Name: live-cell imaging of platelet degranulation and secretion under flow
Uploaded: 2017-07-10T10:00:00.000+00:00
Duration: 11 min 42 s
Description: Watch this Scientific Journal Video about Live-cell Imaging of Platelet Degranulation and Secretion Under Flow at JoVE.com

CM承认国际患者组织对C1抑制剂缺乏症（HAEi），Stichting Vrienden van Het UMC Utrecht和Landsteiner输血研究基金会（LSBR）的财政支持。

乌特勒支大学医学中心的当地医学伦理委员会批准了用于离体研究的血液，包括本研究的血液。 解决方案准备<ol><li> 通过溶解10mM HEPES，0.5mM Na 2 HPO 4，145mM NaCl，5mM KCl，1mM MgSO 4和5.55mM D-吡喃半乳糖苷，制备4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）葡萄糖在蒸馏水中。 <ol><li>制作缓冲液的两个变体:将pH值分别设置为6.5和7.4。为每一天的实验做新鲜的缓冲。补充10ng / mL前列环素。 </li></ol></li><li>通过将50mM Tris和150mM NaCl溶解在蒸馏水中制备TRIS缓冲盐水（TBS）并将pH调节至9.0。 </li><li>通过将85mM柠檬酸三钠，71mM柠檬酸和111mM D-葡萄糖溶解在dis中制备柠檬酸柠檬酸葡萄糖（ACD）耕种水</li><li>通过向HEPES Tyrode缓冲液（pH 7.4）中加入2％（v / v）多聚甲醛（PFA）来制备固定液。加热溶解（50-70℃）并将pH设定为7.4。等分并储存在-20°C。使用前在37°C解冻。 </li><li>通过将1％（m / v）人血清白蛋白（HSA）溶解在HEPES Tyrode缓冲液（pH 7.4）中来制备封闭缓冲液</li><li> 通过向12g甘油中加入4.8g聚乙烯醇制成聚乙烯醇溶液并充分混合。加入12 mL蒸馏水，并在室温下放置在旋转器上过夜。 <ol><li>加入24 mL 0.2 M Tris-HCL，pH 8.5。加热至50℃同时混合30分钟。加入1.25g 1,4-二氮杂双环[2.2.2]辛烷（DABCO）并充分混合。以1,500 xg离心5分钟。将上清液等分（1mL足以用于15-20个载玻片）并保存在-20℃。使用等分试样一次。 </li></ol></li></ol> 2.盖玻璃准备<ol><li>脱脂盖玻璃（25×50mm 2 ）通过在未稀释的染色硫酸中孵育过夜。用蒸馏水冲洗盖玻片，并在烘箱中在60℃下将其干燥过夜。 </li><li>在一个实验台上，用一滴水粘在一块石蜡膜（10 x 15 cm 2 ）上，并通过平滑石蜡膜去除空气。将80μL10μg/ mL VWF或100μg/ mL纤维蛋白滴滴至石蜡膜上。将眼镜放在水滴上;蛋白质溶液将在两个表面之间扩展以覆盖玻璃的整个背面。在室温（RT）孵育1.5小时。 </li><li>取出石蜡膜，然后放置盖玻片，将涂层面朝上放在带有阻塞缓冲液的4孔板中。在37℃下孵育1小时或在4℃下孵育过夜。作为对照，阻挡未涂覆VWF或纤维蛋白原的玻璃罩。 </li></ol> 3.制备富血小板血浆（PRP） </p&gt; <ol><li>通过静脉穿刺收集柠檬酸盐的全血（终浓度:0.32％柠檬酸钠（M / V））。 </li><li>以160 xg离心血液15分钟，无刹车离心。用塑料巴斯德吸管收集上清液（ 即 PRP）。 注意:离心后，可以观察到三层（从上到下）:PRP，白细胞和红细胞。确保不要包含白色和红色的单元格。通常，在离心9-mL血管后可以收集3mL的PRP。 </li><li>如果实验将用洗涤的血小板进行，继续进行第4部分。如果需要PRP，请继续下一步。 </li><li>在收集PRP后，将剩余的血液（含有红细胞颗粒）在2,000 xg下再次离心10分钟，无需制动。收集含血小板血浆（PPP）的上清液。 注意:通常，在第二次离心步骤后可以收集2 mL。 </li><li>计数p的数量在细胞分析仪中的PRP中的小肠，并用PPP（步骤3.4）将其稀释至浓度为150,000血小板/μL。 注:稀释PRP的总体积取决于供体的血小板计数。供体之间的血小板计数差异很大，PRP稀释需要单独评估。血小板计数在15万和45万血小板/μL之间被认为是正常的。 </li><li>继续到第5节。 </li></ol> 4.洗涤血小板的制备<ol><li>使用细胞分析仪确定PRP中的平均血小板体积（MPV）。 注意:这是血小板预激活的指标11 。静息血小板的正常MPV为7.5-11.5 fL 11 。在血小板清洗过程中，MPV应不超过1.5 fL。 </li><li>向PRP添加1/10体积的ACD以防止血小板预激活，并以400 xg离心15分钟，无刹车。删除su并在HEPES Tyrode的缓冲液pH 6.5中小心地将沉淀重新悬浮至PRP的原始体积。 </li><li>通过加入TBS至10μg/ mL的浓度来解冻前列环素的等分试样;保持在冰上。 </li><li>为了防止血小板活化，将最终浓度为10ng / mL的前列腺素加入重悬浮的血小板中，并立即以400xg离心15分钟并在无刹车的情况下离心15分钟。取出上清液，并将细胞小心重新悬浮于HEPES Tyrode缓冲液（pH 7.4）中。 </li><li>计算血小板数（步骤3.5）并确定MPV的变化（步骤4.1）（MPV不应改变超过1.5fL）。 </li><li>使用HEPES Tyrode缓冲液（pH 7.4）调节血小板数，达到150,000血小板/μL的浓度。在实验前，让血小板在室温下恢复30分钟。分离后4小时内用洗涤的血小板进行实验。 </li></ol>流动室总成12 。可以使用各种商业生产的流动室，只要它们可以容纳玻璃盖，玻璃盖优先是可移除的，用于进一步的分析。用于所述实验的流动室由聚（甲基丙烯酸甲酯）制成，以精确地配合显微镜插入台。它包含入口和出口（ 图1A - C ; 1,2），以及真空连接（ 图1A和C ; 3）。定制的硅胶片（ 图1A和D ; 4）放置在顶部。两个切口形成真空通道（ 图1A和D ; 5），以将盖玻璃（ 图1A ; 6）紧紧地连接到腔室。中心切口形成流动通道（ 图1A </strong&gt;和D ; 7; 2.0mm宽和0.125mm高）。入口和出口连接到流动通道，真空连接到真空通道。 <ol><li>用蒸馏水覆盖流动室的顶部，并将定制的硅胶片放置在水面上。通过清除多余的水将片材浮起就位，同时保持片材的位置。 注意:这种硅胶片是可重复使用的（有机硅是惰性的）;一般来说，没有观察到附着于其上的血小板。纸张可以用清洁剂清洁并重新使用，直到材料损坏（ 即撕裂，特别是在通道之间的区域）。通常，片材可以使用20天，每天多次实验。 </li><li>在60℃下将流动室孵育1小时以蒸发残留的水以将硅胶片牢固地粘附到流动室。 </li><li>用塑料巴斯德吸管，加一滴HEPES Tyrode的缓冲液，pH 7.4，流入通道。将盖玻片（见第2部分）与涂层面朝下放在流道上。将真空泵连接到真空接头（ 图1A和C ; 3）。施加〜10 kPa压力。 </li></ol> 6.血小板的预染色和抗体的制备<ol><li> 血小板染色 <ol><li>使用10μg/ mL DAPI在37℃下在黑暗中孵育洗涤的血小板30分钟。为了洗去未结合的DAPI并防止血小板活化，加入终浓度为10 ng / mL的前列环素，立即以400 xg离心15 min，无刹车。 </li><li>将HEPES Tyrode缓冲液（pH 7.4）中的血小板沉淀重建为血小板浓度为150,000血小板/μL。 </li></ol></li><li> 制备抗体 <ol><li>将生物素标记的抗体与Alexa标记的链球菌混合tavidin，摩尔比为1:1。使用前在室温下孵育至少10分钟（步骤7.6）。 注意:生物素标记效率可能因不同批次而异。如果目标分子的可视化存在问题，则应进行对照实验以确认成功的生物素化，以及特异性生物素化抗体的靶结合特性。 </li></ol></li></ol>灌注<ol><li>启动荧光显微镜，以及显微镜软件。使用表1中列出的显微镜设置。 注意:通过在显微镜软件中加载适当的实验，启动显微镜和记录活细胞可视化和记录血小板和选定的荧光标记的抗体和/或染料的设置。在室温下进行流动实验。 </li><li>通过将10 mL注射器连接到管道上并吸入1％HSA blo来阻塞入口管将缓冲液浸入入口管中。在室温下孵育15分钟。通过将HEPES Tyrode缓冲液（pH 7.4）吸入入口管中冲洗入口管。 </li><li>将堵塞的入口管和未处理的出口管连接到腔的入口和出口，直径为1.02mm，长度为〜20-30cm。将10 mL注射器（或更低的体积更精确）连接到出口管。打开注射泵。 注意:注射器的直径与泵上设定的流速相结合确定体积流量。与流路的表面积一起，可以根据公式13计算剪切速率 下面。因此，可以通过改变注射泵的流量或改变流动通道的尺寸来操纵剪切速率。 