这项研究是由NIH的拨款R01HL091020，R01HL093723，R01AI077343，并R01GM096187支持。

1。从全血红细胞隔离<ol><li>准备EAS - 45解决方案。重达从表一的所有成分，并溶于DI水100 200毫升。加水使千毫升解决方案，并调整pH值至8.0。过滤和分装50毫升。冻结储存在-20 ° C。 </li></ol>注：作为一名护士，应当由受过训练的医疗专业等步骤1.2机构审查委员会批准的协议，。 <ol start="2"><li>绘制成10毫升含有EDTA管中位数肘静脉3血5毫升，轻轻混匀血用EDTA，立即彻底避免凝血。 </li><li>尽快处理血液样本。离心除以下步骤应该做的引擎盖下，以保持无菌的准备。转移至50ml离心管中的血液，添加5分钟@ 1000RPM，4 ° C的寒冷和无菌Histopaque 1077和离心机10毫升重复两次。 </li><li>新增10毫升冷，无菌PBS，离心5分钟@ 1000RPM，4 ° C。去除上清。重复4次（共5次）。 </li><li>洗净用无菌EAS - 45（5分钟@ 1000RPM，4℃）的两倍。在过去的洗涤红细胞移动到一个新的无菌15毫升管。 </li><li>悬浮红细胞in10ml EAS - 45。 </li></ol> 2。红细胞生物素<ol><li>以与步骤1中红细胞管。取出后5分钟@ 1000RPM，4 ° C离心上清红细胞沉淀中加入10μl到每个几个小瓶，并添加相应数额的PBS，每小瓶（例如准备六种不同的红细胞生物素浓度表二） 。 </li><li>根据表二 ，做出新的Biotin-X-NHS/DMF 1:10，1:100稀释。 </li><li>添加0.1M硼酸缓冲液中加入10μl每一瓶。 </li><li>添加生物素的解决方案，以每一瓶（见如表二 ）的计算量和涡立即。 </li><li>将每个红细胞小瓶一个50ml的锥形管和肩在室温下孵育30分钟内。 </li><li>每一瓶洗净800μl的EAS - 45为2分钟@ 2000RPM的3倍。 </li><li> EAS - 45100μL每一瓶和储存于4 ° C。现在应该在每一个最终的解决方案的加入1μl〜1mln的红细胞。 </li></ol> 3。 Functionalizing红细胞的生物素联配体* <ol><li>根据制造商的指示，准备2mg/ml链亲和素溶液。 Aliqouted解决方案可能会储存在-20 ° C </li><li>从步骤2.7（10μL）混合等量的红细胞与链霉亲和素溶液，旋涡立即孵育30min后上肩在4 ° C。 </li><li> 2分钟@ 2000RPM的EAS - 45500μL洗3次。加入15μlEAS - 45 / 1％BSA的存储。 </li><li> 20μg/ml配体解决方案，涡（10μL）从步骤3.3混合等量的红细胞和肩在室温下孵育30分钟。 </li><li>洗两次，500μLEAS -45 / 1％BSA为2分钟@ 2000RPM。 EAS - 45 / 1％的存储BSA的加入15μl。 </li></ol> *如果你的蛋白没有生物素的链接，您可以使用市售蛋白质生物素包（例如，Thermo Scientific的＃21955 EZ - Link的微型NHS - PEG4 -生物素包），或使用生物素捕获抗体作为一个中间步骤，如下所示在视频中。 4。受体与配体密度的定量<ol><li>从第3步孵育配体涂层的红细胞，在不同的小瓶，受体含饱和浓度与各自的单克隆抗体的细胞在室温下30分钟。在单独的小瓶无关匹配的同型对照抗体细胞孵育。如果没有荧光标记的抗体，荧光标记的二抗孵育，根据制造商的指示。 </li><li>分析步骤4.1准备流式细胞术样品与相应的荧光CALibration珠。计算图所示的密度。 1。 </li></ol> 5。为微管的制备和细胞室<ol><li>拉使用PN - 30 Narishige“磁性玻璃微电极水平拖轮或萨特仪器微管拖轮毛细管微量。 </li><li>使用与microforge显微拉微量调整到所需的大小（通常所需的直径范围从1 -5μm的，要在研究中使用的细胞的大小而定）的提示。 </li><li>准备削减到所需的大小显微镜盖玻片的细胞室。密封腔，从双方使用矿物油，以避免介质的蒸发，在实验过程中改变渗透压。 </li><li>新增的受体和配体细胞会议厅。 </li></ol> 6。微管粘附频率检测<ol><li>各自移液器吸相互作用的细胞，并用计算机编程piezoelectr<html> IC翻译驱动与控制的接触面积和时间与其他细胞接触和红细胞。检测细胞分离后，通过观察红细胞伸长的粘附事件。 </li><li>对于一个给定的接触时间，重复接触回缩周期50-100倍。在Excel电子表格的列无粘连粘连或“0”，加入“1”，记录所观察到的粘附性事件。您可以使用录音设备，如数字媒体或录像带，显微图像。 </li><li>记录每个接触的时间至少有三个不同的细胞对获得的均值和SEM的附着力频率与接触时间曲线。 </li><li>使用无关配体（如BSA），和/阻止使用其特定的功能封锁单克隆抗体的配体或受体或涂红细胞记录控制的非特异性的结合曲线。在每个接触时间点的特异性粘附频率可以计算出清除非特异性粘附频率 1 。 </li></ol>ove_title“&gt; 7。数据分析<ol><li>适合具体的附着力频率 P A与接触时间T的数据概率模型（公式1），介绍了二阶和一阶扭转，单步之间相互作用的受体和配体 1，品种单一品种单一： <img alt="公式1" src="/files/ftp_upload/3519/3519eq1.jpg" /> 其中 K 2D有效的结合亲和力，K关闭的关闭率，M r 和 M，L，分别是受体与配体密度在第4步测， 和 C的接触面积。曲线拟合有两个参数， 一个 C K 一 K 关闭 ， 作为 C 和 K 一个混为一谈，并呼吁有效2D的亲和力集体。其关闭率的产品是有效的2D率： </e米&gt; C 钾 = A C K 一个X k。 特异性粘附频率P的计算方法是从总的测量粘附性（P）1,21（非特异性）的非特异性粘附分数加减： <img alt="公式（2）" src="/files/ftp_upload/3519/3519eq2.jpg" /></li></ol> 8。代表性的成果： <img alt="图1" src="/files/ftp_upload/3519/3519fig1.jpg" /> 图1整合α 大号β2中性粒细胞的站点密度的测定。中性粒细胞E -选择素，免疫球蛋白为10分钟1μg/ml培养相匹配的实验条件下，在数字3,4或没有E -选择素，免疫球蛋白，然后用饱和浓度PE标记的抗（10μg/ml）人类CD11a单克隆抗体（克隆HI111， 具体表试剂和设备）或无关控制鼠IgG1，洗净，并立即进行分析。样品读取BD LSR与标准的QuantiBRITE PE校准珠的流式细胞仪。 A组荧光直方图显示校准珠（粉红色）与E -选择素免疫球蛋白治疗（蓝色）或未经处理的细胞（绿色）。具体CD11a单克隆抗体染色所示的实线和虚线所示无关亚型对照抗体染色。与E -选择素- IG（所有洗涤步骤，在流式细胞仪缓冲）处理的细胞没有影响CD11a密度，与未经处理的细胞相比。 B组显示密度定量的过程。 LOG10计算的平均荧光强度（FI）的每四个校准珠小组直方图峰值（粉红色圆圈）和（制造商）为每珠的很多特定的PE分子。一个线性回归LOG10 PE分子每珠对LOG10 fluorescENCE绘制。 E -选择素处理的细胞的LOG10 FI（Y）值等于3.99（蓝色实心圆）和2.23（蓝色空心圆），为特定的人与生物圈计划和控制抗体，respectivly。