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/毫升链霉素混合物) 为最后浓度 1 x 106细胞/毫升。</li>
	<li>整除 ~ 2 x 106细胞每井6个井板。</li>
	<li>为了诱导细胞凋亡, 用紫外线照射器去除板盖和照射细胞 150 mJ/厘米2 。这大约需要30-60 秒。 注: 在辐照前, 确保 0.5 x 106细胞被保留用于 "未经处理的" 细胞控制。 注: 细胞凋亡也可通过其他方法 (如抗 Fas 或血清饥饿6) 诱发。</li>
	<li>孵育在 37°c, 5% CO2 2-8 小时, 取决于细胞线。</li>
	<li>使用台式上光显微镜, 可视化细胞来确认凋亡形态的存在, 如起泡、apoptopodia 形成和 ApoBD 形成。 注: 40X 放大倍数足够</li>
	<li>使用 P1000 吸管, 吸管和收集凋亡样本。</li>
	<li>用 1x PBS 冲洗盘子, 并与剩余样品相结合, 以确保最大产量。</li>
	<li>收集 ~ 1/10的 ' 整个凋亡样本 ' (是) 控制。</li>
	<li>收集 "未经处理的" 样本。</li>
	<li>离心机和未经处理的样品在 3000 x g 6 分钟。</li>
	<li>并用重悬在1毫升的 1x PBS, 并预留在冰上。</li>
	<li>对于 ApoBD 隔离, 请继续使用其余的凋亡样本执行步骤2或3。</li>
</ol>2. 通过外地资产管制 ApoBD 隔离
<ol><li>将整个样品离心 3000 x 克, 6 分钟。</li>
	<li>去除大部分上清液而不破坏颗粒。</li>
	<li>并用重悬在染色溶液中含有1毫升 1x A5 结合缓冲器, 75 µL A5-FITC, 2 µL 每 TO-PRO-37细胞。</li>
	<li>在黑暗中孵化样品室温10分钟。</li>
	<li>添加1-2 毫升的 1x A5 结合缓冲和离心机样品在 3000 x g 6 分钟, 以消除多余的污渍。 注: 对于混合细胞种群或组织样本, 执行抗体染色步骤, 使用细胞类型特定标记的组合在 1x A5 结合缓冲和孵化在冰上20分钟 (或根据制造商的协议), 然后离心 3000 x g 6分钟.</li>
	<li>并用重悬样品颗粒在3毫升的外地资产管制署缓冲器 (1x PBS, 1x A5 捆绑缓冲器, 10% FSC, 2 毫米 EDTA) 每 1x107细胞。</li>
	<li>过滤通过70µm 细胞过滤器到一个圆底, 聚丙烯 (流式细胞术) 管, 并保持在冰上和在黑暗中的样品。</li>
	<li>打开µm 机并使用100喷嘴执行标准设置, 执行拖放延迟, 并确保稳定的流。</li>
	<li>加载示例并将捕获速度设置为 ~ 1000 事件/秒。</li>
	<li>调整 FSC, SSC, APC (TO-PRO-3) 和 FITC (A5) 电压和位置事件在外地活动地块内, 以确保人口可以清楚地分开。</li>
	<li>记录2万事件。</li>
	<li>在4部分中设置一个门控策略。</li>
	<li>在排序布局中, 将最终的 ApoBD 门添加为所需的排序填充。</li>
	<li>开始获取样本, 并执行测试排序, 收集 500万 ApoBDs 到一个新的管, 其中包含250µL 的运行情况的缓冲区。</li>
	<li>执行系统回冲洗并加载排序的 ApoBDs。</li>
	<li>获取并记录测试排序 ApoBDs。</li>
	<li>通过比较 FSC (y 轴) vs A5 (x 轴事件), 检查 ApoBD 纯度是否为99%。 注: 由于激光漂白而重新分析样品, A5 染色可能略有减少。</li>
	<li>一旦达到高纯度, 加载原始样本并继续排序, 直到获得所需的 ApoBDs 数。 注意: 如果需要, 可以执行双排序以同时隔离 ApoCells 和 ApoBDs。 注: 在长时间排序时, 建议在4摄氏度处孵化收集管。</li>
	<li>一旦排序完成, 收集后排序 ApoBDs 的一小部分, 后排序 ApoCells, 未经处理, 并验证凋亡和确认后排序纯度。 注意: 虽然测试排序和后排序纯度不应该有显著差异, 但这是基于流的设置和稳定性。</li>
</ol>3. 通过差分离心 ApoBD 隔离
<ol><li>将余下的凋亡样本在 300 x g 处离心10分钟。</li>
	<li>收集上清, 留下500µL, 以避免扰乱细胞颗粒, 并添加到一个新的15毫升圆锥管。</li>
	<li>去除剩余的500µL 和并用重悬细胞颗粒在2毫升 1x PBS (这代表了 ' ApoCell 丰富的分数 '。</li>
	<li>将所收集的上清液离心20分钟, 3000克。</li>
	<li>检查颗粒并仔细清除上清 (上清可能含有小的胞外囊泡, 包括微泡和外来体)。</li>
	<li>并用重悬1毫升 1x PBS 中的颗粒 (这代表了 "ApoBD 富集" 分数)</li>
	<li>收集100µL 的每一个可行的, 是, ApoCell 丰富, 并 ApoBD 丰富的样品在一个新的圆底, 聚苯乙烯 (流式细胞仪) 管。</li>
	<li>添加100µL, 其中包含 2x A5 绑定缓冲区、1:100 A5-FITC 和1:1、000 TO-PRO-3</li>
	<li>在室温下在黑暗中孵育10分钟。</li>
	<li>用流式细胞仪分析样品, 采用上述的浇口策略, 验证 ApoBD 的凋亡和纯度的成功诱导。</li>
</ol>4. 流式细胞术门策略
<ol><li>绘制 TO-PRO-3 (y 轴) 反对 FSC (x 轴), 以分离坏死细胞 (TO-PRO-3高) 从所有其他非透事件 (TO-PRO-3低/中级)。</li>
	<li>选择所有非透事件, 并对 A5 绘制 SSC。门的两个人口, 包括人口 1 (P1), SSC中间/高, A5低/中间细胞和人口 2 (P2), 所有其他事件。</li>
	<li>从 P2, 剧情 TO-PRO-3 反对 A5 和选择 A5 中/高事件排除所有细胞碎片。</li>
	<li>选择所有 A5 积极的事件和阴谋 FSC 对 A5。独立的 ApoBDs (fsc低) 从 ApoCells (fsc中间/高)。 注意: 当 ApoBD 通过基于 ApoBDs 的方法进行隔离时, 我们建议最后一步, 选择 ApoBDs 并比较 TO-PRO-3 A5 和选择所有事件。这可以确保最终的分拣门使用荧光而不是 FSC/SSC 参数。</li>
	<li>对于可行的细胞分析, 选择 P1 并执行两个门控策略之一。对于一般可行的细胞分析, 情节 fsc 对 A5 和选择所有 FSC中间/高细胞, 因此删除剩余的细胞碎片。另外, 为了深入分析, 可行的细胞可以与 A5-早期 ApoCells 分离, TO-PRO-3 对 FSC 进行密谋。选择 TO-PRO-3低, fsc中间/高活性细胞和 TO-PRO-3中间体, FSC中间/高A5-早期 ApoCells。</li>
</ol>