800-1,600 s -1的剪切速度通常是动脉血流模型，而静脉血的模型为25 - 100 s-1流。对于该设置，7.5μL/ min的流速导致剪切速率为25s -1 。 剪切速率= sQ中的6Q / h 2 w 其中:Q =体积流量（mL / s），h =高通道（cm）（0.0125cm;这是硅胶片的厚度），w =宽度通道（cm）（室中0.2cm）。 </li><li>将一滴浸油放在100X物镜上（总放大1000倍），并将玻璃表面放置在显微镜上。 </li><li>将入口管放入含有HEPES Tyrode缓冲液（pH 7.4）的管中。手动拉动连接到出口管的注射器的柱塞，并将溶液拉出室，以从系统中除去空气。将注射器放入注射泵（ 图1B 和C ; 8）中，然后短暂运转，拧紧柱塞，确保稳定的流量。 </li><li>小心地向血小板悬浮液或PRP添加抗体和/或染料与塑料移液器混合，并将混合物转移到1.5-mL管中。 注意:对于剪切速率25 s-1 （7.5μL/ min）的典型30分钟灌注，需要至少225μL血小板悬浮液。用于代表性结果的抗体显示在表2中 。 </li><li>将入口管道从HEPES Tyrode缓冲液（pH 7.4）挤压到含有PRP或洗涤过的血小板以及抗体和/或染料（ 图1B 和C ; 9）的1.5-mL管中。 注意:重要的是，在传送管道时，挤压管道（挤出空气），以防止空气进入管道。通过将入口管以类似的方式移动到另一管，可以在随后的步骤中用各种试剂处理粘附的血小板。 表3中列出了潜在有用的试剂列表。 </li><li>选择"现场可视化"n"选项，将显微镜放置在涂覆的盖玻璃的表面，并以1,600 s-1 （484μL/ min）的剪切速率启动泵，直到血小板到达流动室，此时，通过将注射泵的设置改变为7.5μL/ min的流量，将剪切速率降低到25 s-1 。 </li><li>监控盖玻片的涂层表面，等到第一个血小板开始粘附，将显微镜聚焦在该血小板上，并通过在软件中启动实验开始记录。这里使用的记录设置见表1 。 </li><li>直观地跟踪现场录音。 30分钟后，停止注射泵。 注意:确保在实验过程中血小板保持对焦。通常，20-30个血小板以1000倍的倍率附在视野内;这在整个流动通道中约为2,000-3,000个血小板。 </li><li> 固定使用流动室（可选）。 <ol><li>当注射泵停止后流动停止时，将进样管（无空气）转移到固定液中。重新启动泵，并以与步骤7.8中所述相同的流速将固定缓冲液流过通道至少10分钟。 </li></ol></li><li>从显微镜中取出腔室，清洁玻璃上的浸油，断开真空，并用18 G针小心地从室中取出盖玻片。 注意:防止盖玻片破损和损坏硅片。 </li><li>将盖子滑块（细胞向上）放在组织顶部的4孔板中，用蒸馏水预先润湿。 注意:这样可以防止盖玻片粘附到孔表面并防止干燥。 </li><li>在盖玻片顶部吸取750μL固定液，室温孵育1 h。如果样品不能直接分析，在4℃下将其储存在0.5％PFA（由HEPES Tyrode缓冲液，pH7.4稀释）中，以确保稳定的固定。继续执行第8节处理遮盖玻璃进行成像。 </li><li>使用后，用蒸馏水冲洗腔室，管道和片材，并在洗涤剂溶液中孵育过夜。在重新使用腔室和其他部件之前，用蒸馏水冲洗。 </li><li>保存包含所有元数据的显微镜原始数据文件。需要时，将电影导出为.MOV，h.264压缩或.AVI未压缩。将单独的帧保存为JPEG或TIF文件。 </li></ol> 8.共聚焦荧光显微镜的样品制备<ol><li>将盖玻片转移到新的4孔板上，并用3 mL HEPES Tyrode缓冲液（pH 7.4）冲洗5次。加入3 mL /孔封闭缓冲液，室温孵育1 h。 </li><li>去除阻塞缓冲区;在HEPES Tyrode缓冲液pH 7.4中加入3 mL /孔的0.15％甘氨酸（M / V）;并在R孵育15分钟T. </li><li>去除甘氨酸溶液，并在3 mL /孔封闭缓冲液中洗5次。任选地，加入在封闭缓冲液中稀释的荧光团缀合的抗体以进行染色。在室温下在黑暗中孵育1小时。 </li><li>用阻塞缓冲液洗涤5次，用蒸馏水洗2次。通过用组织（从边缘向内）去除多余的液体，而不用接触细胞来干燥玻璃。 </li><li>将60μL聚乙烯醇溶液移至显微镜载玻片上。将盖玻片放在玻璃瓶的顶部，让聚乙烯醇在两个表面之间展开。在黑暗中在室温过夜孵育。 </li><li>通过共聚焦显微镜分析细胞14 。 </li><li>保存包含所有元数据的记录堆栈数据的原始数据文件。需要时，将原始数据文件处理为MOV，h.264压缩或.AVI未压缩文件。将单独的堆栈保存为JPEG或TIF文件。 </li></ol>