我们解决了X（值B组的绿色和蓝色圆圈绘制）的线性方程X = LOG10 PE /细胞，如PE：单克隆抗体的比例为1:1，α大号β2中性粒细胞总数计算为9587。面密度计算为43个分子/ 微米 2，中性粒细胞直径 228.4μm 。 ICAM - 1的密度同样测流cytometery使用PE抗人CD54的单克隆抗体，这相当于65摩尔/微米2。 <img alt="图2" src="/files/ftp_upload/3519/3519fig2.jpg" /> 图2 微管系统原理图 。我们的微管系统是在内部装配和三个子系统组成：影像子系统，让一个观察，建议条例和分析微量吸气细胞的运动;一个显微子系统，使一个选择的细胞从细胞室，压力子系统，让吸进微量的细胞。成像子系统的核心部分是一个100X的油浸1.25 NA目标倒置显微镜（奥林巴斯IMT - 2 IMT2）。通过电荷耦合器件（CCD）摄像机，图像传送到录像机。视频定时器耦合系​​统的跟踪时间。每个微管可以由机械驱动器安装在显微镜和精细定位与三轴液压显微操纵。以及可用于从新港机械造。微量持有人之一，是安装在压电翻译，其中的驱动程序是通过电压放大器（自制），压电执行器控制计算机LabVIEW代码（根据要求提供），并通过数据采集板的信号转换。这是一个llows之一附着力测试周期的移动吸管，精确和可重复。压力调节子系统是用来控制在实验过程中的吸力。液压线连接到储液罐的微量持有人。一个优良的机械定位，使水库的高度精确的操纵。 微量移液器使用KIMAX熔点高硼硅玻璃（外径1.0 ± 0.07毫米，内径0.7 ± 0.07毫米毛细管生成首先，从毛细管微量使用PN - 30 Narishige“磁性玻璃微电极技术水平拖轮拉（萨特仪器微管拖轮是另一个拉马选项）。二，Microforge系统（造房子）是用来切割到所需的大小开幕微量。Microforge系统的商业模式以及。 为了避免在实验过程中，显微的振动的微量OPE，以及与micromanipulators，被放置在一个空气悬架表。 <img alt="图3" src="/files/ftp_upload/3519/3519fig3.jpg" /> 图3运行粘附频率 F 我具体（一）和非特异性（二）1（红色）和具有约束力10S（蓝色）接触测量与ICAM - 1（A）或hIgG涂层的红细胞粘附之间的反复测试周期的时间（二人中性粒细胞表达整合的α大号β2）F的第 I =（X 1 + X 2 + ... ... + X I）/ I（1≤我≤N），其中 i是试验周期指数，X i等于“1 “ （粘连）或“0”（无粘连），F N（N = 50）被用来作为最佳估计粘连的概率。 <img alt="图4" src="/files/ftp_upload/3519/3519fig4.jpg" /> 图4动力学ICAM - 1结合，以中性粒细胞整合素α 大号β2（ <img alt="广场" src="/files/ftp_upload/3519/3519sq1.jpg" /> ）。附着力的概率测量在每个接触时间的细胞对图3所示是平均，并与接触时间绘制。腔介质为1MM的Ca 2 +和Mg 2 +加1μg/ml的二聚体E -选择素免疫球蛋白的HBSS上调α 大号β2结合。为了捕捉ICAM - 1的免疫球蛋白，对红细胞增加了一个中间步骤，孵化链亲和素孵育阶段后的捕获抗体（生物素化山羊抗人Fc的抗体，eBioscience公司）10μg/ml红细胞。为了控制非特异性结合两种不同的条件：1）红细胞与抗人FC捕获抗体涂层和ICAM - 1 - IG（海外）和2）中性粒细胞到红细胞结合，而不是与人IgG孵育与涂层捕获抗体（Δ）。 10μg/ml人IgG添加到中期，以尽量减少E的有约束力- 选择免疫球蛋白溶液中的红细胞表面上的捕获抗体。非特异性结合人IgG控制曲线记录被用来获得一个特定的粘附概率曲线（ <img alt="广场" src="/files/ftp_upload/3519/3519sq2.jpg" /> ）式。 2。配件特异性粘附EQ概率曲线。 1（实线）返回有效的结合亲和力一个 c K = 1.4•10 -4μm4 和 K关闭= 0.3 秒 -1 。 <table border="1"><tbody><tr><th>试剂</th><th>兆瓦（克/摩尔） </th><th>浓度（毫米） </th><th>量（g） </th></tr><tr><td>腺嘌呤</td><td> 135.13 </td><td> 2 </td><td> 0.27 </td></tr><tr><td> D -葡萄糖（葡萄糖） </td><td> 180.16 </td><td> 110 </td><td> 19.82 </td></tr><tr><td> D -甘露醇</td><td> 182.17 </td><td> 55 </td><td> 10.02 </td></tr><tr><td>氯化钠（NaCl） </td><td> 58.44 </td><td>50 </td><td> 2.92 </td></tr><tr><td>磷酸钠，磷酸氢二铵（NA HPO ） </td><td> 141.95 </td><td> 20 </td><td> 2.84 </td></tr><tr><td> L -谷氨酰胺</td><td> 146.15 </td><td> 10 </td><td> 1.46 </td></tr></tbody></table> 表1的EAS - 45缓冲液配制（1L）。 <table border="1"><tbody><tr><td>在550μL的DMF XHS 25毫克生物素</td><td> 0.1M素的解决方案</td></tr><tr><td> 0.1生物素W / DMF 1:10稀释</td><td> 0.01M素溶液</td></tr><tr><td> 1:100稀释的0.1素W / DMF </td><td> 0.001M素溶液</td></tr></tbody></table> 表2。制备生物素的解决方案。 <table border="1"><tbody><tr><th> 素终浓度（微米） </th><th> 4 </th><th> 10 </th><th> 20 </th><th> 50 </th><th> 100 </th><th> 160 </th></tr><tr><td>红细胞颗粒股票（μL） </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 10 </td></tr><tr><td> 1X PBS（μL） </td><td> 179.2 </td><td> 178 </td><td> 176 </td><td> 179 </td><td> 178 </td><td> 176.8 </td></tr><tr><td> 0.1M硼酸缓冲液（μL） </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 10 </td></tr><tr><td> 0.01M素溶液（μL） </td><td></td><td></td><td></td><td> 1 </td><td> 2 </td><td> 3.2 </td></tr><tr><td> 0.001M素溶液（μL） </td><td> 0.8 </td><td> 2 </td><td> 4 </td><td></td><td></td><td></td></tr></tbody></table> 表3。红细胞生物素。

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D., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+MHC-CD8+interaction+at+the+T+cell+membrane.">Kinetics of MHC-CD8 interaction at the T cell membrane.</a> J. Immunol. 179, 7653-7662 (2007).</li><li>Wasserman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+variant+peptide-mediated+anergy+of+encephalitogenic+T+cells+requires+SHP-1.">MHC variant peptide-mediated anergy of encephalitogenic T cells requires SHP-1.</a> J. Immunol. 181, 6843-6849 (2008).</li><li>Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinetics+of+two-dimensional+TCR+and+pMHC+interactions+determine+T-cell+responsiveness.">The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness.</a> Nature. 464, 932-936 .</li><li>Sabatino, J. J., Huang, J., Zhu, C., Evavold, 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=High+prevalence+of+low+affinity+peptide-MHC+II+tetramer-negative+effectors+during+polyclonal+CD4++T+cell+responses.">High prevalence of low affinity peptide-MHC II tetramer-negative effectors during polyclonal CD4+ T cell responses.</a> J. Exp. Med. 208, 81-90 (2011).</li><li>Jiang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-stage+cooperative+T+cell+receptor-peptide+major+histocompatibility+complex-CD8+trimolecular+interactions+amplify+antigen+discrimination.">Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination.</a> Immunity. 34, 13-23 (2011).</li><li>Spillmann, C. M., Lomakina, E., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesive+contact+dependence+on+impingement+force.">Neutrophil adhesive contact dependence on impingement force.</a> Biophys. J. 87, 4237-4245 (2004).</li><li>Zhu, C., Williams, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+concurrent+binding+of+multiple+molecular+species+in+cell+adhesion.">Modeling concurrent binding of multiple molecular species in cell adhesion.</a> Biophys. J. 79, 1850-1857 (2000).</li><li>Downey, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retention+of+leukocytes+in+capillaries:+role+of+cell+size+and+deformability.">Retention of leukocytes in capillaries: role of cell size and deformability.</a> J. Appl. Physiol. 69, 1767-1778 (1990).</li><li>Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+and+kinetic+analysis+of+Fcgamma+receptor+IIIa+(CD16a)+binding+to+IgG+ligands.">Affinity and kinetic analysis of Fcgamma receptor IIIa (CD16a) binding to IgG ligands.</a> J. Biol. Chem. 282, 6210-6221 (2007).</li><li>Chen, W., Zarnitsyna, V. I., Sarangapani, K. K., Huang, J., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Receptor-Ligand+Binding+Kinetics+on+Cell+Surfaces:+From+Adhesion+Frequency+to+Thermal+Fluctuation+Methods.">Measuring Receptor-Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods.</a> Cell. Mol. Bioeng. 1, 276-288 (2008).</li><li>Thoumine, O., Kocian, P., Kottelat, A., Meister, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+binding+of+fibroblasts+to+fibronectin:+optical+tweezers+experiments+and+probabilistic+analysis.">Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis.</a> Eur. Biophys. J. 29, 398-408 (2000).</li><li>Ounkomol, C., Xie, H., Dayton, P. A., Heinrich, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+horizontal+force+probe+for+mechanical+tests+on+pipette-held+cells,+particles,+and+membrane+capsules.">Versatile horizontal force probe for mechanical tests on pipette-held cells, particles, and membrane capsules.</a> Biophys. J. 96, 1218-1231 (2009).</li><li>Piper, J. W., Swerlick, R. A., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+force+dependence+of+two-dimensional+receptor-ligand+binding+affinity+by+centrifugation.">Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.</a> Biophys. J. 74, 492-513 (1998).</li><li>Li, P., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+competition+binding+between+soluble+and+membrane-bound+ligands+for+cell+surface+receptors.">Analysis of competition binding between soluble and membrane-bound ligands for cell surface receptors.</a> Biophys. J. 77, 3394-3406 (1999).</li><li>Long, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probabilistic+modeling+of+rosette+formation.">Probabilistic modeling of rosette formation.</a> Biophys. J. 91, 352-363 (2006).</li><li>Lou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-enhanced+adhesion+regulated+by+a+selectin+interdomain+hinge.">Flow-enhanced adhesion regulated by a selectin interdomain hinge.</a> J. Cell. Biol. 174, 1107-1117 (2006).</li><li>Yago, T., Zarnitsyna, V. I., Klopocki, A. G., McEver, R. P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+governs+flow-enhanced+cell+tethering+through+L-selectin+at+threshold+shear.">Transport governs flow-enhanced cell tethering through L-selectin at threshold shear.</a> Biophys. J. 92, 330-342 (2007).</li><li>Evans, E., Berk, D., Leung, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detachment+of+agglutinin-bonded+red+blood+cells.+I.+Forces+to+rupture+molecular-point+attachments.">Detachment of agglutinin-bonded red blood cells. I. Forces to rupture molecular-point attachments.</a> Biophys. J. 59, 838-848 (1991).</li><li>Zarnitsyna, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+in+receptor-ligand-mediated+cell+adhesion.">Memory in receptor-ligand-mediated cell adhesion.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 18037-18042 (2007).</li></ol>

没有利益冲突的声明。

要成功使用微管粘附频率检测应考虑的几个关键步骤。首先，确保记录的具体利益的受体 - 配体系统的相互作用。非特异性控制测量（参见图3，4）保证的特殊性。理想的情况下，非特异性黏附概率应低于0.05，对所有接触的时间段，每个时间点之间的特异性和非特异性黏附概率有显著差异。不同的方法可以用来夫妇的红细胞表面的配体。结果表明，氯化铬耦合方法17了非特异性结合的生物...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录＃ </th><th>评论</th></tr><tr><td> 10X PBS </td><td> BioWhittaker </td><td> 17 - 517Q </td><td>用去离子水稀释到1X使用前</td></tr><tr><td> Vacutainer EDTA </td><td>屋宇署</td><td> 366643 </td><td>红细胞隔离</td></tr><tr><td> 10ML PK100 </td><td></td><td></td><td></td></tr><tr><td> Histopaque 1077 </td><td> Sigma - Aldrich公司</td><td> 10771 </td><td>红细胞隔离</td></tr><tr><td>腺嘌呤</td><td> Sigma - Aldrich公司</td><td> A2786 </td><td> EAS - 45准备</td></tr><tr><td> D -葡萄糖（葡萄糖） </td><td> Sigma - Aldrich公司</td><td> G7528 </td><td> EAS - 45准备</td></tr><tr><td> D -甘露醇</td><td> Sigma - Aldrich公司</td><td> 6360 </td><td> EAS - 45准备</td></tr><tr><td>氯化钠（NaCl） </td><td> Sigma - Aldrich公司</td><td> S7653 </td><td> EAS - 45准备</td></tr><tr><td>磷酸三钠，磷酸氢二铵（娜＆＃X2082; HPO₄） </td><td> Fisher Scientific则</td><td> S374 </td><td> EAS - 45准备</td></tr><tr><td> L -谷氨酰胺</td><td> Sigma - Aldrich公司</td><td> G5763 </td><td> EAS - 45准备</td></tr><tr><td>生物素 - X - NHS </td><td> Calbiochem </td><td> 203188 </td><td>红细胞生物素</td></tr><tr><td>二甲基甲酰胺（DMF） </td><td> Thermo Scientific的</td><td> 20673 </td><td>红细胞生物素</td></tr><tr><td>硼酸盐缓冲液（0.1M） </td><td>电子显微镜科学</td><td> 11455-90 </td><td>红细胞生物素</td></tr><tr><td>链霉亲和素</td><td> Thermo Scientific的</td><td> 21125 </td><td>配体functionalizing </td></tr><tr><td>牛血清白蛋白</td><td> Sigma - Aldrich公司</td><td> A0336 </td><td>配体functionalizing </td></tr><tr><td> Quantibrite体育珠</td><td> BD公司</td><td> 340495 </td><td>密度定量</td></tr><tr><td>流式细胞仪</td><td>屋宇署Immunocytometry系统</td><td> BD LSR II </td><td>密度泉tification </td></tr><tr><td>毛细管 0.7 - 1.0MM × 30“ </td><td>金布尔玻璃公司</td><td> 46485-1 </td><td>微管拉</td></tr><tr><td>矿物油</td><td> Fisher Scientific则</td><td> BP2629 - 1 </td><td>商会大会</td></tr><tr><td>显微镜盖玻片</td><td> Fisher Scientific则</td><td> 12 - 544 - G </td><td>商会大会</td></tr><tr><td>聚乙烯α-人类CD11a 克隆您好111 </td><td> eBioscience公司</td><td> 12-0119-71 </td><td>试剂图</td></tr><tr><td> PE抗人类CD54的</td><td> eBioscience公司</td><td> 12-0549 </td><td>试剂图</td></tr><tr><td>鼠IgG1同型对照PE </td><td> eBioscience公司</td><td> 12-4714 </td><td>试剂图</td></tr><tr><td>液压显微</td><td> Narishige </td><td> MO - 303 </td><td>微管系统</td></tr><tr><td>机械手</td><td>新港</td><td> 461 - XYZ - M，SM - 13，DM - 13 </td><td>微管系统</td></tr><tr><td>压电翻译</td><td>玛格力Instrumente </td><td> P - 840 </td><td>微管系统</td></tr><tr><td> LabVIEW的</td><td>美国国家仪器公司</td><td> 8.6版</td><td>微管系统</td></tr><tr><td> DAQ板</td><td>美国国家仪器公司</td><td> USB - 6008 </td><td>微管系统</td></tr><tr><td>光学平台</td><td>动力学系统</td><td> 5200系列</td><td>微管系统</td></tr></tbody></table>

adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface

微管黏附实验是在1998年开发的，衡量的二维（2D）受体-配体结合动力学。检测使用传感器粘附细胞相互作用的分子之一，并提出一个人的红细胞（RBC）的。它采用显微操作与另一个细胞的表达与精确控制的区域和时间，使债券形成的其他相互作用的分子接触，使红细胞。红细胞伸长后发现，除了拉动两个细胞黏附事件。通过控制固定的红细胞表面上的配体的密度，粘连的概率保持在0和1之间的中档。粘连的概率估计在两个细胞之间的一个给定的接触时间的反复接触，周期序列的粘附事件的频率。不同的接触时间，产生一个具有约束力的曲线。拟合概率模型的结合受体-配体反应动力学1曲线返回2D的亲和力和关闭率。 该试剂盒已通过验证，使用与IgG的Fc 段 1-6，选择素与糖缀合物的配体6-9，与配体10-13 homotypical cadherin 的约束力14，T细胞受体与辅助受体肽主要组织相容性复合物15整合Fcγ 受体的相互作用- 19。 该方法已用于生物物理因素，如膜microtopology 5，膜锚2，分子取向和长度为6，承运人刚度9，曲率20，和撞击力20，以及生化因素，量化的二维动力学的规定，如调制的骨架和膜的微环境相互作用的分子， 这些分子表面，15,17,19组织。 该方法也被用来吨Ø研究并发约束力的双种受体-配体3,4，和19三分子相互作用使用修改后的模型 21 。 该方法的主要优点是，它允许在他们的母语膜环境受体的研究。结果可能会非常不同，从获得使用纯化的受体 17 。它还允许在时间分辨率，远远超出了典型的生化方法分一秒的时间尺度的受体 - 配体相互作用的研究。 要说明的微量粘附频率的方法，我们动力学测量间粘附分子-1（ICAM - 1）红细胞约束力的解决方案中的E -选择与二聚体的中性粒细胞激活α大号β2整合素α大号β2功能。

Watch this Scientific Journal Video about Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface at JoVE.com

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

粘附频率检测，用于测量当两个分子相互作用的细胞表面锚定受体 - 配体相互作用的动力学描述。使用一个微管加压作为粘附传感器的人体红血细胞和整合αLβ2和细胞间粘附分子-1受体和配体相互作用，这种机械为基础的检测例证。

粘附频率含量为原位跨交界的分子相互作用，在细胞与细胞界面的动力学分析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

adhesion-frequency-assay-for-situ-kinetics-analysis-cross-junctional

Research

JoVE Journal

Bioengineering

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

14.9K 次观看.  Georgia Institute of Technology. 粘附频率检测，用于测量当两个分子相互作用的细胞表面锚定受体 - 配体相互作用的动力学描述。使用一个微管加压作为粘附传感器的人体红血细胞和整合αLβ2和细胞间粘附分子-1受体和配体相互作用，这种机械为基础的检测例证。

视频: Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

生物电子界面衔接与Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

釉原蛋白 - 壳聚糖水凝胶的发展<em&gt;体外</em&gt;搪瓷愈合了密集接口

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

测量TCR-的pMHC结合<em&gt;原位</em&gt;使用基于FRET的显微镜分析

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

一种三肽稳定的油酸纳米乳液

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

预测神经植入物软化行为的环境动力力学分析

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

澳大利亚同步加速器串行晶体测量的利皮迪科注射协议

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

分子弹簧常量分析由生物元原力探针光谱

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...