作者要感谢金融羚羊的穆里奖（NN00014-06-1-1176）和美国陆军医学研究和装备司令部（批准号W81XWH-09-1-0386和W81XWH-10-1-0806）支持。

1。 RAW 264.7巨细胞培养<ol><li>收获RAW 264.7细胞从他们的保温瓶，当他们到达汇合，他们用细胞刮刀轻轻刮。计数和板块他们到1 5 x 10 5细胞密度/以及在完成0.5毫升Dulbecco的修改鹰中等（cDMEM 24孔细胞培养盘; 10％热灭活胎牛血清（FBS），，2毫米Glutamax 10毫米的肝素钠）在5％CO 2培养箱在37℃孵育过夜。 </li></ol> 2。致病性沙门氏菌沙门伤寒14028转型与文化<ol><li>介绍到 S表达绿色荧光蛋白（GFP）的重组质粒。鼠伤寒由电击。首先，准备在LB肉汤文化和沙门伤寒（ATCC 14028）与曝气，在37℃孵育过夜。 </li><li>第二天，颗粒1毫升细菌微量的最大速度。洗细胞FO你轻轻地重新悬浮颗粒之间每次洗9移液器-冷MOPS /甘油溶液1毫升（（N-吗啉）丙磺酸（MOPS）在20％甘油缓冲1毫米3）倍。最后一次洗涤后，悬浮在50μL拖把/甘油进入哪〜0.5微克质粒DNA pAKgfp1或pAKgfplux1已增加了10的颗粒。 </li><li>细胞悬液转移到预冷藏1毫米的差距电试管和electroporate使用的设置：1800 V，200瓦，25 MF。脉冲传递后，立即加入1 mL LB培养基中，细胞悬液转移到一个新的15 mL管。使细胞在37°C与曝气恢复。一个小时后，集中〜100μL，其余上清离心重悬细胞。 </li><li>最后，在LB板含有100μg/ mL氨苄青霉素，并于37一夜之间板细胞°C。单菌落纯化与抗生素的选择和我的GFP表达restreaking科学也证实了照明用一个标准的异硫氰酸荧光素（FITC标记）滤波器组。 </li><li>接种10毫升，用的沙门氏菌 loopful，已经转化为稳定表达的GFP含有25μg/ mL氨苄青霉素（或适当的抗生素）的LB肉汤。 </li><li>一夜之间生长的细菌在37°C。 </li><li> 沙门氏菌 1:10稀释成含有10毫升LB氨苄青霉素和孵育5小时，在37°C的新鲜文化管</li><li>衡量文化的吸收，在600 nm和使用先前建立的生长曲线，计算出每毫升沙门氏菌的浓度。 </li><li>稀释在cDMEM（0.5毫升/） 沙门氏菌获得的感染复数（MOI），RAW 264.7细胞每100。 </li></ol> 3。制备的纳米粒子悬浮液<ol><li>编造1％FITC加载描述以前11纳米。简单地说，通过酸酐制备颗粒反溶剂nanoencapsulation的，聚合物溶解在二氯甲烷（4°在浓度为25毫克/毫升）和戊烷（-30℃1:200的比例二氯甲烷：戊烷）沉淀。恢复真空过滤纳米。蒸发任何残留的溶剂后，权衡干酸酐，纳米颗粒使用消毒权衡纸。 </li><li> 5毫克的纳米粒子加入0.5毫升的冷磷酸盐缓冲液（PBS，钙和镁的自由，pH值7.4）1.5 mL离心管中，并保持它在冰上直到纳米粒子添加到RAW 264.7细胞。 </li><li>超声纳米粒子悬浮液（保持冰）使用超声波液体处理器，配有微尖约25小号在4至6焦耳。 </li></ol> 4。吞噬含量<ol><li> 5微克/毫升细胞松弛素- D 1小时前引进NP或沙门氏菌预处理RAW 264.7细胞的一个子集，吸中等和REPL抑制剂补充新鲜cDMEM acing。 37°Ç孵化器返回的文化。 </li><li>抑制剂的1小时孵化后，从孵化器中删除板块，涡纳米粒子悬浮液，加入10μl适当的井。 </li><li>涡沙门氏菌和MOI 100的RAW 264.7细胞，通过添加适当的井的细菌感染。 </li><li>点击几次板，混合和孵育37°C或4°C下45分钟（控制）。 </li><li>删除从孵化器或冰箱和冰的地方板块。与冰冷的PBS洗细胞两次（无钙+和Mg 2 +）的吸气式和抛弃旧的介质，以除去未结合的颗粒， 沙门氏菌 ，死亡或独立RAW 264.7细胞。 </li><li>收获后的第二次洗RAW 264.7细胞，加入250μL冷PBS轻轻刮去井。 </li><li>收获细胞吸取到清晰视图单元帽微离心管中，并保持他们在冰上。 </li><li>洗细胞，加入250×Ğ1毫升冷洗涤缓冲液（2％热灭活胎牛血清，0.1％叠氮钠的PBS）离心10分钟，4°C。 </li><li>纸巾上拍打，倒置离心管，弃上清，并删除剩余的缓冲区。轻轻耙通过试管架离心管，重悬细胞沉淀。 </li><li>在PBS中加入100μL的4％多聚甲醛（PFA）和使细胞站立在室温（RT）15分钟修复RAW 264.7细胞。 </li><li>洗RAW 264.7细胞加入250×Ğ1毫升的权限/洗涤缓冲液（BD Biosciences公司）和离心10分钟，4°C。 </li><li>重复步骤4.9。 </li><li>加入100μl含有的Alexa Fluor 660鬼笔环肽（AF 660; 1:150稀释）彼尔姆/清洗缓冲区的染色RAW 264.7细胞的肌动蛋白在室温为15分钟。重复步骤4.11和4.12。 </li><li> RAW 264.7细胞重悬于50μLPBS包含1％PFA和存储他们在黑暗中，在4°C间，直到收购。 </li></ol>提示及注意事项： <ul><li>单一色荧光样品应准备在这一步而设立ImageStream X仪器将用于补偿控制。包括在这个实验中，分别为：RAW 264.7细胞培养与纳米粒子（不标记的肌动蛋白）; 沙门氏菌培养RAW 264.7细胞（肌动蛋白标记）;肌动蛋白只标RAW 264.7细胞的补偿控制。 </li><li>为抑制剂的研究，它是重要的，包括对车辆的控制，作为控制溶解抑制剂。在这个实验中，细胞松弛素D的100％DMSO溶解。因此，我们在cDMEM含DMSO和评估孵育细胞内部的纳米粒子或沙门氏菌 。我们没有发现任何中型组和，二甲基亚砜控制（数据未显示）之间的显着性差异。 </li><li> SA的mple准备指南提供的Amnis网站（ <a href="https://www.amnis.com/images/1__Sample_Prep_Guide_3L_-_0418081.pdf" target="_blank">https://www.amnis.com/images/1__Sample_Prep_Guide_3L_-_0418081.pdf</a> ）是一个检查的ImageStream X荧光的兼容性很好的资源。要访问此文件，用户必须先注册（免费的负责人）与阿美族。 </li></ul> 5。样品采集ImageStream X <ol><li>最多ImageStream X和发射功率激励。 </li><li>初始化射流。的这个脚本Speed​​Beads结束在应该运行。 </li><li>在文件菜单中，选择加载默认的模板。 </li><li>在图片廊视图菜单，选择和按运行安装程序开始成像的珠子。 </li><li>调整的核心跟踪中心的图像横向（如有必要）。 </li><li>选择明场（BF）的通道，并单击“设置强度。 </li><li>等待，直到流动速度的简历一直是乐SS超过0.2％。 </li><li>每天校准仪器。在助攻“选项卡，单击”全部启动运行校准和测试，并确认所有已经过去了。 </li><li>按冲洗锁和负载（FLL）加载的第一个样本。载入中最亮的样本，每个荧光荧光实验。运行此示例，您首先要建立的仪器设置，然后不要更改它们在整个实验，这是关键。 </li><li>打开在实验中使用的每个激光和激光功率设置，所以每个荧光最大像素值在100和4000之间的罪名，在散点图。 </li><li>设置单元格的分类标准，以消除不必要的对象的集合。只有细胞收集数据，高炉到50微米的通道选择区域下的极限。面积小于50微米的对象将被视为碎片并不会被收购。选择要收集的渠道。 </li><li>输入文件名，目标文件夹，设置序列＃1和收购活动。 </li><li>点击运行采集，收集和保存的第一个实验数据文件。 </li><li>点击FLL和运行下的实验样品。重复，直到所有实验样本已收集。 </li><li>点击比赛设置（关闭明和散射的激光，使所有渠道收集），并积极从每个单个的染色样品500每个荧光细胞收集在实验中，以制定一个补偿矩阵。 </li></ol>总结，样品可以运行在以下顺序： <ul><li>明亮的样本。 </li><li>其余测试样品。 </li><li>含有单双色荧光补偿控制。 </li></ul>提示及注意事项： <ul><li>一个的INSPIRE模板可以保存和重新加载设置的仪器设置。一旦模板被保存，自动进样器可用于无人操作运行一个模板和合作的实验样品mpensation控制每个单一色彩控制模板。 </li><li>这些实验：488和658 nm激发激光器的ImageStream X配置; 785纳米散射激光; 40X（0.75 NA）的目标，ISX一个标准的过滤器堆栈6通道系统。使用自动进样器选项允许无人值守采集样品。 </li><li>以下设置被用于实验。纳米粒子被设置到10MW的488纳米激光成像，658 nm激光设置为50毫瓦，785 nm激光设置为2兆瓦和BF在通道1设置为20兆瓦的488纳米激光成像沙门氏菌感染的细胞， 658 nm激光设置为50毫瓦，785 nm激光通道1设置为2兆瓦，和BF。补偿控制在高炉和785的情况下收集。至少5000事件（即细胞），每个测试样品的成像。 </li></ul> 6。图像分析<ol><li>推出的创意和国际化的向导和负载上双击E的测试样品。RIF文件。 </li><li>点击“新矩阵”在第2步创建一个补偿矩阵。赔偿向导启动。在实验中添加单一色彩控制文件。通过点击以下方向的向导下，直到补偿矩阵文件保存和装入箱的国际化向导的步骤2。 </li><li>单击“下一步”，并按照方向，直到产生。DAF文件。 </li><li>所选择的图像采集过程中使用的通道设置图像显示属性。点击CH 2（FITC）和CH（自动对焦，660）5。 （BF和SSC是默认选中）。 </li><li>选择图像通道，使细胞边界（CH01）和纳米粒子或细菌就被收集在通道（CH02）。 </li><li>生成一个散点图区明视与明视所有细胞的长宽比。按一下个别的点和门控周围汗衫定义的单个细胞的人口。单细胞的宽高比第1轮和双峰0.5左右（ 图1A）。 </li><li>明场梯度根直方图的意思是明的形象广场（RMS）的生成和人口在图片库视图设置选择彬（ 图1B）。点击垃圾箱，以确定最佳焦点细胞开始画线区域门集中细胞。较高的RMS梯度，更好地集中。除非有其他你想门上的污渍，跳过下一步。 </li><li>产生一个新的分散强度频道2 x轴与最大像素y轴CH2情节（ 图1C）。点击点和查看图像，以帮助你周围绘制，积极为纳米粒子或细菌细胞的区域。 </li><li>一个国际化的特征直方图产生的一个地区，从0开始，通过观察图像（ 图1D）应调整。国际化的特点是比整个细胞的强度在细胞内的强度。据缩放等，约一半的强度为0的价值就在里面。向导创建了一个区域内指定一个从第5步使用发现细胞表面的细胞图像输入和侵蚀这个由4个像素的面具。请注意，此面膜可以手动为不同类型的细胞在必要时调整。在这个实验中，我们先明图像上创建一个对象的遮罩，手动调整功能，并削弱由这4个像素。国际化的特征，然后计算这4个像素的侵蚀对象面具。此功能使我们能够区分内在的细菌和微粒，其中有大部分遮罩边界内的荧光信号，从表面结合的微粒和细菌，其中有多数外面罩的边界（ 图2）荧光信号。 </li><li>创建一个新的国际化的有限元分析的新的直方图TURE侵蚀的对象遮罩的基础上。通过查看在选定的bin模式的图像，绘制一个区域内在细胞的门。在我们的实验中，我们设置在0.3这个门。得分比0.3更低的细胞被认为是阳性细胞表面结合粒子。 </li><li>最后，消除背景标签的细胞，并确定具体的内在纳米粒子或细菌，思想现货计数功能使用。现货计数是一个功能，连接组件或图像中的小口罩的数量计数。遮罩功能，现货，山顶和强度来定义的斑点。被列入当场面具发现有用户指定的半径和阈值高于当地背景的图像中明亮的细节，面具被分裂成个别景点，山顶的功能，然后点以上200计数强度高强度细化。看到的Amnis点掩蔽指南的进一步资料（ 图1E）。