Name: high-throughput nitrobenzoxadiazole-labeled cholesterol efflux assay
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay at JoVE.com

这项工作得到了西班牙马德里 salud carlos iii 研究所 fis (ps12/00866) 研究赠款的部分支持;fondo euroo para el desarrollo 区域 (fder);red de research ación en sida (ris)、isciii-retic (rd16/h500/0002) 和 cerca 方案/generalitat de catalunya。作者感谢生物研究研究所的逆转录病毒学和病毒免疫病理学实验室。我们感谢特塞兹帕、罗维拉和 c. hurtado 的协助, 并感谢知识和技术转让办公室的 s. cufí在保护发明方面的指导。

为这项研究, 获得了道德委员会的批准 (巴塞罗那医院诊所, 刑事调查委员会; 批准号 hcb/2012-0756) 和所有主体的书面知情同意。
1. nbd-胆固醇制剂
<ol><li>将 nbd 胆固醇 (mM 494.63; 见材料表) 中溶解, 以获得库存 (2 mm)。对于10毫克小瓶, 将整个小瓶中的整个小瓶中的乙醇体积溶解, 以获得 2 mm 的库存。</li>
	<li>从 rpmi 1640 中的库存中稀释 nbd 胆固醇, 再加上10% 的胎牛血清和5...

