Name: sensitive detection of proteopathic seeding activity with fret flow cytometry
Uploaded: 2015-12-08T13:00:00.000+00:00
Duration: 12 min 31 s
Description: Watch this Scientific Journal Video about Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry at JoVE.com

This work was supported by the Tau Consortium (M.I.D); National Institutes of Health Grant 1R01NS071835 (M.I.D.), a Department of Defense Grant PT110816 (to M.I.D.), 1F32NS087805 (to J.L.F.), and 1F31NS079039 (to B.B.H.).

注:该协议强调利用FRET的流式细胞仪从小鼠生物样本检测播种活动。它还与重组原纤和人类生物样品兼容。按照IACUC批准的程序进行鼠标安乐死与脑收获。 1.脑提取<ol><li>以下用异氟烷（2％）深麻醉，灌注的小鼠用含有0.03％肝素冰冷的PBS，并提取在以下量具等描述的细节的大脑 。18 </li><li>放置提取的组织在一个冷冻小瓶中，并通过将在液氮中冷冻单元。可选择地，冷冻在干冰上的组织。一旦冻结，如果有必要转移组织至-80℃，并存储用于在延长的时间周期。 注:此协议是与全脑匀浆或微观解剖的大脑区域兼容。见萩原等人进行了详细的显微解剖协议19。 </li></ol&gt; 2.制备生物种材料<ol><li>通过将蛋白酶抑制剂（不含EDTA）到1X TBS（每10ml 1片）准备同质化的缓冲区。涡大力，和存储在冰上。蛋白酶抑制剂必须是不含EDTA，以保持生物传感器细胞活力。 </li><li>权衡冷冻的脑组织中的一次性称重舟，并转移到一个5毫升锥形管中。添加冰冷的均化缓冲液，使得最终溶液是10％重量/体积。暂时储存在冰。组织块和随后的均化缓冲液量将根据所使用的脑大小和/或区域而有所不同。这里，成年小鼠hemibrain称量0.2克悬浮在2毫升10％重量/体积溶液。 </li><li>转移样品到寒冷的房间，并调整探头超声仪来进行相应的设置。为探针超声处理用的Omni Ruptor（此处示出），设置功率至20％，其对应于约75 W·使用一个脉冲模式，以确保样品不OV在超声ER-加热。脉冲发生器设置为30％，相当于约500毫秒。 </li><li>通过与水，异丙醇，和水再次漂洗，拭去探针在每种溶液之间清洁探头超声仪的尖端。是一定的冲洗和擦拭两侧和探头与实验室擦拭物底部。 </li><li>与一个样本的工作的时间，淹没探针针尖插入匀浆缓冲，并启动超声发生器。使用上述的功率和脉冲发生器的设置，提供25总脉冲。确保组织是完全悬浮。请注意，以避免在该步骤中样品的泡沫。 注:样品易于发泡如果一个）的总体积为低或b）将匀浆升温。有了这个特殊的仪器，不要用超声处理体积小于250微升。为了确保样本留冷藏，管可以在该步骤中保存在冰上。 </li><li>清洁探头由抹着剩余的裂解液，然后交付10个脉冲到烧杯中Øf清洁时的水。冲洗的侧面和探头与水，异丙醇，和水再次的底部（如在步骤2.4），擦拭干在每个步骤之间。为了避免污染，彻底冲洗探头样本之间。 注意:对于tau聚集，未发现更严格的清洗方法是必要的，以防止样品到样品的污染。其他淀粉样蛋白，然而，可能要求已在文献20,21被广泛描述的附加 ​​服务。 </li><li>商店匀浆在冰上直至所有样本都被处理。 </li><li>自旋匀浆在21300×g离心15分钟，在4℃。 </li><li>上清转移到一个干净的试管，注意不要打扰颗粒。弃沉淀。等分上清液，并在-80℃用于进一步使用商店裂解物。然而避免匀浆冷冻/解冻循环，因为这会减少播种效率。 </li></ol> 3. R​​eplating生物传感器细胞注意:使用四种细胞系用于该测定:HEK 293T（株＃1），则RD-P301S-CFP（细胞系＃2），的RD-P301S-YFP（细胞系＃3），和RD，P301S-CFP / YFP（细胞系＃4）。请参考表1来测定每个细胞系的贡献。 <ol><li>在无菌环境中，吸培养基。细胞冲洗用温水PBS，并吸出。 Trypsinize细胞（3 ml的胰蛋白酶-EDTA（0.05％）的）下3分钟，淬火用温培养基（9 ml的DMEM中，10％胎牛血清，1％青霉素/链霉素，1％Glutamax的），并立即细胞转移到一个锥形管中。 </li><li>离心细胞在1000×g离心5分钟，室温。吸出培养基，在温暖的培养基悬浮细胞沉淀。 </li><li>使用血细胞计数器，确定细胞密度为每个细胞系。细胞密度将根据汇合在收获和再悬浮体积时间变化。从10cm皿在10ml培养基收获在90％汇合，细胞密度悬浮细胞为约100万个细胞/毫升。 </li><li>使细胞的一个主混合物+媒体，使得一个96孔板的各孔将包含35,000个细胞的130微升培养基（例如，使一个主混合物为100孔，悬浮3.5百万细胞在13ml培养基）。修改手机号码​​，以适应个人的实验设计和时间表。用于细胞治疗〜铺板后18小时，加入35,000个细胞到96孔板的每个孔中。 </li><li>使用多通道移液管，慢慢吸取130微升主混合物的细胞悬浮液到一个平底的每个孔中，组织培养处理的96孔板中。