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G., van Zanten, H., IJsseldijk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+perfusion+chamber+to+detect+platelet+adhesion+using+a+small+volume+of+blood.">A new perfusion chamber to detect platelet adhesion using a small volume of blood.</a> Thromb Res. 92, S43-S46 (1998).</li><li>Slack, S. M., Turitto, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+chambers+and+their+standardization+for+use+in+studies+of+thrombosis.+On+behalf+of+the+Subcommittee+on+Rheology+of+the+Scientific+and+Standardization+Committee+of+the+ISTH.">Flow chambers and their standardization for use in studies of thrombosis. On behalf of the Subcommittee on Rheology of the Scientific and Standardization Committee of the ISTH.</a> Thromb Haemost. 72 (5), 777-781 (1994).</li><li>Oreopoulos, J., Berman, R., Browne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinning-disk+confocal+microscopy:+present+technology+and+future+trends.">Spinning-disk confocal microscopy: present technology and future trends.</a> Methods Cell Biol. 123, 153-175 (2014).</li><li>Nishibori, M., Cham, B., McNicol, A., Shalev, A., Jain, N., Gerrard, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+CD63+is+in+platelet+dense+granules,+is+deficient+in+a+patient+with+Hermansky-Pudlak+syndrome,+and+appears+identical+to+granulophysin.">The protein CD63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlak syndrome, and appears identical to granulophysin.</a> J. Clin. Invest. 91 (4), 1775-1782 (1993).</li><li>Ruiz, F. A., Lea, C. R., Oldfield, E., Docampo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+dense+granules+contain+polyphosphate+and+are+similar+to+acidocalcisomes+of+bacteria+and+unicellular+eukaryotes.">Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes.</a> J. Biol. Chem. 279 (43), 44250-44257 (2004).</li><li>Tersteeg, C., Fijnheer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+von+Willebrand+Factor+under+Control:+Alternatives+for+ADAMTS13.">Keeping von Willebrand Factor under Control: Alternatives for ADAMTS13.</a> Semin Thromb Hemost. 42 (1), 9-17 (2016).</li><li>Tersteeg, C., de Maat, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmin+cleavage+of+von+Willebrand+factor+as+an+emergency+bypass+for+ADAMTS13+deficiency+in+thrombotic+microangiopathy.">Plasmin cleavage of von Willebrand factor as an emergency bypass for ADAMTS13 deficiency in thrombotic microangiopathy.</a> Circulation. 129 (12), 1320-1331 (2014).</li><li>Basir, A., de Groot, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Hemocompatibility+Testing+of+Dyneema+Purity+Fibers+in+Blood+Contact.">In Vitro Hemocompatibility Testing of Dyneema Purity Fibers in Blood Contact.</a> Innovations (Phila). 10 (3), 195-201 (2015).</li></ol>