<ol>
	<li>Hansson, G. K., Hermansson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+system+in+atherosclerosis.">The immune system in atherosclerosis.</a> Nature Immunology. 12 (3), 204-212 (2011).</li><li>Libby, P., Ridker, P. M., Hansson, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+challenges+in+translating+the+biology+of+atherosclerosis.">Progress and challenges in translating the biology of atherosclerosis.</a> Nature. 473 (7347), 317-325 (2011).</li><li>Ilhan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis+and+the+role+of+immune+cells.">Atherosclerosis and the role of immune cells.</a> World Journal of Clinical Cases. 3 (4), 345 (2015).</li><li>Greaves, D. 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C., Rotllan, N., Julve, J., Blanco-Vaca, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+macrophage-specific+RCT+and+antioxidant+and+antiinflammatory+HDL+activity+measurements:+New+tools+for+predicting+HDL+atheroprotection.">In vivo macrophage-specific RCT and antioxidant and antiinflammatory HDL activity measurements: New tools for predicting HDL atheroprotection.</a> Atherosclerosis. 206 (2), 321-327 (2009).</li><li>Santos-Gallego, C. G., Ibanez, B., Badimon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL-cholesterol:+Is+it+really+good?+Differences+between+apoA-I+and+HDL.">HDL-cholesterol: Is it really good? Differences between apoA-I and HDL.</a> Biochemical Pharmacology. 76 (4), 443-452 (2008).</li><li>Rothblat, G. H., Phillips, M. C. <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+heterogeneity+and+function+in+reverse+cholesterol+transport.">High-density lipoprotein heterogeneity and function in reverse cholesterol transport.</a> Current Opinion in Lipidology. 21 (3), 229-238 (2010).</li><li>Hafiane, A., Genest, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL-mediated+cellular+cholesterol+efflux+assay+method.">HDL-mediated cellular cholesterol efflux assay method.</a> Annals of Clinical and Laboratory Science. 45 (6), 659-668 (2015).</li><li>Szalaia, R., Hadzsiev, K., Melegh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P450+Drug+Metabolizing+Enzymes+in+Roma+Population+Samples:+Systematic+Review+of+the+Literature.">Cytochrome P450 Drug Metabolizing Enzymes in Roma Population Samples: Systematic Review of the Literature.</a> Current Medicinal Chemistry. 23, 1-26 (2016).</li><li>Mcintosh, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Sterols+for+the+Study+of+Cholesterol+Trafficking+in+Living+Cells.">Fluorescent Sterols for the Study of Cholesterol Trafficking in Living Cells.</a> Probes and Tags to Study Biomolecular Function: for Proteins, RNA, and Membranes. , 1-33 (2008).</li><li>Osta&#353;ov, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIM+studies+of+22-+and+25-NBD-cholesterol+in+living+HEK293+cells:+Plasma+membrane+change+induced+by+cholesterol+depletion.">FLIM studies of 22- and 25-NBD-cholesterol in living HEK293 cells: Plasma membrane change induced by cholesterol depletion.</a> Chemistry and Physics of Lipids. 167, 62-69 (2013).</li><li>Song, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+fluorescent+NBD-cholesterol+efflux+in+THP-1-derived+macrophages.">Characterization of fluorescent NBD-cholesterol efflux in THP-1-derived macrophages.</a> Molecular Medicine Reports. 12 (4), 5989-5996 (2015).</li><li>Liu, N., Wu, C., Sun, L., Zheng, J., Guo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sesamin+enhances+cholesterol+efflux+in+RAW264.7+macrophages.">Sesamin enhances cholesterol efflux in RAW264.7 macrophages.</a> Molecules. 19 (6), 7516-7527 (2014).</li><li>Qin, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+THP-1+cells+as+a+model+for+mimicking+the+function+and+regulation+of+monocytes+and+macrophages+in+the+vasculature.">The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature.</a> Atherosclerosis. 221 (1), 2-11 (2012).</li><li>Low, H., Hoang, A., 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=Cholesterol+Efflux+Assay.">Cholesterol Efflux Assay.</a> Journal of Visualized Experiments. (61), 5-9 (2012).</li><li>Gimpl, G., Gehrig-Burger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+reporter+molecules.">Cholesterol reporter molecules.</a> Bioscience Reports. 27 (6), 335-358 (2007).</li><li>Zhang, J., Cai, S., Peterson, B. R., Kris-Etherton, P. M., Heuvel, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vanden+Development+of+a+Cell-Based,+High-Throughput+Screening+Assay+for+Cholesterol+Efflux+Using+a+Fluorescent+Mimic+of+Cholesterol.">Vanden Development of a Cell-Based, High-Throughput Screening Assay for Cholesterol Efflux Using a Fluorescent Mimic of Cholesterol.</a> ASSAY and Drug Development Technologies. 9 (2), 136-146 (2011).</li><li>Rader, D. D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+HDL+metabolism+and+function:+implications+for+novel+therapies.">Molecular regulation of HDL metabolism and function: implications for novel therapies.</a> Journal of Clinical Investigation. 116 (12), 3090-3100 (2006).</li><li>Davidson, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+apolipoprotein+B+depletion+on+HDL+subspecies+composition+and+function.">The effects of apolipoprotein B depletion on HDL subspecies composition and function.</a> Journal of Lipid Research. 57 (4), 674-686 (2016).</li><li>Park, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sage+weed+(Salvia+plebeia)+extract+antagonizes+foam+cell+formation+and+promotes+cholesterol+efflux+in+murine+macrophages.">Sage weed (Salvia plebeia) extract antagonizes foam cell formation and promotes cholesterol efflux in murine macrophages.</a> International Journal of Molecular Medicine. 30 (5), 1105-1112 (2012).</li><li>Litvinov, Y., Savushkin, E. V., Garaeva, E. A. D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+Efflux+and+Reverse+Cholesterol+Transport:+Experimental+Approaches.">Cholesterol Efflux and Reverse Cholesterol Transport: Experimental Approaches.</a> Current Medicinal Chemistry. 371 (25), 2383-2393 (2016).</li><li>Phillips, A., Mucksavage, M. L., Wilensky, R. L., Mohler, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+efflux+capacity,+high-density+lipoprotein+function,+and+atherosclerosis.">Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.</a> N Engl J Med. 364 (2), 127-135 (2011).</li><li>Rohatgi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL+Cholesterol+Efflux+Capacity+and+Incident+Cardiovascular+Events.">HDL Cholesterol Efflux Capacity and Incident Cardiovascular Events.</a> New England Journal of Medicine. 371 (25), 2383-2393 (2014).</li></ol>

荧光标记胆固醇是分析和研究天然胆固醇体外特性和代谢的一种很有前途的策略。它的主要优点是, 它可以被细胞吸收, 允许细胞内和膜内的分布研究, 并可以应用于胆固醇外排检测, 如在本协议中 (图 7)。一些被荧光标记的甾醇允许在体外跟踪胆固醇, 包括身体胆固醇, 丹西胆固醇, 脱氢雌酚,和 22-和25-nbd-低压12。特别是, 22-nbd-胆固醇是适当的研究不同细胞类?...