<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>

作者没有什么可透露的。

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。
我们的新的流式细胞仪分析方法允许 ApoBDs, 这往往被忽视使用传统的分析方法, 被检测和量化20。此外, 这种改进的流式细胞术方法, 利用染色 TO-PRO-3, 揭示了额外的 A5 早期凋亡阶段, 从而更好地划定凋亡进展20。传统上, ApoBD 检测在很大程度上依赖于基于图像的技术, 如共焦显微镜和组织学, 而我们的流式细胞术方法为定量 ApoBD 的形成提供了高通量的方法。虽然看似复杂, 该程序是相对容易的, 只需要商业上可用的试剂和基本流式细胞仪。ApoBDs 的逻辑检测和精确量化将进一步了解凋亡细胞微环境, 从而决定细胞清除和免疫应答。事实上, 通过将流式细胞术与细胞特异染色相结合的方法, 我们最近报道了 ApoBDs24中胞质含量的异构分布。这些发现表明, ApoBDs 可以归类为不同的子集, 每个 ApoBD 子集可能表现出不同的功能。
我们最近开发的 ApoBD 隔离技术也将有助于在细胞外囊泡领域的进展。传统上, 用于 ApoBD 隔离的差分离心方法可能包括大量较小的细胞, 这可能会影响下游功能化验。然而, 我们改进的差分离心法可用于分离 ApoBDs 到97% 纯度。虽然差速离心可实现高纯度, 但这种方法可能不适合从复杂样品中分离 ApoBDs。与此相反, 我们的基于 ApoBDs 的方法可以丰富99% 纯度, 并基于 ApoBDs 的独特的生物学特性, 包括颗粒大小, 粒度和 PtdSer 暴露, 而不是完全依赖于粒子密度。这种方法也有可能同时识别和隔离 ApoBDs 的不同细胞起源使用细胞类型特定的标记24。尽管有一项长期的资产管制程序, 可能需要1-8 小时, 这取决于所需 ApoBDs 的数量, 准确的 ApoBD 隔离和随后的下游分析将允许直接归因于 ApoBDs 的分子特征和功能作用。对于这种方法, 关键的是 ApoBD 隔离方法与技术, 如流式细胞术 (如这里概述), 以验证诱导的凋亡和纯度的 ApoBD 的样品。此外, 通过基于 ApoBD 的方法, 建立适当的外地资产管制是必不可少的, 因为不稳定的流或不正确执行的下降延迟可能导致低纯度。
总的来说, 我们概述了用于量化凋亡诱导、ApoBD 检测和高度纯 ApoBDs 分离的尖端方法。我们的方法可以为研究凋亡细胞的拆卸过程提供一个新的工具, 并阐明这一过程在疾病设置中的作用。

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, 确定单核细胞凋亡和 ApoBD 形成的诱导作用 (图 2)。同时, 流式细胞仪分析表明, 紫外线治疗结果在 20% ApoCells (图 3)。
THP-1 单核细胞 ApoBDs 是通过两种方法分离出来的。首先, 为一种高纯度的基于流式燃料的方法制备了样品, 在染色和微流化之前, 只需要单离心步骤就能对整个凋亡样品进行颗粒状。这种方法适用于功能性化验, 当需要显着较高的样品纯度 (例如, qPCR 分析), 当需要特定数量的 ApoBDs, 或从复杂的样本获取特定于细胞类型的 ApoBDs。使用这种方法, ApoBDs 被隔离为 ~ 99% 纯度 (图 4)。
其次, THP-1 单核细胞 ApoBDs 也通过两步差分离心法分离。第一步包括分离存活细胞、ApoCells 和坏死细胞。第二步包括从小的胞外囊泡 (如微泡和外来体) 中分离出较大的 ApoBDs, 这些细胞无法在 3000 x g 上进行颗粒化. 然后进行流式细胞术以确认凋亡诱导和 ApoBD 样品纯度, 并展示了一个 ApoBD 丰富的样本, 包含 ~ 97% ApoBDs (图 4)。这是一种快速有效的技术, 将 ApoBD 分离为相对较高的纯度, 并且在从含有单个细胞类型的样品中纯化 ApoBDs 时是适当的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58317/58317fig1.jpg"> 图 1.ApoBD 隔离的示意图, 通过一个基于外地或差分离心方法.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58317/58317fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58317/58317fig2.jpg"> 图 2.流式细胞术门策略.确定了六个细胞/微粒子集 (包括可行细胞、A5 早期 ApoCells、A5+ ApoCells、坏死细胞、ApoBDs 和碎片), 并用于选择 ApoBD 以进行基于外地资产管制的隔离。(a) 膜 permeabilised 坏死细胞与非 permeabilised 事件分离。(b) A5低中间、SSC低-高细胞与 A5低高度事件分离。(b. i.) 深入分析, TO-POR-3低活性细胞可与 TO-PRO-3中间A5-早期 ApoCells 分离。(二) 另外, 在简单计算 ApoBD 纯度时, 可将可行细胞与 FSC低事件分离。(c) 排除 A5低碎片。(d) fsc中间/高ApoCells 与 fsc低ApoBDs 分离。(e) 选择所有 A5-TO-PRO-3中间/高ApoBDs 进行排序。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58317/58317fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a> 
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58317/58317fig3.jpg"> 图 3.THP-1 单核细胞凋亡的验证.对未经治疗或紫外线照射的 THP-1 单核细胞进行了流式细胞术分析, 以确定活体细胞、A5 早期 ApoCells、A5+ ApoCells 和坏死细胞的水平。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58317/58317fig4.jpg"> 图 4.THP-1 单核细胞衍生 ApoBDs 的纯化.流式细胞术分析是在孤立的 THP-1 单核细胞衍生 ApoBDs 通过一个基于流场法或差异离心的方法, 显示了丰富的 ApoBDs 从可行的细胞, ApoCells 和坏死细胞。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58317/58317fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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. ...

detection-and-isolation-of-apoptotic-bodies-to-high-purity

Nanyang Technological University

Research

JoVE Journal

Biochemistry

10.5K 次观看.  La Trobe University. 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。

视频: Detection and Isolation of Apoptotic Bodies to High Purity

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

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

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

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

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 ...

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

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

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

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

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

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 and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

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

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

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

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

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

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

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

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Presented is a protocol to fabricate a paper-based device for the effective enrichment 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 ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

高分辨率中子光谱研究蛋白质和水合水的皮秒纳秒动力学

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

小细胞外囊泡高通量分离纯化的流式细胞术分选参数优化

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...