作者没有什么可以披露的。

在世界范围内，血栓形成是死亡和发病的主要原因，血小板在其发展中起着核心作用。这项工作描述了流动下血小板脱颗粒活细胞成像的方法。通常假设当血小板活化时，所有颗粒内容物直接释放到溶液中。伴随的结果表明，情况并非如此。在粘附和脱颗粒过程中，血小板保留了大量的多磷酸盐（ 图4 ）。另外，多核蛋白VWF在脱粒后与血小板表面相关（ 图5 ）。这表明控...

血小板是在血液中循环的无核细胞。它们的主要功能是在损伤部位密封血管破裂并防止失血。在这些损伤部位，内皮下胶原纤维变得暴露，随后被多聚体蛋白质von Willebrand因子（VWF）覆盖。 VWF以依赖于细胞表面1上的糖蛋白Ibα-IX-V复合物的机制循环中与血小板相互作用，降低血小板的速度。这在高剪切速率下尤其重要。随后血小板在从胶原接受活化脉冲的同时进行形态学变化。这导致不可逆的扩散，最终导致血小板聚集。这两个过程都取决于颗粒物质的分泌以促进血小板 - 血小板的串扰。其中，血小板α颗粒含有纤维蛋白原和VWF，以帮助血小板粘附和桥接血小板以整联蛋白依赖的方式结合在一起。血小板致密颗粒含有无机化合物2 ，包括钙和二磷酸腺苷（ADP），有助于增强血小板活化。此外，血小板含有（过敏）炎症3 ，补体控制蛋白4和血管生成因子5,6的介质，提出了在不同条件下这些内容是否以及如何差异释放的问题。 自20世纪80年代以来，血流动力学模型的研究对血栓形成机制的研究具有重要意义。从那时起，已经取得了很大的技术进步，并且目前开发了包括纤维蛋白形成的流动模型来测定治疗性血小板浓缩物的离体电位离体 8或研究剪切速率扰动对血栓形态的影响9 。驱动稳定粘附和生理性血栓形成（止血）与病理性血栓形成（血栓形成）的分子和细胞 - 生物学机制的差异可能非常微妙，并且促进了允许这些亚细胞的实时可视化的流动模型的发展流程。 这种设置将有价值的过程的一个例子是细胞内多磷酸盐的（再）分布和凝血因子的募集，以揭示其对纤维蛋白超微结构的时间依赖性影响10 。研究往往局限于终点分析。所描述的方法的主要目的是使得能够在流动期间血小板激活期间进行的动态亚细胞过程的实时视觉调查。