根据世界卫生组织的数据, 全世界目前的主要死亡原因是缺血性心脏病和中风 (死亡总人数为 1 520万人) 1.两者都是心血管疾病 (cvd), 可在动脉粥样硬化和血管破裂 2, 3.
动脉粥样硬化是一种血管壁炎症性疾病, 其中巨噬细胞、t 细胞、肥大细胞和树突状细胞浸润内皮细胞并从血液中积累, 最终形成动脉粥样硬化斑块。动脉粥样硬化斑块呈现脂质核和胆固醇晶体, 证明了高分辨率 b 超测量颈动脉内膜介质厚度4,5。在巨噬细胞中, 胆固醇流出对脂质受体颗粒是通过 atp 结合盒 (abc) 受体 abca1, atp 结合盒子家族 g 成员 1 (abcg1), 和清除剂受体 sr-bi 进行的。巨噬细胞中胆固醇流入和流出的不平衡被认为是动脉粥样硬化发生的一个关键过程6。胆固醇流出被认为是消除胆固醇从外周组织到血浆和肝脏的关键一步, 这个过程被称为反向胆固醇转运 (rct)。胆固醇主要从巨噬细胞转移到高密度脂蛋白 (hdl) 颗粒表面发现的载脂蛋白 a1 (apoa1)。然后, 高密度脂蛋白将胆固醇输送到肝脏进行排泄和再利用7,8,9。
传统上, (3h) 放射性标记的胆固醇已被用于胆固醇排出10。放射性同位素发射信号高度敏感10;然而, 放射性标记的胆固醇存在明显的障碍, 如长期的规程、电离辐射照射的风险, 以及需要特殊的放射性设施和设备来确保安全处理放射性排放。相反, 荧光已成功地纳入诊断技术, 因为它在荧光信号检测简单, 荧光种类繁多, 并具有安全性11。几种荧光标记的甾醇已被用于研究胆固醇代谢, 包括脱氢雌酚 (具有内在荧光)、丹西胆固醇、4, 4-二氟-3, 4adia-s-indacene (body y)-胆固醇和 22-(n-(7-硝基苯甲酸-2-oxa-1, 3-diazol-4-基)-23, 24-bisnor-5 choren-3β-ol (nbd-胆固醇)。特别是, nbd 胆固醇在人的细胞中具有有效的吸收12。目前有两种不同的 nbd 标签胆固醇:22-(n-(n-(7-硝基苯-2-o理事会-1, 3-二唑-4-基) 氨基)-23, 24-双苯-5-胆碱-3b-ol (22-nbd) 和 25-(n-[(7-硝基苯-2-氧-1, 3-二唑-4-基)-甲基] 氨基)-27-不高胆固醇 (25-nbd;图 1)。标记为22-nbd 的胆固醇可能最适合胆固醇外排研究, 而25-NBD-cholesterol 胆固醇主要用于细胞膜动力学研究13,14。
细胞系通常用于体外胆固醇排出分析是单核细胞样细胞, 如人类白血病衍生的 thp-1 细胞, 小鼠原始264.7 细胞15, 或 j774.1。所有这些细胞都可以用 phorbol 12-mristate 13 乙酸 (pma) 在体外分化为巨噬细胞, 但 thp-1 衍生巨噬细胞 (dthp-1) 最能反映和模仿人类巨噬细胞16。
在本研究中, 我们利用22-NBD-cholesterol 胆固醇作为 [3h]-胆固醇的替代品, 优化和规范荧光高通量法, 以确定 dmthp-1 上血清样品的胆固醇流出能力。此外, 我们还将优化后的荧光技术与标准放射性模拟技术进行了比较。