而电镀，将吸管尖在井的中心，而不是接触板的底部。 注意:虽然镀格式可以被修改，使得n = 4的孔中的每一个细胞系的1％至3板被推荐的。该板（N = 84）的其余部分为细胞系＃4（生物传感器细胞）保留。 </li><li>使细胞通过离开该板静置在室温10分钟以沉淀。孵育O / N在37℃，5％的CO 2，和与＃8805; 80％的相对湿度。 </li></ol> 4.处理细胞注意:以下天，当tau蛋白生物传感器细胞为60-65％汇合，制备种子转导复合物如下: <ol><li>在一个管中，结合转染试剂，如脂质体，具有减少的血清培养基，如的Opti-MEM，以使一个主混合物。每口井，增加1.25微升转染试剂和8.75微升减少血清培养基。轻轻一抖管搭配，简单地降速，孵化5分钟，在室温。 </li><li>在另一个管中，结合种子材料（来自步骤2.9的裂解物等分试样）具有降低血清培养基。 注:晶种材料的量会根据个体实验而变化。当选择种子体积，考虑到:丰种子样本和潜在毒性的。原油匀浆可能是有毒的细胞。因此，使用最低量可以监视你想要的效果。裂解液/孔范围FR以前测试卷嗡1-5微升，对应于:5-20微克总蛋白。确保每孔的总体积（种子+还原血清培养基）是10微升。 </li><li>从4.1和4.2，说明甩尾轻轻混合两管相结合的内容，简要降速（1000 XG，5秒），并在室温下孵育至少20分钟，最多2小时。 </li><li>轻轻吸取个别生物传感器孔的侧20微升转导复合物。使用三个技术重复，如果可能的话。返回处理的细胞以孵化24-48小时。在相同条件下孵育，如步骤3.6中所述。 注:合适的阴性对照条件，这种设置是空脂质体（ 即脂质体试剂+降低血清培养基）处理的生物传感器细胞，因为应用程序磷脂引入了荧光剖面相对生物传感器细胞未曝光的脂质体试剂的轻微变化。 </li></ol> 5.收获细胞FRET流式细胞仪<p类="jove_content"&gt;注意:前收获细胞-通常24-48小时的后处理，也可以使用一个标准的倒置荧光显微镜将GFP过滤器得到播种活性的初步读数。没有籽料（ 即空脂质体）处理的细胞显示弥漫性荧光，而与籽料处理的细胞会表现出强烈的点状和网状细胞内的夹杂物（ 图1A - B）。 <ol><li>使用多通道移液管，吸所有细胞培养基（150微升）。 Trypsinize（50微升）细胞5分钟，并骤冷以150微升冷却的培养基。不包括以胰蛋白酶在这一步，因为它可能会导致细胞的提升和随后的细胞丢失前一个PBS冲洗。通过门控策略，流式细胞仪过程中排除任何污染的死细胞和碎片。 </li><li>立即淬火，转移细胞到96孔圆底板，和离心机在1000×g离心5英里后n将在RT。 </li><li>吸并丢弃中，注意避免干扰细胞沉淀。 </li><li>轻轻地，彻底，重悬在50μl的2％多聚甲醛的细胞沉淀，并孵育10分钟。或者，运行细胞活着，虽然固定提供更清洁，更一致的结果。 </li><li>离心细胞在1000×g离心5分钟，室温。吸并丢弃多聚甲醛，注意避免干扰细胞沉淀。 </li><li>轻轻地，彻底，重悬细胞沉淀在200微升冷却的流式细胞仪缓冲液（HBSS，1％FBS，1mM EDTA）中，并尽快运行板，以避免细胞聚集。 </li></ol> 6. FRET流式细胞仪注意:使用流式细胞仪如MACSQuant VYB，其配备的FRET-兼容激光线和过滤器组（ 表2）。在本节中的每个步骤中，单击感兴趣的以及使用软件96孔模板，单击"玩"开始样品提升和流量。通过点击"新分析窗口"图标，让绘图或统计信息表。通过点击标题的X或Y轴，并选择适当的过滤器更换个别二元地块轴参数。要改变细胞群或荧光信号，增加或减少与适当的过滤器相关联的电压。有了这个仪器，运行&lt;1000次/秒，以确保精确的单细胞的监控。 <ol><li>做一个侧面散射面积（SSC-A）与前向散射面积（FSC-A）二元情节，并与小区1号线的运行，调整SSC和FSC电压，直到细胞群体是在左下象限。单击多边形工具，并定义细胞群（门P1）。对于所有的门控参数见图 2。 </li><li>做一个FSC-H（高度）与FSC-A二元情节，并申请门P1到该小区通过点击"活"以上的情节，并选择P1。随着细胞系＃1运行后，单击多边形工具，并定义单个细胞（门P2）。 </li><li>使3直方图，每个所关心的过滤器:CFP，YFP和FRET。通过点击"活"上述地块，并选择P2应用门P2到所有这些地块。同细胞系＃1的运行，调整电压使得该位数细胞群中在CFP，YFP和FRET直方图都是0和1之间要测量的CFP和FRET，激发细胞用405nm的激光，和捕获荧光带五十分之四百五十nm和50分之525纳米过滤器，分别为。为了测量YFP，激发细胞与488 nm激光和捕捉荧光带50分之525纳米过滤器。激光和过滤器设置在表2中进一步描述。 </li><li>对于CFP外溢到FRET和YFP渠道进行补偿。 注:补偿是荧光溢出校正的过程。每当1荧光染料的所述荧光发射内的过滤器检测DESI发生荧光外溢gned从另一个荧光染料测量信号。有了适当的补偿，CFP溢出的荧光可以从FRET和YFP的通道被删除。 注:补偿既需要荧光阳性和阴性细胞的存在。因此，为了补偿在CFP阳性细胞（细胞系＃2），阴性细胞（细胞系＃1）必须被添加到之前的运行样品。 <ol><li>穗在30微升的细胞系＃1时从单井到200微升的细胞系＃2停牌前夕补偿。 </li><li>点击"仪器设置"图标，然后"补偿"选项卡，并选中"矩阵"，打开补偿表。 </li><li>做一个FRET-A对CFP-A二元情节，并申请门P2，如步骤6.3中所述。随着细胞系1 + 2的运行，点击"象限"图标，并绘制象限，使得荧光阴性和阳性的人口是由下面的两个象限分离（盖茨LL3一个第二LR3，分别）。 </li><li>创建显示FRET为LL3和LR3门中位数荧光强度（MFI）一个统计表。调整的补偿矩阵的FRET参数，直到FRET信号的MFI是LL3和LR3之间的等价物。在此步骤之后，FRET的信号的MFI等于未染色的细胞和CFP阳性细胞之间，这表明不存在CFP溢出到FRET通道。 </li><li>做一个YFP-A对CFP-A二元情节，并申请门P2，如步骤6.3中所述。绘制象限（LL4和LR4）相似，在步骤6.4.3完成。 </li><li>创建显示YFP的MFI为114和LR4一个统计表。调整在补偿矩阵YFP的参数，直到YFP信号的MFI为114和LR4之间的等价物。在该步骤之后，一个信号的YFP的MFI是未染色等于细胞和CFP-阳性细胞之间，这表明由于缺乏CFP溢出进入YFP通道。 </li></ol></li><li> FOLL由于门的设置和补偿，突出感兴趣的水井和运行板的其余部分。 </li><li>毕竟样品已运行，导出数据文件，通过点击"文件"，然后"复制"。选择"数据文件"选项卡，选中实验文件夹，然后单击"复制"。 </li></ol> 7.数据分析注:点击标题对X或Y轴，并选择适当的过滤个别二元地块更改轴参数。 <ol><li>在流式细胞仪分析程序流程打开FCS文件。 </li><li>基于散射特性，使用多边形门从空脂质体处理的细胞定义的细胞群体（SSC VS FSC）和单群（FSC-H VS FSC-A），同样，在第6节。 </li><li>如果在安装过程中进行CFP补偿，不进一步调整CFP。 </li><li>创建FRET VS YFP二元情节。使用细胞系＃3，通过绘制多边形栅极沿FRET的阳性细胞群的斜率运行定义一个虚假的FRET栅极。此门排除YFP单阳性细胞发射FRET的过滤器内的信号，并绘制类似于先前由禁止等人22所示</li><li>通过在FRET VS CFP二元阴谋策划空脂质体处理的CFP / YFP细胞定义FRET门。沿着向上延伸，并向左远离人口的人口的斜率引入一个多边形栅极。此门应排除大多数细胞（〜99％），以使得背景FRET是细胞≥1％。即转入这个门的任何事件都被认为FRET阳性。 注意:上述每个单独的栅极被施加到所有样品构建后续入口处。下面的分析中，四个独立的门被定义（细胞群;单;假FRET; FRET）。 </li><li>感兴趣的记录分析参数，其中包括:％的FRET积极性和MFI的FRET阳性细胞。通过测量％的积极性和小额信贷机构的产品手动计算综合FRET密度。 注意:如果集成FRET密度用作衡量的结果，设定背景FRET和MFI，以避免计算产品是小于其两个个别因素（如在步骤7.5中定义）为大于或等于1。 </li></ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeding+of+normal+tau+by+pathological+tau+conformers+drives+pathogenesis+of+Alzheimer-like+tangles.">Seeding of normal tau by pathological tau conformers drives pathogenesis of Alzheimer-like tangles.</a> Journal of Biological Chemistry. , (2011).</li><li>Holmes, B. B., 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=Heparan+sulfate+proteoglycans+mediate+internalization+and+propagation+of+specific+proteopathic+seeds.">Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds.</a> Proc Natl Acad Sci U S A. 110 (33), E3138-E3147 (2013).</li><li>de Calignon, 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=Propagation+of+Tau+Pathology+in+a+Model+of+Early+Alzheimer's+Disease.">Propagation of Tau Pathology in a Model of Early Alzheimer's Disease.</a> Neuron. 73 (4), 685-697 (2012).</li><li>Liu, 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=Trans-Synaptic+Spread+of+Tau+Pathology+In+Vivo.">Trans-Synaptic Spread of Tau Pathology In Vivo.</a> PLoS One. 7 (2), e31302 (2012).