<table><tbody><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)&amp;nbsp;</td><td>VWR</td><td>441476L</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S-0876</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>31434</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>31248</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck KGaA</td><td>1.05886</td><td></td></tr><tr><td>D-glucose</td><td>Merck KGaA</td><td>1.04074</td><td></td></tr><tr><td>Prostacyclin&amp;nbsp;</td><td>Cayman Chemical</td><td>18220</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>Merck KGaA</td><td>1.06448</td><td></td></tr><tr><td>Citric acid&amp;nbsp;</td><td>Merck KGaA</td><td>1.00244</td><td></td></tr><tr><td>Cover glasses</td><td>Menzel-Gl&amp;auml;ser</td><td>BBAD02400500#A</td><td>24 x 50 mm, No. 1 = 0.13 - 0.16 mm thickness.</td></tr><tr><td>Chromosulfuric acid (2% CrO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Riedel de Haen</td><td>07404</td><td>CAS [65272-70-0].</td></tr><tr><td>Von Willebrand factor (VWF)</td><td>in-house purified</td><td></td><td></td></tr><tr><td>Fibrinogen</td><td>Enzyme Research Laboratories</td><td>FIB3L</td><td></td></tr><tr><td>4 well dish, non-treated</td><td>Thermo Scientific</td><td>267061</td><td></td></tr><tr><td>Human Serum Albumin Fraction V</td><td>Haem Technologies Inc.</td><td>823022</td><td></td></tr><tr><td>Blood collection tubes, 9 mL, 9NC Coagulation Sodium Citrate 3.2%</td><td>Greiner Bio-One</td><td>455322</td><td></td></tr><tr><td>Cell analyser&amp;nbsp;</td><td>Abbott Diagnostics</td><td>CELL-DYN hematology analyzer</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>30525-89-4&amp;nbsp;</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus, Holliston, MA</td><td>Harvard apparatus 22</td><td></td></tr><tr><td>10 mL syringe with 14.5 mm diameter</td><td>BD biosciences</td><td>305959</td><td>Luer-Lok syringe</td></tr><tr><td>Anti-CD63-biotin&amp;nbsp;</td><td>Abcam&amp;nbsp;</td><td>AB134331</td><td></td></tr><tr><td>Anti-CD62P-biotin&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>Dy137</td><td></td></tr><tr><td>4&amp;rsquo;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>Polysciences Inc.&amp;nbsp;</td><td>9224</td><td></td></tr><tr><td>Streptavidin, Alexa Fluor 488 conjugate</td><td>Thermo Scientific</td><td>S11223&amp;nbsp;</td><td></td></tr><tr><td>Immersion oil</td><td>Zeiss</td><td>444963-0000-000</td><td></td></tr><tr><td>Detergent solution</td><td>Unilever, Biotex</td><td></td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>56-40-6&amp;nbsp;</td><td></td></tr><tr><td>Polyvinyl alcohol</td><td>Sigma</td><td>9002-89-5</td><td>Mowiol 40-88.</td></tr><tr><td>Tris hydrochloride</td><td>Sigma</td><td>1185-53-1&amp;nbsp;</td><td></td></tr><tr><td>1,4-Diazabicyclo[2.2.2]octane (DABCO)</td><td>Sigma</td><td>280-57-9</td><td></td></tr><tr><td>Sheep Anti-hVWF pAb</td><td>Abcam&amp;nbsp;</td><td>AB9378</td><td></td></tr><tr><td>Alexa fluor 488-NHS</td><td>Thermo Scientific</td><td>A20000</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>15523-1L-R</td><td></td></tr><tr><td>Parafinn film</td><td>Bemis</td><td>PM-996</td><td>4 in. x 125 ft. Roll.</td></tr><tr><td>Silicone sheet non-reinforced</td><td>Nagor</td><td>NA 500-1</td><td>200 mm x 150 mm x 0.125 mm.</td></tr><tr><td>Customized cut silicone sheet with perfusion and vacuum channels</td><td>in-house made</td><td></td><td>Made of Silicone sheet non-reinforced (Nagor, NA 500-1)</td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf AG</td><td>T9661-1000AE</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Zeiss Observer Z1&amp;nbsp;</td><td></td><td>Equiped with LED excitation lights.</td></tr><tr><td>Microscope software</td><td>Zeiss ZEN 2</td><td>blue edition</td><td></td></tr><tr><td>18 G needle (18 G x 1 1/2&quot;)</td><td>BD biosciences</td><td>305196</td><td></td></tr><tr><td>NaCl</td><td>Riedel de Haen</td><td>31248375</td><td></td></tr><tr><td>Tris</td><td>Roche</td><td>10708976</td><td></td></tr><tr><td>Plastic pasteur pipet</td><td>VWR</td><td>612-1681</td><td>&amp;nbsp;7 mL non sterile, graduated up to 3 mL.</td></tr><tr><td>Silicone tubing</td><td>VWR</td><td>228-0656</td><td>Inner diameter. x Outer diameter x Wall thickness = 1.02 x 2.16 x 0.57 mm.</td></tr><tr><td>Microscope slides</td><td>Thermo Scientific</td><td>ABAA000001##12E</td><td>76 x 26 x 1 mm, ground edges 45 &amp;deg;, frosted end.</td></tr></tbody></table>