统计重<html>端口模板生成的，包括“报告”菜单中的各种功能。 </li><li>此文件被保存为模板文件，将一批文件，所有的实验分析。 </li><li>在思想的软件，单击“工具→批量数据文件和输入所有。RIF文件。加入补偿矩阵文件（CTM）和模板文件中的相应部分（AST）。提交一批加工。处理步骤后，所有。RIF文件进行了分析和DAF文件为每个个别的原始文件生成。最后报告文件生成与所有样本的统计数据。 </li></ol>提示及注意事项： <ul><li>图像分析进行思想软件4.0版和一些修改的国际化向导。向导是自我启发和产生的ouptput。DAF文件可以按照向导中的说明。 </li></ul> 7。代表结果“&gt; 图2中的代表图像，证明是的MIFC可以被用来成功区分内在（左图）与表面结合（右图），NP（ 图2A）或沙门氏菌 （ 图2B）减少了国际化的NP和沙门氏菌 。既抑制肌动蛋白和温度降低到4°C（ 图3A）。的NP势必表面呈阳性的细胞百分比增加约8％，在37°C大于35％后，无论是松弛素D或4°C处理（ 图3B）。相比之下，与细胞表面结合沙门氏菌的比例从35％降低至15％后，细胞松弛素-D治疗。 沙门氏菌的RAW 264.7细胞的潜伏期在4°C降低内部没有明显增加金额表面约束的细菌相比，到37°C控制。一起，这些数据表明，&lt;EM&gt;沙门氏菌和NP内部由一个类似的肌动蛋白细胞的过程，需要是温度依赖。此外，数据表明， 沙门氏菌巨噬细胞的持续附件需要肌动蛋白聚合。 <img alt="图1" src="/files/ftp_upload/3884/3884fig1.jpg" /> 图1。用来确定内部与表面结合的纳米粒子和沙门氏菌浇注战略示意图 （a）限制单细胞分析，重要的是要消除碎片和多细胞活动。单细胞和双峰分离从思想功能区和明（M01）的长宽比的多细胞聚集。面积是平方微米的图像宽高比的大小是短轴，长轴，因此衡量一个圆（一个完美的圆圈，将有1宽高比分为双峰通常有ASPE克拉比0.5左右，是典型的多细胞聚集小于0.5）。一个地区被吸引到门单细胞活动（步骤6.6）。 （B）为焦点细胞的大门，思想具有梯度明形象的RMS在绘制直方图。通过检测图像中的像素值的变化梯度RMS功能测量图像的清晰度质量。梯度较高的RMS值表明一个更集中的图像（步骤6.7）。 （三）选择绿色荧光阳性细胞细胞上具有较高的最大像素值和强度在绿色荧光通道（步骤6.8）的门。 （四）具有内在的NP或沙门氏菌细胞通过选择一个国际化的得分等于或大于0.3的细胞群。细胞接收分数低于0.3被认为是表面的约束（步骤6.9）。 （五）在“内在的”闸门细胞的进一步特点的基础上斑点的数量（NP或STM），因为疗法E是一些背景染色细胞计数为内在的，但有一个点值为零（步骤6.11）。代表性的图像，每个门。 <a href="http://www.jove.com/files/ftp_upload/3884/3884fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3884/3884fig2.jpg" /> 图2。内在的和表面的束缚粒子（NP）或沙门氏菌的细胞图像。（一）内在NP（左侧面板）和细胞的NP绑定到其表面，而不是内在（右图）的RAW 264.7细胞的代表图像。 （二）内在沙门氏菌 （左图）和细胞沙门氏菌绑定到其表面而不是内在（右图）的RAW 264.7细胞的代表图像。 <img alt="图3" src="/files/ftp_upload/3884/3884fig3.jpg" /> 图3。 cytochala罪三维细胞治疗抑制NP和沙门氏菌的国际化。（一）预处理RAW 264.7细胞与细胞松弛素-D 或潜伏期在4°C降低发病率相比，在37°C培养基中培养的RAW 264.7细胞的NP或沙门氏菌的内在。 NP和沙门氏菌的RAW 264.7细胞中含有二甲基亚砜（即车辆控制）培养基表明类似的国际化水平相比，中等单独（数据未显示）。 （二）增加的RAW 264.7细胞与细胞松弛素-D预处理细胞表面的NP约束，同时减少与细胞表面结合沙门氏菌 ％％。