</li><li>Holmes, B. B., 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=Proteopathic+tau+seeding+predicts+tauopathy+in+vivo.">Proteopathic tau seeding predicts tauopathy in vivo.</a> Proc Natl Acad Sci U S A. 111 (41), E4376-E4385 .</li><li>Yoshiyama, 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=Synapse+loss+and+microglial+activation+precede+tangles+in+a+P301S+tauopathy+mouse+model.">Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model.</a> Neuron. 53 (3), 337-351 (2007).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. 65 (65), (2012).</li><li>Hagihara, H., Toyama, K., Yamasaki, N., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+hippocampal+dentate+gyrus+from+adult+mouse.">Dissection of hippocampal dentate gyrus from adult mouse.</a> J Vis Exp. (33), (2009).</li><li>Yan, Z. X., Stitz, L., Heeg, P., Pfaff, E., Roth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectivity+of+prion+protein+bound+to+stainless+steel+wires:+a+model+for+testing+decontamination+procedures+for+transmissible+spongiform+encephalopathies.">Infectivity of prion protein bound to stainless steel wires: a model for testing decontamination procedures for transmissible spongiform encephalopathies.</a> Infect Control Hosp Epidemiol. 25 (4), 280-283 (2004).</li><li>McDonnell, G., 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=Cleaning,+disinfection+and+sterilization+of+surface+prion+contamination.">Cleaning, disinfection and sterilization of surface prion contamination.</a> J Hosp Infect. 85 (4), 268-273 (2013).</li><li>Banning, C., 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+flow+cytometry-based+FRET+assay+to+identify+and+analyse+protein-protein+interactions+in+living+cells.">A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells.</a> PLoS One. 5 (2), e9344 (2010).</li><li>Morozova, O. A., March, Z. M., Robinson, A. S., Colby, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Features+of+Tau+Fibrils+from+Alzheimer's+Disease+Brain+Are+Faithfully+Propagated+by+Unmodified+Recombinant+Protein.">Conformational Features of Tau Fibrils from Alzheimer's Disease Brain Are Faithfully Propagated by Unmodified Recombinant Protein.</a> Biochemistry. , (2013).</li><li>Sui, D., Liu, M., Kuo, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+aggregation+assays+using+hyperphosphorylated+tau+protein.">In vitro aggregation assays using hyperphosphorylated tau protein.</a> J Vis Exp. (95), e51537 (2015).</li></ol>

This assay has been licensed to Janssen Pharmaceuticals.

此处所描述的FRET流式细胞仪系统是一个功能强大的工具，快速和定量地评估tau蛋白播种活性。它只需要适度的细胞培养的经验和FRET的应用知识和流式细胞仪。其他播种测定法，如硫磺素T - 这表现出增强的荧光当结合β片层结构 - 是费力的，需要一个纯的，重组蛋白质底物。此外， 在体外播种测定法的tau仅半定量的并且通常不敏感种子材料23,24的亚纳摩尔水平。 FRET流式细胞仪，但...

细胞内的tau淀粉样蛋白的积累定义τ病变如阿尔茨海默氏病。在早期疾病阶段，病理通常定位于大脑的离散区域，但随着疾病进展，病理学总是沿不同的神经网络1-5传播。越来越多的证据表明，有毒蛋白聚集体跨细胞繁殖underlies此病理（6-10综述）。在这个模型中，proteopathic种子（例如，tau蛋白）释放从供体细胞，并进入邻近细胞，转化天然tau蛋白成经由模板化的构象变化11-15错误折叠形式。此处所描述的测定法的开发灵敏检测这种播种活性。它是用重组蛋白和生物样品相容并允许proteopathic播种活动16分钟程度的定量。 HEK 293T细胞稳定表达tau蛋白重复ðomain（RD）含有与疾病相关的突变P301S融合到CFP或YFP（以下简称为tau蛋白-RD-CFP / YFP的细胞）作为播种活性的稳定的生物传感器。在没有proteopathic种子，细胞保持tau蛋白作为水溶性单体，和具有没有明显的背景的FRET。自然吸收或牛头种子脂质体介导转导到细胞内，但是，结果在RD-CFP和RD-YFP聚集，产生的是单个细胞内通过流式细胞仪测量的FRET信号。 该测定的众多成分设计，以提高灵敏度和降低可变性。带1的单克隆细胞系:1的RD-CFP / YFP表达比率被选择的，因为它提供了最佳的信号:噪声。为了增加灵敏度，磷脂用来引入种子直接导入细胞（尽管研究摄取生物学机制，这可以省略）。最后，流式细胞仪监测FRET在群体水平和单个细胞<html>平，与其他蛋白质聚集测定。最终的测量结果，集成的FRET密度，是高度的定量和两对细胞凝集的数量，和聚集已经发生向其中每个单元内的程度帐户。所有这些优化的参数，提高了灵敏度，并确保重现性。 该系统最近采用了全面的研究转基因P301S tau蛋白病小鼠17进行评估的时间开始和相关头播种活动进展到其他常用的tau病理学标志（ 例如 ，MC1，AT8，PG5，并ThioflavinS）。播种活动是迄今为止头病理的最早和最强大的标志物进行评估，组织学检测由至少6周之前。播种活性似乎在1.5个月，并逐渐随着年龄增大，这表明proteopathic种子在其发作和/或神经变性16的级数的因果作用。 e_content"&gt;从生物样品中的种子材料分钟水平的精确定量可以促进监测早期疾病进展的研究。通过缩短测试时间并且使得能够使用较年轻的动物，这可能增加的临床前动物试验的效率和准确性。例如，在在P301S鼠标如前所述，铅化物可作为交付早在4-6周，2-4周后进行疗效监测。该法应准确量化播种的任何削减（，或在发病播种活动的前夕）活性。FRET流式细胞仪已经在体外筛选的应用，以及，例如，抗tau蛋白的试剂（例如，抗体，小分子等），可以迅速地对它们阻断直接在培养接种诱导，使用任一重组tau蛋白的能力进行测试聚集体或脑源性裂解物作为种子源（图5）。采用这种设置，一旦晶种材料制备，一个前periment只需要三天完成，包括数据分析。 proteopathic播种活动的快速定量因此可以促进神经退行性疾病的许多研究。

<table><tbody><tr><td>TBS</td><td>Sigma</td><td>T5912</td><td></td></tr><tr><td>cOmplete Protease Inhibitors (EDTA-free)</td><td>Roche</td><td>4693159001</td><td></td></tr><tr><td>Cryo-vials</td><td>Sarstedt</td><td>72.694.006</td><td></td></tr><tr><td>Analytical Balance</td><td>Mettler Toledo</td><td>XSE 105DU</td><td></td></tr><tr><td>Weighing Boats</td><td>Fisher Scientific</td><td>13-735-743</td><td></td></tr><tr><td>15 ml conical tube</td><td>USA Scientific</td><td>1475-0501</td><td></td></tr><tr><td>Omni Sonic Ruptor Ultrasonic Homogenizer</td><td>Omni International</td><td>18-000-115</td><td></td></tr><tr><td>Micro-Tip for Ultrasonic Homogenizer</td><td>Omni International</td><td>OR-T-156</td><td></td></tr><tr><td>2-Propanol</td><td>Fisher Scientific</td><td>A451</td><td></td></tr><tr><td>Noise Cancelling Ear Muffs</td><td>Fisher Scientific</td><td>19-145-412</td><td></td></tr><tr><td>Kimwipes</td><td>Fisher Scientific</td><td>S47299</td><td></td></tr><tr><td>1.5 ml tubes</td><td>USA Scientific</td><td>&lt;a href=&quot;http://www.usascientific.com/Seal-Rite-1.5-ml-tube-sterile.aspx&quot;&gt;1615-5510&lt;/a&gt;</td><td></td></tr><tr><td>Microcentrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5424 000.215</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-136</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11965-084</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>HyClone</td><td>SH30071.03</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>GlutaMax</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>50 ml Conical Tubes</td><td>Phenix Research</td><td>SS-PH15</td><td></td></tr><tr><td>25 ml reagent resevoirs</td><td>VWR</td><td>41428-954</td><td></td></tr><tr><td>Multi channel pipet</td><td>Fisher Scientific</td><td>TI13-690-049</td><td></td></tr><tr><td>96 well flat bottom plates</td><td>Corning</td><td>3603</td><td></td></tr><tr><td>Opti-MEM</td><td>Life Technologies</td><td>31985-070</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668019</td><td></td></tr><tr><td>96 well round bottom plates</td><td>Corning</td><td>3788</td><td></td></tr><tr><td>16% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>RT 15710</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>P5493</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>ED2SS</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies</td><td>14185-052</td><td></td></tr><tr><td>Sorvall ST 40 Centrifuge</td><td>Thermo Scientific</td><td>75004509</td><td></td></tr><tr><td>BIOLiner Swinging Bucket Rotor</td><td>Thermo Scientific</td><td>75003796</td><td></td></tr><tr><td>Hemacytometer</td><td>VWR</td><td>15170-172</td><td></td></tr><tr><td>MACSQuant VYB Flow Cytomter</td><td>Miltenyi Biotec</td><td>130-096-116</td><td></td></tr><tr><td>Chill 96 Rack</td><td>Miltenyi Biotec</td><td>130-094-459</td><td></td></tr><tr><td>Flow Jo analysis software</td><td>Flow Jo</td><td></td><td></td></tr><tr><td>20 &amp;mu;l pipet tips</td><td>Rainin</td><td>GPS-L10</td><td></td></tr><tr><td>200 &amp;mu;l pipet tips</td><td>Rainin</td><td>GPS-250</td><td></td></tr><tr><td>1 ml pipet tips</td><td>Rainin</td><td>GPS-1000</td><td></td></tr><tr><td>200 &amp;mu;l pipet tips</td><td>USA Scientific</td><td>1111-1800</td><td></td></tr><tr><td>5 ml serological pipett</td><td>Phenix Research</td><td>SPG-606180</td><td></td></tr><tr><td>10 ml serological pipett</td><td>Phenix Research</td><td>SPG-607180</td><td></td></tr><tr><td>25 ml Serological pipett</td><td>Phenix Research</td><td>SPG-760180</td><td></td></tr></tbody></table>

sensitive detection of proteopathic seeding activity with fret flow cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

FRET流式细胞仪能敏感，定量，并从重组或生物样本快速检测播种活动。实验设置是容易的:表达的tau-RD-CFP / YFP单克隆来源的稳定细胞系转导的种子材料，孵育24-48小时，并进行流式细胞术分析（图1A）。在没有种子，生物传感器细胞保持tau蛋白以可溶，单体形式（图1B）。在种子的情况下，然而，生物传感器细胞将tau蛋白为凝聚状态（图1C）中，产生一个是针?...

Watch this Scientific Journal Video about Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry at JoVE.com

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

sensitive-detection-proteopathic-seeding-activity-with-fret-flow

Research

JoVE Journal

Bioengineering

14.9K 次观看.  University of Texas Southwestern Medical Center. Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

视频: Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

科研

发育生物学

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

免疫与感染

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

一种基于流式细胞术的细胞表面蛋白结合测定，用于评估抗癌适配体的选择性和特异性

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

癌症研究

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM：免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

生物学

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

细胞 Bead-based 免疫分析平台对受激小鼠脾多重细胞因子的研究

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal ...

人中性粒细胞中 β2 整合素激活抑制剂的筛选

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. &#946;2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling &#946;2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of &#946;2 integrins, is utilized to quantify &#946;2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil &#946;2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on &#946;2 integrin inhibition are assessed within 3 h. Molecules that directly target &#946;2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

作者聚焦:鉴定 β2 整合素激活小分子拮抗剂的方法的开发 

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit &#946;2 integrin activation on human neutrophils.

一种基于流式细胞术的高通量技术筛选整合素抑制药物

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

一种基于流式细胞仪的检测方法, 用于识别破坏荧光标记的 CXC 趋化因子配体12到 CXC 趋性因子受体4的化合物

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

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IP - FCM：免疫沉淀，流式细胞仪检测

细胞 Bead-based 免疫分析平台对受激小鼠脾多重细胞因子的研究

一种基于流式细胞术的高通量技术筛选整合素抑制药物

一种基于流式细胞术的高通量技术筛选整合素抑制药物

一种基于流式细胞术的高通量技术筛选整合素抑制药物

一种基于流式细胞仪的检测方法, 用于识别破坏荧光标记的 CXC 趋化因子配体12到 CXC 趋性因子受体4的化合物

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定