<table>
	<tbody>
		<tr>
			<td>96-well collecting plate</td>
			<td>Corning Inc</td>
			<td>Costar 3912</td>
			<td>white 96-well plate with opaque clear bottom</td>
		</tr>
		<tr>
			<td>96-well culture plate</td>
			<td>Corning Inc</td>
			<td>Costar 3610</td>
			<td>white 96-well plate with flat clear bottom</td>
		</tr>
		<tr>
			<td>Cholesterol Efflux Assay Kit (Cell-based)</td>
			<td>Abcam</td>
			<td>ab196985</td>
			<td>commercial high-throughput cell-based assay kit aimed to determine the cholesterol efflux</td>
		</tr>
		<tr>
			<td>colorless RPMI 1640</td>
			<td>Sigma-Aldrich</td>
			<td>R7509</td>
			<td>RPMI 1640 with no pehnol-red</td>
		</tr>
		<tr>
			<td>Gen5 Data Analysis Software</td>
			<td>BioTek</td>
			<td></td>
			<td>Version 2.0</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8790-100G</td>
			<td>Glycine, non-animal use</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Biotek</td>
			<td>SYNERGY HT</td>
			<td>Multi-Detection Microplate Reader</td>
		</tr>
		<tr>
			<td>Lysis Solution 1</td>
			<td>in-house</td>
			<td></td>
			<td>50 mM Tris Buffer, 150 mM NaCl and H2O</td>
		</tr>
		<tr>
			<td>Lysis Solution 2</td>
			<td>in-house</td>
			<td></td>
			<td>Pure ethanol and Cell Lysis Solution 1:1 (v:v)</td>
		</tr>
		<tr>
			<td>NBD-cholesterol</td>
			<td>Thermo-Fisher</td>
			<td>N1148</td>
			<td>22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-23,24-Bisnor-5-Cholen-3&#946;-Ol</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td>Phosphate-buffered&#160;saline</td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>Sigma-Aldrich</td>
			<td>202452-250G</td>
			<td>polyethylene glycol</td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma-Aldrich</td>
			<td>79346</td>
			<td>Phorbol 12-merystate B-acetate.</td>
		</tr>
		<tr>
			<td>R10</td>
			<td>in-house</td>
			<td></td>
			<td>RPMI 1640 supplemented with 10 % fetal bovine serum and 5 % Penicillin/Streptomycin.</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td>RPMI 1640 with 2mM L-glutamine containing 1.5 g/L sodium bicarbonate and 4.5 g/L glucose</td>
		</tr>
		<tr>
			<td>THP-1 cells</td>
			<td>Sigma-Aldrich</td>
			<td>ATCC, #TIB-202</td>
			<td>monocyte-like line derived from leukemia from a one year old baby</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma-Aldrich</td>
			<td>P1754</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput nitrobenzoxadiazole-labeled cholesterol efflux assay

动脉粥样硬化导致心血管疾病 (cvd)。目前尚不清楚胆固醇-高密度脂蛋白 (chdl) 浓度是否在动脉粥样硬化的发展中起因果作用。然而, 动脉粥样硬化斑块形成早期的一个重要因素是胆固醇流出能力的 hdl (hdl 颗粒接受胆固醇从巨噬细胞), 以避免泡沫细胞的形成。这是避免胆固醇在内皮细胞中积累的关键一步, 也是通过肝脏消除胆固醇的反向胆固醇转运 (rct) 的一部分。巨噬细胞模型中的胆固醇流出能力对血清或血浆的影响是一种很有前途的工具, 可作为动脉粥样硬化的生物标志物。传统上, [3h]-胆固醇已用于胆固醇外排分析。在这项研究中, 我们的目标是开发一个更安全和更快的策略, 使用荧光标签胆固醇 (nbd-胆固醇) 在细胞检测, 以跟踪胆固醇的吸收和流出过程中的 thp-1 衍生的巨噬细胞。最后, 我们对 nbd-胆固醇流出法进行了优化和规范, 并开发了一种利用96孔板进行高通量分析的方法。

胆固醇流出法的目的是在体外测定含有 hdl 颗粒的特定血清、血浆或上清液的胆固醇流出能力。该方法包括将标签胆固醇加载到标准巨噬细胞系的培养中, 并诱导与与细胞稀释为无 fbs 介质的测试样品接触。最后, 在培养基和细胞裂解液中测量 nbd 的荧光水平。为了优化测量, 从细胞到培养基的流出胆固醇与含有乙醇的溶剂的一个体积混合。在测量之前, 细胞中残留的 nbd 胆固醇会重新悬浮到含有乙醇...

Watch this Scientific Journal Video about High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay at JoVE.com

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

在巨噬细胞模型中测量血清或血浆的体外胆固醇流出能力是动脉粥样硬化的一个很有前途的工具。在本研究中, 我们优化和规范了荧光 nbd-胆固醇外排法, 并开发了一个使用96孔板的高通量分析。

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 ...