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P., Chitkara, D., Kumar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyanhydrides+as+localized+drug+delivery+carrier:+an+update.">Polyanhydrides as localized drug delivery carrier: an update.</a> Expert. Opin. Drug. Deliv. 5, 889-907 (2008).</li><li>Pfeifer, B. A., Burdick, J. A., Little, S. R., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ester-anhydride):poly(beta-amino+ester)+micro-+and+nanospheres:+DNA+encapsulation+and+cellular+transfection.">Poly(ester-anhydride):poly(beta-amino ester) micro- and nanospheres: DNA encapsulation and cellular transfection.</a> Int. J. Pharm. 304, 210-219 (2005).</li><li>Ahmed, F., Friend, S., George, T. C., Barteneva, N., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numbers+matter:+quantitative+and+dynamic+analysis+of+the+formation+of+an+immunological+synapse+using+imaging+flow+cytometry.">Numbers matter: quantitative and dynamic analysis of the formation of an immunological synapse using imaging flow cytometry.</a> J. Immunol. Methods. 347, 79-86 (2009).</li><li>Hampton, M. B., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+quantifying+phagocytosis+and+bacterial+killing+by+human+neutrophils.">Methods for quantifying phagocytosis and bacterial killing by human neutrophils.</a> J. Immunol. Methods. 232, 15-22 (1999).</li><li>Rieger, A. M., Hall, B. E., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+differentially+modulates+particle+binding,+phagocytosis+and+downstream+antimicrobial+mechanisms.">Macrophage activation differentially modulates particle binding, phagocytosis and downstream antimicrobial mechanisms.</a> Dev. Comp. Immunol. 34, 1144-1159 (2010).</li><li>Murphy, K. C., Campellone, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lambda+Red-mediated+recombinogenic+engineering+of+enterohemorrhagic+and+enteropathogenic+E.+coli.">Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli.</a> BMC. Mol. Biol. 4, 11 (2003).</li><li>Karsi, A., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+host+range+fluorescence+and+bioluminescence+expression+vectors+for+Gram-negative+bacteria.">Broad host range fluorescence and bioluminescence expression vectors for Gram-negative bacteria.</a> Plasmid. 57, 286-295 (2007).</li><li>Ulery, B. D., Phanse, Y., Sinha, A., Wannemuehler, M. J., Narasimhan, B., Bellaire, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+chemistry+influences+monocytic+uptake+of+polyanhydride+nanospheres.">Polymer chemistry influences monocytic uptake of polyanhydride nanospheres.</a> Pharm. Res. 26, 683-690 (2009).</li><li>Doherty, G. J., McMahon, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+endocytosis.">Mechanisms of endocytosis.</a> Annu. Rev. Biochem. 78, 857-902 (2009).</li><li>Vercauteren, D., Vandenbroucke, R. E., Jones, A. T., Rejman, J., Demeester, J., De Smedt, S. C., Sanders, N. N., Braeckmans, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+inhibitors+to+study+endocytic+pathways+of+gene+carriers:+optimization+and+pitfalls.">The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls.</a> Mol. Ther. 18, 561-569 (2010).</li><li>Di Marzio, L., Marianecci, C., Cinque, B., Nazzarri, M., Cimini, A. M., Cristiano, L., Cifone, M. G., Alhaique, F., Carafa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-sensitive+non-phospholipid+vesicle+and+macrophage-like+cells:+binding,+uptake+and+endocytotic+pathway.">pH-sensitive non-phospholipid vesicle and macrophage-like cells: binding, uptake and endocytotic pathway.</a> Biochim. Biophys. Acta. 1778, 2749-2756 (2008).</li><li>Torres, M. P., Vogel, B. M., Narasimhan, B., Mallapragada, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+novel+polyanhydrides+with+tailored+erosion+mechanisms.">Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms.</a> J. Biomed. Mater. Res. A. 76, 102-110 (2006).</li></ol>

