Name: detection and isolation of apoptotic bodies to high purity
Uploaded: 2018-08-12T15:00:00
Description: Watch this Scientific Journal Video about Detection and Isolation of Apoptotic Bodies to High Purity at JoVE.com

这项工作得到了国家卫生和医学研究理事会 (GNT1125033 和 GNT1140187) 和澳大利亚研究理事会 (DP170103790) 提供给 i.k.h. P 的赠款的支持。

1. 诱导凋亡
<ol><li>离心细胞样品在 300 x g 5 分钟, 并丢弃上清, 以清除任何预先存在的细胞碎片。 注: 当使用粘附细胞时, 先用1x 磷酸缓冲溶液 (PBS) 在凋亡诱导之前进行种子细胞的冲洗。</li>
	<li>确定单元格编号并收集单元格。 注: 根据化验后的隔离, 我们建议至少 1 x 107细胞的起始细胞数。</li>
	<li>并用重悬在完全媒介 (各自的媒介包含 10% (卷/卷) 胎儿小牛血清, 50 毫升青霉素, 50 µg/毫...

<ol>
	<li>Poon, I. K., Lucas, C. D., Rossi, A. G., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cell+clearance:+basic+biology+and+therapeutic+potential.">Apoptotic cell clearance: basic biology and therapeutic potential.</a> Nature Reviews. Immunology. 14, 166-180 (2014).</li><li>Atkin-Smith, G. K., Poon, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disassembly+of+the+Dying:+Mechanisms+and+Functions.">Disassembly of the Dying: Mechanisms and Functions.</a> Trends in Cell Biology. 27, 151-162 (2017).</li><li>Tixeira, R., 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=Defining+the+morphologic+features+and+products+of+cell+disassembly+during+apoptosis.">Defining the morphologic features and products of cell disassembly during apoptosis.</a> Apoptosis: An International Journal on Programmed Cell Death. 22, 475-477 (2017).</li><li>Sebbagh, M., 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=Caspase-3-mediated+cleavage+of+ROCK+I+induces+MLC+phosphorylation+and+apoptotic+membrane+blebbing.">Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing.</a> Nature Cell Biology. 3, 346-352 (2001).</li><li>Coleman, M. 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=Membrane+blebbing+during+apoptosis+results+from+caspase-mediated+activation+of+ROCK+I.">Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I.</a> Nature Cell Biology. 3, 339-345 (2001).</li><li>Atkin-Smith, G. K., 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=A+novel+mechanism+of+generating+extracellular+vesicles+during+apoptosis+via+a+beads-on-a-string+membrane+structure.">A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure.</a> Nature Communications. 6, 7439 (2015).</li><li>Poon, I. K., 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=Unexpected+link+between+an+antibiotic,+pannexin+channels+and+apoptosis.">Unexpected link between an antibiotic, pannexin channels and apoptosis.</a> Nature. 507, 329-334 (2014).</li><li>Moss, D. K., Betin, V. M., Malesinski, S. D., Lane, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+microtubules+in+apoptotic+chromatin+dynamics+and+cellular+fragmentation.">A novel role for microtubules in apoptotic chromatin dynamics and cellular fragmentation.</a> Journal of Cell Science. 119, 2362-2374 (2006).</li><li>Kerr, J. F., Wyllie, A. H., Currie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+a+basic+biological+phenomenon+with+wide-ranging+implications+in+tissue+kinetics.">Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.</a> British journal of cancer. 26, 239-257 (1972).</li><li>Witasp, E., 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=Bridge+over+troubled+water:+milk+fat+globule+epidermal+growth+factor+8+promotes+human+monocyte-derived+macrophage+clearance+of+non-blebbing+phosphatidylserine-positive+target+cells.">Bridge over troubled water: milk fat globule epidermal growth factor 8 promotes human monocyte-derived macrophage clearance of non-blebbing phosphatidylserine-positive target cells.</a> Cell Death and Differentiation. 14, 1063-1065 (2007).</li><li>Orlando, K. A., Stone, N. L., Pittman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+kinase+regulates+fragmentation+and+phagocytosis+of+apoptotic+cells.">Rho kinase regulates fragmentation and phagocytosis of apoptotic cells.