该协议是我们在脑血浆中胆固醇回流的胆固醇终止的基于细胞的方法。特别是在筛选中。这可能用于诊断心血管风险，并识别或评估胆固醇降低维持。该技术避免了风险和负担，因为安全处理辐射材料提供的结果具有可比的质量水平，胆固醇回流已报告与胆固醇回流关联，因此，血浆或陶瓷胆固醇回流的接受可以比较，我们的参考控制，并可能提供风险遭受这种方法也将应用于细胞系，像其他人类细胞系，甚至主要宏相。宏相或和人类诱导喷雾按钮干细胞。为了开始这个程序，在纯乙醇中溶解NPD胆固醇，以获得最终浓度为2微摩尔的库存溶液。稀释AR10介质中25微升的NBD胆固醇库存，最终浓度达到5微摩尔。培养thp-1细胞在我们的10个介质在37摄氏度与5%的二氧化碳。每三天将细胞密度调整为每毫升30万个细胞。然后获得一个96井板与平坦的透明底部。为了更好的均质化，在15毫升管中准备10毫升的thp-1细胞，并加入200微升的PMA库存。轻轻地将混合物的100微升混合到板井中，每孔有20万个细胞。在37摄氏度下孵育5%的二氧化碳48至72小时，将thp-1细胞分化成DM thp-1细胞。首先用无菌水稀释10%PBS中的甘氨酸，浓度为200微摩尔，浓度为pH7.4。将40毫升稀释的甘氨酸加入10克钉8000，以准备20%的钉溶液，并大力混合以均质化。接下来，在1.5ml管中，将四个部分（每10个样本的20%）涂抹到每个血清或血浆样品中。将混合物留在冰上25分钟。离心钉脂蛋白B沉淀在13000倍G，并在4摄氏度15分钟。丢弃沉淀物，将上看液转移到新管中。检索包含分化 thp-1 细胞的板。取出并丢弃培养介质，然后用 1X PBS 清洗两次细胞。在每个井中加入100微升5微摩尔MVD胆固醇。在37摄氏度下孵育，二氧化碳含量为5%。第二天丢弃，丢弃介质。用 PBS 清洗细胞两次，然后加入 100 微升 ABDS，或纯脂接受物在 rpmi 1630 介质中稀释，以达到所需的浓度。包括一个阴性控制，其中不包含胆固醇接受器在阳性控制，如文本协议中概述，并在37摄氏度孵育4至6小时。同时，准备200毫升的细胞解解溶液，如文本中所述。将该溶液与乙醇混合，体积比为一比，获得解液。对于介质和BD胆固醇检测，从收集到的新的白色96孔板中收集的细胞介质中去除细胞介质，其底部不透明。在每个介质样品的100微升中加入100微升纯乙醇，获得新板材的一对一比例。使用光照计，测量荧光灯的强度在463纳米的激发和536纳米的发射。对于细胞内NBD胆固醇检测，用PBS清洗细胞两次。将100微升的解液也加入到每井中，并在室温下孵育，同时摇晃25分钟。在此之后，测量荧光的强度，同时在软件中将灵敏度参数调整为 50。然后确定文本协议中概述的胆固醇出血的最终测量中的胆固醇出血率。在纯乙醇中稀释NBD胆固醇会产生最高的荧光强度值，而水溶液中的高含量则会降低强度。这表明，荧光的这种分子的发射高度依赖于介质，其中含有它。当以一比一的比例使用介质和乙醇的混合物时，荧光的强度似乎按比例增加，胆固醇的浓度与胆固醇类似，这表明探针在这些条件下行为得当。为了确定用胆固醇接受器孵育加载的细胞所需的时间，细胞培养在不同时间点采集。胆固醇的流出从零到六小时呈线性变化，最大信号在ABD被添加到细胞中6小时后被捕获。通过测量含高密度脂蛋白不同百分比的胆固醇流出，测试饱和阈值和动态范围。在高通量资产中，流出量从 1% 线性演变为 7%ABDS，达到 7% 的胆固醇出血能力峰值，浓度高于 7%，荧光的强度与 ABDS 百分比成反比。然后，通过比较荧光方法与标准无线电标记技术，对基于荧光的方法的性能进行了评价。当使用不同的 ABDS 浓度时，这两种技术都高度相关。所述方法对胆固醇出血值内接受物浓度的增加敏感，在 5 到 15% 之间，确保纤维素不含聚合物非常重要。临界点是对含有环境的聚合乙醇进行分级，以均匀和最佳重量测量荧光。按照这个程序，可以测量名称技术胆固醇，并确定药物在胆固醇流走途径的影响。此外，学校最终被用作诊断评估心血管风险。我们认为，这种方法比放射性标准方法更容易和安全得多。然而，它仍然是依赖于细胞。请记住，乙醇是易燃的，应相应地存储和处理。

Research

JoVE Journal

Medicine

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

一种高通量自动化的平台，制造蛋白质治疗的细胞系发展

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

科研

医学

Mouse Eye Enucleation for Remote High-throughput Phenotyping

老鼠的眼睛剜除术的远程高通量的表型

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Cholesterol Efflux Assay

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 ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

高通量顺序酶联免疫吸附试验的急性移植物抗宿主病的生物标志物的验证

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

一个器官高通量系统原发性多发性骨髓瘤细胞的药物敏感性的表征

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

高通量，复用和有针对性的蛋白质组学分析脑脊液量化神经退行性生物标志物和载脂蛋白E亚型状态

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients