作者想感谢来自衣阿华州立大学流式细胞设施美国陆军医学研究和装备司令部（格兰特W81XWH-09-1-0386和W81XWH-10-1-0806号）的财政支持和肖恩·里格比博士他的专家技术援助。

1。鼠标采用肺灌洗收获（AMɸ）肺泡巨噬细胞<ol><li>穿白大褂，一次性手套，和适当的保护眼睛，如适当的个人防护装备（PPE）。 </li><li>开始收获前准备完整AMɸ（cAMɸ）培养基。新增2.5青霉素/链霉素（笔/链球菌）解决方案，250β-巯基乙醇微升（巯基乙醇）和25热灭活胎牛血清（FBS），毫升222.25 Dulbelcco的修改鹰培养基（DMEM培养基）毫升毫升。过滤消毒cAMɸ介质在真空条件下使用0.2微米的孔大小瓶顶过滤器。 </li><li>安乐死二氧化碳窒息在euthanization室的一个健康的鼠标。虽然C57BL / 6小鼠（6-8周龄），用于该项目的目的，这种肺灌洗技术，可以进行任何的小鼠品系。使用压缩气瓶为1分钟，让气体进入腔流前鼠标放置在CH琥珀色，以保证室完全与CO 2收费。在会议厅内放置至少一分钟安乐死鼠标鼠标。此外，作为一个物理的方法如1.4中所述确认鼠标死亡，各组小鼠心脏穿刺抽血通过。 </li><li>躺在它的背部（背侧），喷用70％乙醇的鼠标，找到胸骨，并在30度角从腹部到胸腔的23号针（1毫升注射器插入50毫米左右） 。针妥善安置，应尽可能收集血液取决于鼠标的大小介于0.4至1毫升。检查停止生命迹象的动物。 </li><li>将鼠标在实验室工作台上，以便它躺在其腹部（腹面）。喷雾用70％乙醇消毒工作环境的鼠标。约一半，脊柱鼠标捏皮肤。使用夹层已用70％乙醇消毒的剪刀，通过皮肤小切口。&lt;/ LI&gt; <li>抢在切口两侧的皮肤和鼠标颅尾轴沿相反的方向拉的皮肤。正确完成后，皮肤会被删除从底层的胴体。 </li><li>现在应该显示鼠标的脖子外露的肌肉和腺体。仔细削减该组织和拉对前钳使用鼠标，从而暴露喉，气管，食管鼠标。避免切割颈内静脉或颈内动脉。 </li><li>现在可见气管和识别是与它周围的软骨环的组织。微妙的用钳子举行气管，轻轻抬起，并分开的，这是位于背侧的食管气管。 </li><li>做一个小切口前，其中一个持有喉气管后。应在约45°度角的切口。另外，不完全横切气管，因为这将防止从收回体腔。 </li><li>填充用无菌PBS汤姆猫准备的导管插入导管，气管管腔装有1毫升注射器。使用镊子握住气管，并与你的手按住导管尖端，以提高控制和轻轻插入气管切开网站。轻轻插入到气管导管约20毫米。夹住气管导管，大约是释放另一方面。 </li><li>使用装有导管1毫升的注射器，轻轻注入肺部0.75毫升无菌PBS室温，然后轻轻地吸的PBS后进入注射器。三次重复此过程，同时外部按摩胸部。从导管拔下注射器和分配到15 mL离心管灌洗液。 0.75毫升的新鲜PBS填充注射器和导管重新连接注射器后，重复步骤1.9和1.10两次，每次鼠标。液体复苏可能是局部的，取得比VO卢梅注入。如果从多个鼠标收集细胞，15 mL管放置在冰上收获细胞，肺灌洗，直到所有已执行。 </li></ol> 2。肺灌洗AMɸ收获加工<ol><li> 10分钟在4°C离心​​250×Ğ肺灌洗液</li><li>到适当的废物容器内的生物安全柜使用无菌技术，倒出上清液。轻轻耙整个试管架顶部的离心管，重悬细胞沉淀。加入1毫升cAMɸ培养基悬浮细胞。 </li><li>枚举的使用1库尔特颗粒计数器Z1的第二稀释剂Isoton到10毫升，库尔特计数器瓶吸取20μL细胞的细胞。添加到小瓶3至4滴ZAP-oglobin的第二裂解试剂，轻轻倒置3至4倍，然后加载到柜台样品瓶获得细胞的浓度。一定编程与稀释倍数为2×10 4的计数器显示细胞浓度为每毫升细胞数。 </li><li>使用cAMɸ介质稀释细胞以2.5×每毫升10 5。 </li><li>免除2 mL 2.5×10 5细胞/毫升到每个井的6孔的组织培养皿中。 </li><li>用5％的CO 2为6小时的气氛，以丰富为37℃加湿孵化器内孵化AMɸ，培养皿，使细胞坚持到井底。轻轻吸出上清液，以不破坏贴壁细胞。 </li><li>使用无菌PBS（钙，镁的自由），除去非贴壁细胞和碎片冲洗井。 cAMɸ培养基中加入2毫升，每口井。 </li><li>孵育前5％的CO 2气氛，37℃培养箱培养箱内过夜细胞除了兴奋剂。这个浓缩步骤后，约87％的细胞是阳性的巨噬细胞标记CD11b和F4/80的（ 图2c）。 </li></ol> 3。新增的Polyanhydridé纳米粒子和控制处理<ol><li>编造通过酸酐反溶剂nanoencapsulation的15纳米。在这个过程中，聚合物溶解在二氯甲烷（4°在浓度为25毫克/毫升）和戊烷（-20°C〜1:200的比例二氯甲烷:戊烷）沉淀。 </li><li>掂量出酸酐纳米粒子，用消毒纸张重量上的平衡，可以准确地测量微克量。加入到1.5 mL离心管中的颗粒。 </li><li>悬浮纳米颗粒在冰冷cAMɸ的。计算不超过0.3毫升培养基添加到每口井，以获得所需浓度的纳米粒子。置于冰上直到纳米粒子添加到文化的再悬浮颗粒。 </li><li>添加纳米的AMɸ文化之前，使用微尖板凳顶sonicator超声颗粒。消毒喷洒70％乙醇和wipin的微尖g与无菌Kimwipe事先向超声。超声在10-20焦耳为30至45秒，同时保持在冰上管。肉眼观察到纳米粒子悬浮。如果颗粒没有得到充分的分散，使悬挂设置1至2分钟，上冰，以确保纳米粒子聚合物的玻璃化转变温度低于前再次超声分散为30至45秒。这种超声一步的目的是充分分散纳米粒子，作为50:50 CPTEG的:青山医院纳米粒子往往聚集时，接触到水环境。然而，一些小的纳米粒子聚集可能仍然暂停。 </li><li>纳米粒子悬浮液（充分分散）应保持在冰上，并立即使用，以防止过早表面糜烂或聚集。 </li><li>取出培养皿中含有从孵化AMɸ在生物安全柜的地方。倾斜板在约45度角和关闭的Medi量移液器UM将被添加到纳米粒子悬浮液（它不应该超过0.3毫升）。移除介质时，请勿打扰的贴壁细胞。 </li><li>涡酸酐纳米粒子悬浮简要之前将它添加到相应的井。吸取到具体的井（S）（不超过0.3毫升）的纳米粒子悬浮量，但是，不吸取了上下混合。这个动作可以分离附着AMɸ从井里。这些粒子将落户到井底，接触的细胞。对于在本报告所述的研究，纳米粒子与AMɸ合作培养的最终浓度为125微克/毫升。随后的水井和理想的治疗组进行。包括正面和负面的控制，如中等只有200毫微克/毫升脂多糖（LPS）到指定的井组。 </li><li>返回的培养皿中，以37°C间湿润的孵化器与5％CO 2 48小时的气氛。在我们laborator动力学研究y有证明48小时内是最佳培养时期的细胞表面标记的表达和分泌细胞因子的转录和蛋白合成发生，因为它允许有足够的时间评估。虽然扩展文化时期（即72小时）期间积累更高水平的细胞因子，评估在这个时间点的细胞表面标志的表达，可以混淆的细胞因子水平升高的贡献。观察表面标志表达的变化可能并不直接归属与纳米粒子本身的刺激。 </li></ol> 4。评价AMɸ激活使用流式细胞仪<ol><li>准备荧光激活细胞分选（FACS）缓冲液（0.1％叠氮化钠和0.1％牛血清白蛋白（BSA））在0.1 M磷酸缓冲液（pH值7.4）之前收获镀巨噬细胞与纳米Ø已共同培养ŕ兴奋剂。 </li><li>准备工作阻断缓冲区和单克隆抗体的解​​决方案。准备块缓冲，稀释抗体（Sigma公司）和anti-CD16/32（eBioscience）为1 mg / mL和10μg/ ml的大鼠，分别在流式细胞仪缓冲区。稀释表面标记抗体在流式细胞仪缓冲面板制造商建议的浓度或用户优化浓度。在这项工作中提出的结果，表面标志面板组成的MHC II类，CD86，CD40和CIRE（CD209）抗体。面板中还包括抗鼠抗体对巨噬细胞标记F4/80的和CD11b积极识别数据分析过程中的巨噬细胞的表型。时，选择对MHC II类分子的抗体，为近交系小鼠品系，往往在他们的MHC二单体型不同，必须小心。选定的抗体必须与您感兴趣的小鼠品系表达了特定的MHC同种抗原反应。 </li><li>经过48小时的孵化与纳米粒子和/或兴奋剂，冰20分钟的地方细胞培养皿。从板块中删除的贴壁的巨噬细胞，24厘米细胞刮刀轻轻刮去井。 </li><li>使用5毫升血清吸管，轻轻吸细胞和中等上下五次产生单细胞悬液。成独立的5毫升聚苯乙烯管吸取个别井的内容，所以不能混在一起从不同的试验性治疗的细胞。 </li><li>每管加入2毫升流式细胞仪缓冲。 </li><li>管细胞在4°C离心​​10分钟，在250×Ğ。 </li><li>倒出上清，在剩余液轻轻重悬细胞沉淀，耙管穿过一个试管架的顶部。 </li><li>为了防止非特异性结合的单克隆抗体，每管细胞添加10μL封闭液（4.2准备）。涡流管和孵育30分钟冰。 </li><li>每个管和incub的加入适当稀释的单克隆抗体体积在冰上15分钟的暗吃。包含要分析的样品管应该得到的polycromatic面板（应个别实验室进行抗体结合以前的优化）抗体结合。其他对照组应包括正确设置流式细胞仪上采集参数，包括一管没有标记的细胞，并补偿管对应单独使用每一个单一的颜色来标记细胞。要正确设置流式细胞仪分析的大门，荧光减一（FMO）的控制，应包括在收购。鱼类统营处的控制面板上除了一个用来取代其各自的同型对照与所有的荧光标记的抗体标记细胞。正面和负面的人群（即，与阳性对照兴奋剂和中等细胞单独分装），也应包括在鱼类统营处和补偿控制管。 </li><l我&gt;加入2毫升流式细胞仪缓冲区为10分钟，每个管，旋涡和离心250×Ğ4°C间。 </li><li>耙管穿过一个试管架的顶部，倒出上清液和残余液重悬细胞沉淀。 </li><li>如果抗体面板组成，但不直接结合到荧光抗体，生物素，而不是共轭，荧光标记的链霉亲和需要形象化的表面标志。在此步骤中，加入稀释的荧光标记的链霉亲和适当的音量，并在黑暗中孵育15分钟冰。在4.2，将稀释的链霉制造商推荐的浓度或用户优化。这一步只需要含有生物素化抗体的面板。 </li><li>执行在4.10中所述的洗涤步骤。 </li><li>屋宇署稳定在去离子水1:3的固定液稀释适当的音量。每管加入100μL稀释固定液而轻轻VOrtexing，以防止细胞聚集。标记和固定细胞可以稳定储存在4°C大约一个星期之前收购流式细胞仪在黑暗中。 </li></ol> 5。具有代表性的数据在步骤3.1制备纳米粒子的平均直径163±24 nm和形态的与5,16以往的研究结果一致。在解决前，后超声纳米如图1所示，证明超声的需要，以确保足够分散的颗粒。通过肺灌洗获得的收获AMɸs流仪分析如图2所示。 CD11b和抗鼠抗小鼠F4/80的抗体以及相应的鱼类统营处控制相结合的细胞标记允许建立背景标签，，确定AMɸs门作进一步的分析。与酸酐的肺泡巨噬细胞治疗纳米粒子增强活化增加，平均荧光强度（ 图3）MHC II类分子CD40，CD86，CIRE所示。 <img alt="图1" src="/files/ftp_upload/3883/3883fig1.jpg" /> 图1 50:50 CPTEG:本院纳米（一）之前和之后（二）30超声小号。 <img alt="图2" src="/files/ftp_upload/3883/3883fig2.jpg" /> 图2。流（一）的Alexa Fluor 700抗CD11b和PE-Cy7鱼类统营处控制（B）的Alexa Fluor 700鱼类统营处控制和PE-Cy7 anti-F4/80标记的收获肺泡巨噬细胞的流式细胞仪分析和（C ）的Alexa Fluor 700抗CD11b和PE-Cy7 anti-F4/80。在右上角的数字代表双阳性细胞的百分比。 <img alt="图3" src="/files/ftp_upload/3883/3883fig3.jpg" /> 图3。直方图显示荧光INTEN增加电视大学50:50 CPTEG肺泡巨噬细胞共培养后表面的表达，CD86，MHC II类（甲），CD40分子（乙）（C）和CIRE（四）:青山医院酸酐纳米为48小时。直方图描绘为鱼类统营处控制为AMɸ标记（ <img alt="图线1" src="/files/ftp_upload/3883/3883line1.jpg" /> ），未经处理的AMɸ对所有的细胞表面标志的抗体标记（ <img alt="图线3" src="/files/ftp_upload/3883/3883line3.jpg" /> ）和AMɸ纳米粒子和培养，对所有的细胞表面标志的抗体标记（ <img alt="图线2" src="/files/ftp_upload/3883/3883line2.jpg" /> ）。

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Immun. 79, 1842-1847 (2011).</li><li>Morimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+Macrophages+that+Phagocytose+Apoptotic+Neutrophils+Produce+Hepatocyte+Growth+Factor+during+Bacterial+Pneumonia+in+Mice.">Alveolar Macrophages that Phagocytose Apoptotic Neutrophils Produce Hepatocyte Growth Factor during Bacterial Pneumonia in Mice.</a> Am. J. Respir. Cell Mol. Biol. 24, 608-615 (2001).</li><li>Gordon, S., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+and+macrophage+heterogeneity.">Monocyte and macrophage heterogeneity.</a> Nat. Rev. Immunol. 5, 953-964 (2005).</li><li>Ulery, B. D. <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>Petersen, L. K., Sackett, C. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+analysis+of+protein+stability+in+polyanhydride+nanoparticles.">High-throughput analysis of protein stability in polyanhydride nanoparticles.</a> Acta Biomater. 6, 3873-3881 (2010).</li><li>Irvin, C. G., Bates, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+lung+function+in+the+mouse:+the+challenge+of+size.">Measuring the lung function in the mouse: the challenge of size.</a> Respir. Res. 4, 4 (2003).</li></ol>

没有利益冲突的声明。

酸酐纳米疫苗平台显示效果时，给予单剂量疗法5滴鼻。在此疫苗交付平台引起的肺部测量驻地吞噬细胞群的激活，允许评估其潜在的能力，最终促进适应性免疫反应。 具体来说，从肺灌洗液中收获肺泡巨噬细胞和不同配方的纳米粒子治疗提供了不同的粒子化学能力的见解，激活巨噬细胞，导致抗原提呈6,8。此外， 在体外研究中，这些都是用于?...

<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> CAM媒体 </td><td></td><td></td><td></td></tr><tr><td> DMEM培养液</td><td> cellgro </td><td> 15-013-CV </td><td></td></tr><tr><td> 50毫米2 - 巯基乙醇</td><td>西格玛</td><td> M3148-25ML </td><td></td></tr><tr><td>青霉素/ 10000微克/毫升溶液</td><td> cellgro </td><td> 30-002-CI </td><td></td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S11150 </td><td></td></tr><tr><td> 流式细胞仪缓冲 </td><td></td><td></td><td></td></tr><tr><td>氯化钠</td><td> Fisher Scientific则</td><td> S671-500 </td><td></td></tr><tr><td>磷酸钠</td><td> Fisher Scientific则</td><td> MK7868500 </td><td></td></tr><tr><td>氯化钾</td><td> Fisher Scientific则</td><td> P217500 </td><td></td></tr><tr><td>磷酸钾</td><td> Fisher Scientific则</td><td> P288-200 </td><td></td></tr><tr><td>牛血清白蛋白（牛血清白蛋白） </td><td>西格玛</td><td> A7888 </td><td></td></tr><tr><td>叠氮化钠</td><td>西格玛</td><td> S2002 </td><td></td></tr><tr><td> 抗体 </td><td></td><td></td><td></td></tr><tr><td>鼠IgG </td><td>西格玛</td><td> I4341 </td><td></td></tr><tr><td>反女士CD16/32 </td><td> eBioscience </td><td> 16-0161 </td><td></td></tr><tr><td>抗MHC II类女士单体IA / IE浏览器，M5/114.15.2克隆，共轭异硫氰酸荧光素（FITC标记） </td><td> EBIOS新增5Y科学</td><td> 11-5321 </td><td></td></tr><tr><td>抗CD86的鼠标，克隆GL-1，共轭变藻蓝蛋白（APC）Cy7 </td><td> Biolegend </td><td> 105030 </td><td></td></tr><tr><td>抗小鼠CD40，克隆1C10，结合到APC </td><td> eBioscience </td><td> 17-0401 </td><td></td></tr><tr><td>抗鼠CD209，克隆5H10，结合生物素</td><td> eBioscience </td><td> 13-2091 </td><td></td></tr><tr><td>抗小鼠CD11b的克隆M1/70，共轭的Alexa Fluor 700 </td><td> eBioscience </td><td> 56-0112 </td><td></td></tr><tr><td> F4/80的反鼠标，克隆BM8，结合藻红蛋白（PE）Cy7 </td><td> eBioscience </td><td> 25-4801 </td><td></td></tr><tr><td> PE得克萨斯红色标记的链霉</td><td> BD公司</td><td> 551487 </td><td></td></tr><tr><td> 其他耗材和试剂 </td><td></td><td></td><td></td></tr><tr><td>乙醇</td><td> Fisher Scientific则</td><td> A405-20 </td><td>作为70％（V / V） </td></tr><tr><td>压缩的CO 2 </td><td> linweld </td><td> 16000060 </td><td></td></tr><tr><td> 1毫升注射器</td><td> BD公司</td><td> 309659 </td><td></td></tr><tr><td>主权3½"神父汤姆Catcatheter的</td><td>肯德尔</td><td> 703021 </td><td></td></tr><tr><td>生物安全柜</td><td> NUAIRE </td><td>系列22 </td><td></td></tr><tr><td>解剖剪刀</td><td>费舍尔</td><td> 138082 </td><td></td></tr><tr><td>镊子</td><td> roboz </td><td> RS-8254 </td><td></td></tr><tr><td> PBS，1X无钙，镁</td><td> cellgro </td><td> 21-040-CM </td><td></td></tr><tr><td> 15 mL离心管，带螺旋盖</td><td> VWR国际</td><td> 21008-216 </td><td></td></tr><tr><td>治疗板六以及组织文化</td><td> COSTAR </td><td> 3516 </td><td></td></tr><tr><td>塑料管架</td><td> NALGENE </td><td> 5970 </td><td></td></tr><tr><td>细胞刮刀24厘米</td><td>卟啉</td><td> 99002 </td><td></td></tr><tr><td> 5 mL聚苯乙烯回合中筒底部</td><td>鹘</td><td> 352008 </td><td></td></tr><tr><td>吸管，加大援助</td><td>德拉蒙德</td><td> 4-000-105 </td><td></td></tr><tr><td> 10，5，2毫升移液器</td><td>费舍尔</td><td> 13-675 </td><td></td></tr><tr><td> 200和10μL的micropipettors </td><td>吉尔森Pipetman </td><td> F123601 </td><td></td></tr><tr><td> 200和10μL枪头</td><td>费舍尔</td><td> 02-707 </td><td></td></tr><tr><td>屋宇署稳定固色剂</td><td> BD公司</td><td> 338036 </td><td></td></tr><tr><td> isoton第二稀释剂</td><td>贝克曼 - 库尔特</td><td> 8546719 </td><td></td></tr><tr><td> ZAP-oglobin II裂解试剂</td><td>贝克曼 - 库尔特</td><td> 7546138 </td><td></td></tr><tr><td>库尔特计数器的聚苯乙烯瓶</td><td>贝克曼 - 库尔特</td><td> 14310-684 </td><td></td></tr><tr><td>试管</td><td> BD公司</td><td> 352008 </td><td></td></tr><tr><td> 设备 </td><td></td><td></td><td></td></tr><tr><td>冷冻离心机</td><td> labnet </td><td> 50075040 </td><td></td></tr><tr><td>湿润的孵化器的CO 2 </td><td> nuaire </td><td>模型自动流8500 </td><td></td></tr><tr><td> FACSCanto流式细胞仪</td><td> BD公司</td><td> 338960 </td><td></td></tr><tr><td>库尔特颗粒计数器Z1的</td><td>贝克曼 - 库尔特</td><td> WS-Z1DUALPC的</td><td></td></tr><tr><td> sonicator液体微尖加工设备</td><td> misonix </td><td>型号的S-4000 </td><td></td></tr></tbody></table>

harvesting murine alveolar macrophages and evaluating cellular activation induced by polyanhydride nanoparticles

可生物降解的纳米粒子已经出现作为一个通用平台的设计和实施新鼻内疫苗对呼吸道传染病。具体来说，酸酐纳米脂肪癸二酸（SA），芳香1,6 -双（P-羧基苯氧）正己烷（CPH），或双亲1,8 -双（P-羧基苯氧）-3,6 - dioxaoctane组成（CPTEG）显示独特的体积和表面侵蚀动力学1,2和可利用缓慢释放功能的生物大分子（如蛋白抗原，免疫球蛋白等） 在体内 3,4,5。这些纳米粒子也具有内在的辅助活动，使他们6,7,8疫苗交付平台的一个很好的选择。 为了阐明机制激活先天免疫后鼻腔粘膜接种，必须评估的分子和细胞反应的抗原P怨恨细胞（APC）负责启动免疫反应。树突状细胞是进行气管中发现的主要装甲运兵车，而在肺实质9,10,11（AMɸ）肺泡巨噬细胞为主。 AMɸ是非常有效地清除病原微生物和细胞碎片12,13肺部。此外，这种细胞类型发挥宝贵的作用在运输微生物抗原的淋巴结，这是一个重要的第一步启动适应性免疫反应9。 AMɸ也表达水平升高的先天模式识别和清道夫受体，分泌促炎介质，和总理幼稚T细胞12,14。一个人口相对纯的AMɸ（例如，大于80％），可以很容易地获得通过肺灌洗在实验室中研究。从免疫动物AMɸ收获的居民提供了一个代表性的表型的巨噬细胞，将encounter的粒子在体内的疫苗。在此，我们描述了从小鼠到收获和文化AMɸ使用的协议和检查酸酐纳米粒子在体外治疗后的巨噬细胞的激活型。

Watch this Scientific Journal Video about Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles at JoVE.com

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

在此，我们描述了小鼠肺泡巨噬细胞，居住在肺先天免疫细胞，并检查其激活，共培养酸酐纳米收获的协议。

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 ...

harvesting-murine-alveolar-macrophages-evaluating-cellular-activation

Research

JoVE Journal

Bioengineering

19.8K 次观看.  Iowa State University. 在此，我们描述了小鼠肺泡巨噬细胞，居住在肺先天免疫细胞，并检查其激活，共培养酸酐纳米收获的协议。

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

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

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

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 ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

微生物诱导方解石沉淀通过介导<em&gt; Sporosarcina巴斯德</em

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中脉冲激光烧蚀制备金属纳米粒子:研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

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 ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

水样组织的采集装置

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...