sherree研究的朋友受雇于Amnis公司，该公司生产ImageStream X系统。

有研究表明，聚（乳酸 - 乙醇酸共聚物（PLGA）或酸酐为基础的可生物降解的纳米粒子可以被用于运载包裹抗原或药物靶细胞，其成效是非常重要的吞噬细胞，这些纳米粒子吸收，从而使定量国际化，设计新颖的纳米给药系统的关键，通过使用这种方法。分析，由多种细胞类型的纳米粒子吸收差可以轻松地分析至目前为止，已量化粒子吸收传统的显微镜和流式细胞仪。然而，各自的替代的方法来研...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>的RAW 264.7细胞株</td><td>美国菌种保藏中心（ATCC） </td><td> TIB-71 </td><td></td></tr><tr><td>杜尔贝科的改良Eagle培养基（DMEM培养基） </td><td> cellgro </td><td> 10-013-CV </td><td></td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td>小号11150 </td><td>优质甲级</td></tr><tr><td> glutamax </td><td> Gibco公司</td><td> 35050-061 </td><td></td></tr><tr><td>肝素钠</td><td> Gibco公司</td><td> 15630-080 </td><td></td></tr><tr><td> 24孔板</td><td>卟啉</td><td> 92024 </td><td></td></tr><tr><td>细胞培养瓶</td><td>卟啉</td><td> 90151 </td><td></td></tr><tr><td>细胞刮刀</td><td>卟啉</td><td> 99002 </td><td> 24厘米</td></tr><tr><td>血清型鼠伤寒沙门氏菌 </td><td> ATCC </td><td> 14028 </td><td></td></tr><tr><td> A型肉毒毒素ECM630电的电池机器人</td><td> A型肉毒毒素哈佛仪器</td><td></td><td></td></tr><tr><td>公开资讯观测站</td><td> Fisher Scientific则</td><td> BP308 </td><td></td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td> cellgro </td><td> 21-040-CV </td><td></td></tr><tr><td>超声波液体处理器</td><td>管理信息系统ONIX </td><td>的S-4000 </td><td></td></tr><tr><td>细胞松弛素 - D </td><td> Sigma-Aldrich公司， </td><td> C8273 </td><td></td></tr><tr><td>甲醛</td><td> polysciences </td><td> 04018 </td><td></td></tr><tr><td>洗缓冲区</td><td> 2％热灭活胎牛血清，0.1％叠氮钠的PBS。 </td><td></td><td></td></tr><tr><td>烫发/洗缓冲区</td><td> BD公司</td><td> 554714 </td><td></td></tr><tr><td>清除查看单元帽微管</td><td>西格玛</td><td> T4816 </td><td></td></tr><tr><td>鬼笔660的Alexa Fluor </td><td> Invitrogen公司</td><td> A22285 </td><td></td></tr><tr><td> ImageStream X </td><td> amnis语料库TION </td><td> 100200 </td><td>选项​​：658nm激光，自动进样器</td></tr><tr><td>叠氮化钠</td><td> Fisher Scientific则</td><td>小号227I-500 </td><td></td></tr></tbody></table>

analyzing cellular internalization of nanoparticles and bacteria by multi-spectral imaging flow cytometry

纳米粒子系统已经出现在疫苗交付有价值的工具，通过他们的能力，有效地交付货物，包括蛋白质，抗原提呈细胞1-5。国际化的纳米粒子的抗原提呈细胞（NP），是在封装的抗原产生有效的免疫反应的关键步骤。为了确定在纳米制定影响功能如何变化，我们寻求开发一种高通量，定量实验的协议，该协议是兼容内在的纳米颗粒以及细菌检测。到今天为止，显微镜和流式细胞仪，两个独立的技术，已用于研究纳米粒子的吞噬功能的方法。流式细胞仪的高通量的性质产生了强大的统计数据。然而，由于分辨率低，不能准确地量化与细胞结合纳米粒子内部。显微镜生成高空间分辨率的图像，H贷款本息偿还期中，它是费时，涉及小样本6-8。多光谱成像流式细胞仪（MIFC）是一种新技术，采用显微镜和流式细胞仪同时执行层的核心，通过多种颜色的光谱荧光和亮场成像方面。此功能提供了一个准确的分析荧光信号强度和高速蜂窝不同的结构和功能之间的空间关系。 在此，我们描述了一个方法，利用MIFC特征的细胞群体的内在酸酐纳米粒子或沙门伤寒。我们还描述了制备纳米粒子悬浮液，细胞标记，ImageStream X系统的采集和分析数据的使用理念的应用。我们也证明了一种技术的应用，可以用来区分国际化带够纳米粒子和细菌athways使用的肌动蛋白介导的吞噬作用的抑制剂细胞​​松弛素 - D。

Watch this Scientific Journal Video about Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry at JoVE.com

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

在这篇文章中，我们描述了一种方法，利用多光谱成像流式细胞仪量化酸酐纳米粒子或RAW 264.7细胞细菌的国际化。

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 ...

analyzing-cellular-internalization-nanoparticles-bacteria-multi

Research

JoVE Journal

Bioengineering

16.2K 次观看.  Iowa State University. 在这篇文章中，我们描述了一种方法，利用多光谱成像流式细胞仪量化酸酐纳米粒子或RAW 264.7细胞细菌的国际化。

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

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

科研

生物工程

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

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 ...

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, ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

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 ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

悬浮中金纳米粒子尺寸的不对称流场-流分数

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...