</a> Experimental Cell Research. 312, 5-15 (2006).</li><li>Holmgren, 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=Horizontal+transfer+of+DNA+by+the+uptake+of+apoptotic+bodies.">Horizontal transfer of DNA by the uptake of apoptotic bodies.</a> Blood. 93, 3956-3963 (1999).</li><li>Zernecke, 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=Delivery+of+microRNA-126+by+apoptotic+bodies+induces+CXCL12-dependent+vascular+protection.">Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection.</a> Science Signaling. 2, ra81 (2009).</li><li>Schiller, M., 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=Autoantigens+are+translocated+into+small+apoptotic+bodies+during+early+stages+of+apoptosis.">Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis.</a> Cell Death and Differentiation. 15, 183-191 (2008).</li><li>Elamin, M. 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=Curcumin+inhibits+the+Sonic+Hedgehog+signaling+pathway+and+triggers+apoptosis+in+medulloblastoma+cells.">Curcumin inhibits the Sonic Hedgehog signaling pathway and triggers apoptosis in medulloblastoma cells.</a> Molecular Carcinogenesis. 49, 302-314 (2010).</li><li>Sagulenko, V., 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=AIM2+and+NLRP3+inflammasomes+activate+both+apoptotic+and+pyroptotic+death+pathways+via+ASC.">AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC.</a> Cell Death and Differentiation. 20, 1149-1160 (2013).</li><li>Crescitelli, R., 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=Distinct+RNA+profiles+in+subpopulations+of+extracellular+vesicles:+apoptotic+bodies,+microvesicles+and+exosomes.">Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes.</a> Journal of Extracellular Vesicles. 2, (2013).</li><li>Turiak, 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=Proteomic+characterization+of+thymocyte-derived+microvesicles+and+apoptotic+bodies+in+BALB/c+mice.">Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice.</a> Journal of Proteomics. 74, 2025-2033 (2011).</li><li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labelled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.</a> Journal of Immunological Methods. 184, 39-51 (1995).</li><li>Jiang, 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=Monitoring+the+progression+of+cell+death+and+the+disassembly+of+dying+cells+by+flow+cytometry.">Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.</a> Nature Protocols. 11, 655-663 (2016).</li><li>Berda-Haddad, Y., 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=Sterile+inflammation+of+endothelial+cell-derived+apoptotic+bodies+is+mediated+by+interleukin-1alpha.">Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1alpha.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 20684-20689 (2011).</li><li>Lleo, 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=Shotgun+proteomics:+identification+of+unique+protein+profiles+of+apoptotic+bodies+from+biliary+epithelial+cells.">Shotgun proteomics: identification of unique protein profiles of apoptotic bodies from biliary epithelial cells.</a> Hepatology. 60, 1314-1323 (2014).</li><li>Atkin-Smith, G. K., 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=Isolation+of+cell+type-specific+apoptotic+bodies+by+fluorescence-activated+cell+sorting.">Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting.</a> Scientific Reports. 7, 39846 (2017).</li><li>Jiang, 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=Determining+the+contents+and+cell+origins+of+apoptotic+bodies+by+flow+cytometry.">Determining the contents and cell origins of apoptotic bodies by flow cytometry.</a> Scientific Reports. 7, 14444 (2017).</li></ol>

