Efflux.">Reconstituted High-Density Lipoprotein Attenuates Platelet Function in Individuals With Type 2 Diabetes Mellitus by Promoting Cholesterol Efflux.</a> Circulation. 120, 2095-2104 (2009).</li></ol>

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?...

<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

Cholesterol Efflux Assay | Medicine | Video

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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29.5K 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。

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Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

实验转移的含量

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

科研

医学

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

在犬体内的肌肉功能测定

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

一个小鼠模型角膜血管生成研究掌上含量

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

球体的检测，以测量TGF -β诱导的入侵

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

一个<em在体内</em分析测试血管通透性

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

该<em&gt;在OVO</em&gt; CAM作为一个肉瘤Xenograftmodel的检测

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

模拟胰腺神经可塑性：<em&gt;体外</em&gt;双神经元可塑性分析

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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 ...

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.