live-cell imaging of platelet degranulation and secretion under flow

血小板是止血的重要参与者，形成血栓以密封血管性破裂。他们也参与血栓形成，形成阻塞脉管系统并损伤器官的血栓，具有威胁生命的后果。这激发了血小板功能的科学研究以及在流动条件下发生时跟踪细胞 - 生物过程的方法的发展。 血小板粘附和聚集研究的各种流动模型可用于血小板生物学的两个关键现象。这项工作描述了一种在激活期间研究流动下的实时血小板脱颗粒的方法。该方法利用与注射器 - 泵装置耦合的流动室，该注射器 - 泵装置放置在宽场倒置的基于LED的荧光显微镜下。这里描述的装置允许同时激发由荧光标记的抗体或荧光素递送的多个荧光团ent染料。在活细胞成像实验之后，可以使用静态显微镜（ 即共焦显微镜或扫描电子显微镜）进一步处理和分析覆盖玻璃。

图1显示了流动室的图像和实验装置;硅片的位置和尺寸;和管道连接。 图2提供了流动室的尺寸的细节。 图3和电影1显示血小板粘附和扩散在固定化VWF上的时间序列图像。 CD63是一种跨膜蛋白，其被插入静止血小板15的细胞内致密颗粒的膜中。其在血小板表面上的时间依赖性动?...

Watch this Scientific Journal Video about Live-cell Imaging of Platelet Degranulation and Secretion Under Flow at JoVE.com

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

这项工作描述了基于荧光显微镜的方法研究血小板粘附，扩散和分泌的流动。这种多功能平台能够调查血小板功能，用于血栓形成和止血的机械研究。

活细胞成像血小板脱水和分泌流量

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

live-cell-imaging-of-platelet-degranulation-and-secretion-under-flow

Research

JoVE Journal

Biology

11.5K 次观看.  University Medical Center Utrecht. 这项工作描述了基于荧光显微镜的方法研究血小板粘附，扩散和分泌的流动。这种多功能平台能够调查血小板功能，用于血栓形成和止血的机械研究。 

视频: Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

科研

生物工程

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

免疫与感染

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

根据流量的血小板粘附和聚集，使用微流控流细胞

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

生物学

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

用螺旋粘度计对人血小板和细胞表面受体进行均匀剪切检测

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

在维沃 小鼠头骨骨髓中巨细胞和前列腺的双光子成像

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

<meta charset="utf8"></head><body>评估创伤和输血中血小板功能的平台

Microfluidics in Assessing Platelet Function

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

<meta charset="utf8"></head><body>微流体评估血小板功能

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

微流体评估血小板功能

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying ...

活细胞成像血小板脱水和分泌流量

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此视频中的章节

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

根据流量的血小板粘附和聚集，使用微流控流细胞

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

用螺旋粘度计对人血小板和细胞表面受体进行均匀剪切检测

在维沃小鼠头骨骨髓中巨细胞和前列腺的双光子成像

微流体评估血小板功能

微流体评估血小板功能

活细胞成像血小板脱水和分泌流量

摘要

探索更多视频

此视频中的章节

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

根据流量的血小板粘附和聚集，使用微流控流细胞

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

用螺旋粘度计对人血小板和细胞表面受体进行均匀剪切检测

在维沃 小鼠头骨骨髓中巨细胞和前列腺的双光子成像

微流体评估血小板功能

微流体评估血小板功能

在维沃小鼠头骨骨髓中巨细胞和前列腺的双光子成像