自二十世纪五十年代早期的描述以来, 细胞凋亡领域已取得显著进展, 成为一个突出的研究领域。尽管有广泛的兴趣和广泛的努力, 但由于缺乏适当的方法, 细胞凋亡的某些方面, 特别是 ApoBDs 的形成, 没有得到很好的研究。这些主要包括通过传统的流式细胞仪 A5/PI 分析和 ApoBD 分离的杂质同时跟踪凋亡进展和 ApoBD 形成的局限性。我们最近制定了解决这些方法缺陷的办法。
我们的...

细胞凋亡是一种精心研究的程序性细胞死亡形式, 需要保持生理平衡, 并清除人体内潜在的有害细胞1。细胞凋亡诱导后, 凋亡细胞 (ApoCells) 可进行一系列形态学改变, 并分解成小的膜状囊泡, 称为 ApoBDs。总的来说, 这个过程被称为凋亡细胞拆卸, 可分为3个不同的步骤, 根据形态学2,3。步骤 1 (等离子膜起泡) 的特点是形成的气球状结构在细胞表面称为泡4,5。步骤 2 (凋亡突起形成) 包括长膜突起的形成, 如 apoptopodia, 串珠状 apoptopodia 和微管尖峰6,7,8。最后, 步骤 3 (ApoBD 形成) 包括凋亡突起和/或 ApoCells 的碎片生成 ApoBDs6,9。先前的研究结果表明, ApoBDs 在帮助细胞凋亡和调节细胞间沟通方面起着重要作用。例如, 建议将 ApoCell 的碎片分解为 ApoBDs 可以生成小的 "咬大小" 的碎片, 可以很容易地被周围的吞噬2,10,11。此外, ApoBDs 可能会窝藏一系列生物分子, 如 DNA、RNA 和蛋白质, 这些生物大分子可能被贩运到周围细胞, 以促进细胞通讯12、13、14。为了在功能上调查这些过程, 必须确认三关键参数, 包括 (i) 验证凋亡诱导和 ApoBD 形成, (ii) 分离 ApoBDs, (iii) 确认 ApoBD 纯度。
以前, 许多方法, 包括流式细胞术和电子显微镜已经被用来研究细胞凋亡和 ApoBDs15,16,17,18。然而, ApoBD 检测和量化往往是困难或忽视。例如, 最常规使用的流式细胞术细胞凋亡试验采用蛋白 V (A5, 一种结合外部 "吃-我" 信号磷脂 (PtdSer)) 和核酸染色碘碘化物 (PI)19的蛋白质。然而, 通过使用这种普遍的染色组合, 分析假设只有三种类型的细胞子集 (可行细胞, ApoCells 和坏死细胞) 在样本中。此外, 虽然被许多研究人员认为是 "金本位" 的细胞凋亡, 流式细胞仪检测和随后的数据分析往往排除 ApoBDs 通过一个初始浇口步骤选择 FSC/SSC中间高事件。因此, 我们最近开发了一种新的流式细胞仪检测, 使用 A5 和 TO-PRO-3, 另一个核酸染色, 可选择性地占去 caspase 3/7 激活 pannexin 1 (PANX1) 通道7,20。caspase 3 诱导的 PANX1 活化在细胞凋亡早期 PtdSer 暴露之前, TO-PRO-3 差异性染色凋亡和坏死细胞。此外, 这种方法结合我们的新的门控策略包括所有获得的事件在数据分析和结果, 六细胞/粒子子集被确定, 包括: (i) 可行的细胞 (FSC/SSC中间/高, A5低, TO-PRO-3低), (二) A5-早期 ApoCells (FSC/SSC中间/高, A5低, TO-PRO-3中间), (iii) A5+ ApoCells (FSC/SSC中间/高, A5高, TO-PRO-3中间体), (iv) 坏死细胞或晚 ApoCells (FSC/SSC 中/高, A5高, TO-PRO-3高), (v) ApoBDs (FSC/SSC低, A5中间, TO-PRO-3低/中间体) 和 (vi) 碎片 (FSC/SSC低, A5低, TO-PRO-3低)20。我们的方法强调分析所有细胞/粒子子集的重要性, 更重要的是从细胞和碎片20分离 ApoBDs。因此, 该方法证明了一种有效的方法来验证诱导的凋亡和 ApoBD 形成同时。
传统上, ApoBDs 是通过各种不同的离心方法分离出来的, 即 ApoBDs 可以从细胞或其他基于密度的胞外囊中分离出来。然而, 这种离心方法往往受到低 ApoBD 纯度, 缺乏一个量化步骤, 以确认样品纯度, 和/或无法分离细胞类型特定的 ApoBDs17,21,22。因此, 我们最近开发了两种方法, 一种基于资产管制和一种新的差分离心方法, 可以与我们以前建立的流式细胞术方法相结合, 以验证诱导的凋亡和样品纯度23。ApoBD 隔离通过我们的基于生物传感器的方法可以丰富 ApoBDs 高达99% 纯度, 并可以结合各种细胞类型特异抗体, 以隔离 ApoBDs 从混合细胞种群, 组织样本和体液23。此外, 我们经修正的差分离心法证明了一种有效的分离 ApoBDs > 90% 纯度23的方法。
本文详细介绍了验证凋亡诱导的实验过程, 并对 ApoBD 的形成进行了检测和量化。本文还对采用基于流式和差分离心法的 ApoBD 隔离工作流进行了详细的阐述和比较。代表的数据表明, 所描述的方法为未来的 ApoBD 研究提供了一个有效的前沿工具。

<table border="0" cellpadding="0" cellspacing="0" style="width:1309px;" width="1309">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Cells e.g. cultured human THP-1 monocytes (clone number: TIB-202)</td>
			<td style="width:149px;">ATCC</td>
			<td style="width:119px;">-</td>
			<td style="width:513px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">RPMI 1640 medium</td>
			<td style="width:149px;">Life Technologies</td>
			<td style="width:119px;">22400-089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Penicillin-streptomycin mixture</td>
			<td style="width:149px;">Life Technologies</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">FSC</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:119px;">10099-141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x PBS</td>
			<td style="width:149px;">-</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Annexin V FITC</td>
			<td style="width:149px;">BD Bioscience</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TO-PRO-3 iodide</td>
			<td style="width:149px;">Life Technologies</td>
			<td style="width:119px;">T3605</td>
			<td>TO-PRO-3 may cause skin, eye and respiratory irration. Avoid direct contact.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x Annexin V binding buffer</td>
			<td style="width:149px;">BD Bioscience</td>
			<td style="width:119px;">556454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:119px;">1001710526</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Centrifuge tube (15 mL)</td>
			<td style="width:149px;">Cellstar</td>
			<td style="width:119px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Microcentrifuge tube (1.5 mL)</td>
			<td style="width:149px;">Sarstedt</td>
			<td style="width:119px;">72.690.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Tissue culture incubator (37 &#176;C, 5% CO2)</td>
			<td style="width:149px;">-</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Centrifuge</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:119px;">392932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS ARIA III Flow cytometer, configured with two lasers for FITC and APC detection</td>
			<td style="width:149px;">BD Bioscience</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Canto II Flow cytometer, configured with two lasers for FITC and APC detection</td>
			<td style="width:149px;">BD Bioscience</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">FACS Diva 6.1.1 software</td>
			<td style="width:149px;">BD Bioscience</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">FlowJo 8.8.6 software</td>
			<td style="width:149px;">-</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;UV Stratalinker 1800</td>
			<td style="width:149px;">Stratagene</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection and isolation of apoptotic bodies to high purity

凋亡体 (ApoBDs)、微泡和外来体是胞外囊泡家族的关键成员, ApoBDs 是最大的类型之一。有人建议, ApoBDs 可以通过贩运生物分子来帮助细胞清除以及细胞间通讯。传统的 ApoBDs 识别和分离方法往往由于缺乏准确的定量和低样品纯度而受到限制。在这里, 我们描述了一个工作流, 以确认诱导的凋亡, 验证 ApoBD 的形成, 并隔离 ApoBDs 到高纯度。我们还将概述和比较荧光活化细胞分选 (ApoBDs) 和差分离心法分离的方法。此外, 使用以前建立的基于流式细胞仪的染色和分析方法, 可以确定分离 ApoBDs 的纯度。结合上述方法, THP-1 单核细胞凋亡和凋亡细胞的拆卸被诱导和验证, THP-1 单核细胞产生的 ApoBD 被隔离成纯度为97-99%。

使用这里概述的过程, THP-1 单核细胞凋亡被诱导和 ApoBDs 检测和隔离通过一个基于外地资产管制或差异离心方法 (图 1)。首先, 通过紫外线照射诱导细胞凋亡, 并在2-3 小时孵化后采集标本, 起泡、凋亡膜突形成和 ApoBDs6的生成。采用 TO-PRO-3 和 A5-based 流式细胞仪方法, 通过分离存活细胞、坏死细胞、早期 ApoCells、ApoCells、ApoBDs 和碎屑, 确?...

Watch this Scientific Journal Video about Detection and Isolation of Apoptotic Bodies to High Purity at JoVE.com

Detection and Isolation of Apoptotic Bodies to High Purity

利用流式细胞仪或差速离心的工作流来检测、定量和分离凋亡样本中的凋亡体, 以高纯度。

高纯度凋亡体的检测与分离

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Research

JoVE Journal

Biochemistry

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

高密度脂蛋白的分离的非编码小RNA定量

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

科研

生物化学

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

一个定制HPLC纯化协议，产量高纯度淀粉样蛋白β42和贝塔淀粉样蛋白肽40，有能力寡聚物形成的

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

高通量荧光素酶法评价单周转蛋白酶 PCSK9 的蛋白质水解

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

在氧化应激期间细胞粘附和纤维蛋白扩散动力学

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

微量和外显子的纸基预集中和分离

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...