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>

<ol>
	<li>Seeley, W. W., Crawford, R. K., Zhou, J., Miller, B. L., Greicius, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+diseases+target+large-scale+human+brain+networks.">Neurodegenerative diseases target large-scale human brain networks.</a> Neuron. 62 (1), 42-52 (2009).</li><li>Zhou, J., Gennatas, E. D., Efstathios, D., Kramer, J. H., Miller, B. L., Seeley, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+Regional+Neurodegeneration+from+the+Healthy+Brain+Functional+Connectome.">Predicting Regional Neurodegeneration from the Healthy Brain Functional Connectome.</a> Neuron. 73 (6), 1216-1227 (2012).</li><li>Raj, A., Kuceyeski, A., Weiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Network+Diffusion+Model+of+Disease+Progression+in+Dementia.">A Network Diffusion Model of Disease Progression in Dementia.</a> Neuron. 73 (6), 1204-1215 (2012).</li><li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+stageing+of+Alzheimer-related+changes.">Neuropathological stageing of Alzheimer-related changes.</a> Acta Neuropathol. 82 (4), 239-259 (1991).</li><li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+of+Alzheimer's+disease-related+neurofibrillary+changes.">Staging of Alzheimer's disease-related neurofibrillary changes.</a> Neurobiol Aging. 16 (3), 271-278 (1995).</li><li>Frost, B., Diamond, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-like+mechanisms+in+neurodegenerative+diseases.">Prion-like mechanisms in neurodegenerative diseases.</a> Nat Rev Neurosci. 11 (3), 155-159 (2009).</li><li>Holmes, B. B., Diamond, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+protein+aggregate+propagation.">Cellular mechanisms of protein aggregate propagation.</a> Current Opinion in Neurology. 25 (6), 721-726 (2012).</li><li>Kaufman, S. K., Diamond, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-like+propagation+of+protein+aggregation+and+related+therapeutic+strategies.">Prion-like propagation of protein aggregation and related therapeutic strategies.</a> Neurotherapeutics. 10 (3), 371-382 (2013).</li><li>Holmes, B. B., Diamond, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-like+properties+of+Tau+protein:+the+importance+of+extracellular+Tau+as+a+therapeutic+target.">Prion-like properties of Tau protein: the importance of extracellular Tau as a therapeutic target.</a> J Biol Chem. 289 (29), 19855-19861 (2014).</li><li>Guo, J. L., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+transmission+of+pathogenic+proteins+in+neurodegenerative+diseases.">Cell-to-cell transmission of pathogenic proteins in neurodegenerative diseases.</a> Nat Med. 20 (2), 130-138 (2014).</li><li>Frost, B., Jacks, R. L., Diamond, M. I. <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+misfolding+from+the+outside+to+the+inside+of+a+cell.">Propagation of tau misfolding from the outside to the inside of a cell.</a> J Biol Chem. 284 (19), 12845-12852 (2009).</li><li>Guo, J. L., Lee, V. M. 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>
		</tr>
		<tr>
			<td>cOmplete Protease Inhibitors (EDTA-free)</td>
			<td>Roche</td>
			<td>4693159001</td>
		</tr>
		<tr>
			<td>Cryo-vials</td>
			<td>Sarstedt</td>
			<td>72.694.006</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo</td>
			<td>XSE 105DU</td>
		</tr>
		<tr>
			<td>Weighing Boats</td>
			<td>Fisher Scientific</td>
			<td>13-735-743</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>USA Scientific</td>
			<td>1475-0501</td>
		</tr>
		<tr>
			<td>Omni Sonic Ruptor Ultrasonic Homogenizer</td>
			<td>Omni International</td>
			<td>18-000-115</td>
		</tr>
		<tr>
			<td>Micro-Tip for Ultrasonic Homogenizer</td>
			<td>Omni International</td>
			<td>OR-T-156</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A451</td>
		</tr>
		<tr>
			<td>Noise Cancelling Ear Muffs</td>
			<td>Fisher Scientific</td>
			<td>19-145-412</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>S47299</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>USA Scientific</td>
			<td><a href="http://www.usascientific.com/Seal-Rite-1.5-ml-tube-sterile.aspx">1615-5510</a></td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424 000.215</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11965-084</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
		</tr>
		<tr>
			<td>50 mL Conical Tubes</td>
			<td>Phenix Research</td>
			<td>SS-PH15</td>
		</tr>
		<tr>
			<td>25 mL reagent resevoirs</td>
			<td>VWR</td>
			<td>41428-954</td>
		</tr>
		<tr>
			<td>Multi channel pipet</td>
			<td>Fisher Scientific</td>
			<td>TI13-690-049</td>
		</tr>
		<tr>
			<td>96 well flat bottom plates</td>
			<td>Corning</td>
			<td>3603</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668019</td>
		</tr>
		<tr>
			<td>96 well round bottom plates</td>
			<td>Corning</td>
			<td>3788</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 15710</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>ED2SS</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14185-052</td>
		</tr>
		<tr>
			<td>Sorvall ST 40 Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004509</td>
		</tr>
		<tr>
			<td>BIOLiner Swinging Bucket Rotor</td>
			<td>Thermo Scientific</td>
			<td>75003796</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>VWR</td>
			<td>15170-172</td>
		</tr>
		<tr>
			<td>MACSQuant VYB Flow Cytomter</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-116</td>
		</tr>
		<tr>
			<td>Chill 96 Rack</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-459</td>
		</tr>
		<tr>
			<td>Flow Jo analysis software</td>
			<td>Flow Jo</td>
			<td></td>
		</tr>
		<tr>
			<td>20 uL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-L10</td>
		</tr>
		<tr>
			<td>200 uL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-250</td>
		</tr>
		<tr>
			<td>1 mL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-1000</td>
		</tr>
		<tr>
			<td>200 uL pipet tips</td>
			<td>USA Scientific</td>
			<td>1111-1800</td>
		</tr>
		<tr>
			<td>5 mL serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-606180</td>
		</tr>
		<tr>
			<td>10 mL serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-607180</td>
		</tr>
		<tr>
			<td>25 mL Serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-760180</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?...

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

14.8K 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

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

科研

生物工程

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

一个手机上的宽视场荧光显微镜和荧光成像流式细胞仪

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

飞行昆虫检测与分类用便宜的传感器

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

蛋白质的MS检测快速酶法处理与流通型微反应器

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

使用光声流细胞测定卵巢癌检测

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.