这项工作是由来自瑞士国家科学基金会资助（310030_162579 / 1布伦达雷娜塔郭某和320030_144150皮埃尔丰塔纳），以及由来自瑞士心脏基金会的资助。

五名例健康志愿者招募采血进行血小板分离和聚集测试。获得书面知情同意书和协议批准了日内瓦大学医院的中央伦理委员会。认证所有的志愿者是健康的，并在至少试验前10天都没有采取任何血小板干扰药物。 1.缓冲液配制人类血液收集和洗涤过的血小板分离<ol><li>通过溶解1.4克柠檬酸一水合物（C 6 H 8 O 7•H 2 O，66.6毫摩尔），将2.5g柠檬酸三钠二水合物（的Na 3 C 6 H 5制备酸-柠檬酸盐-葡萄糖（ACD）的100毫升水溶液O 7•2 H 2 O，85毫摩尔）和2克无水D（+） -对葡萄糖。该溶液的pH为约4.5。 </li><li> 为蒂罗德缓冲准备股票方案如下<ol><li>通过溶解80克的NaCl，2g的氯化钾，10g的碳酸氢钠和0.58克的NaH 2 PO 4 * H 2 O在500ml的蒸馏水H 2 O的各自的最终浓度为2.73男，53.6毫米，238制备储备溶液1 mM和8.4毫米。保持在4℃下该溶液中。 </li><li>通过在500毫升10.15克的MgCl 2 * 6 H 2 O（100毫摩尔）蒸馏水H 2 O溶液保持在4℃下溶解制备储备溶液2。 </li><li>通过溶解10.95克（100毫米）的CaCl 2 * 6 H在500ml蒸馏水H 2 2 O O.保持溶液在4℃下制备储备溶液3。 </li></ol></li><li>通过在50毫升蒸馏水H 2 O的最终体积。这对应于136.5毫氯化钠，2.68毫米氯化钾，终浓度稀释2.5毫升的储备溶液1准备蒂罗德缓冲液11.9毫米的NaHCO 3和0.42毫摩尔的NaH 2 PO 4 * H 2 O.将pH调节至7.35，并通过与0.22μm的过滤˚F消毒ilters。 </li><li> 通过稀释5毫升储备溶液1准备台氏白蛋白0.35％缓冲液（TA缓冲器7），1毫升的储备溶液2,2毫升的储备溶液3，将0.5mL 1M HEPES，1.8毫升200g / L的人血清白蛋白的和0.1克无水D（+） -对在100毫升的最终体积葡萄糖蒸馏H 2 O. <ol><li>将pH调节至7.35，用1N的HCl，并通过加入蒸馏水H 2 O（总体积的10％）设定的摩尔渗透压浓度至295毫渗量/ L。最终浓度在该溶液中有:124毫米的NaCl，2.44毫摩尔的KCl，10.82毫的NaHCO 3，0.38毫摩尔的NaH 2 PO 4 * H 2 O，0.91毫摩尔MgCl 2 * 6 H 2 O，1.82毫摩尔CaCl 2的* 6 H 2 O ，保持在37℃TA缓冲器在整个实验期间。 </li></ol></li></ol> 2.采血车<ol><li> 收集45-50毫升静脉血，从使用19号针和无或低的止血带肘前静脉，到50 mL管CON泰宁ACD抗凝剂（1个体积ACD为6个体积的血液）。丢弃的血液第一1-2毫升，以避免凝血酶和组织因子的存在。 <ol><li>收集后，轻轻颠倒混合管与ACD血。在37℃下孵育10分钟的样品。 </li></ol></li></ol> 3.人的制备洗涤血小板<ol><li>预加热离心机至37℃。所有离心步骤如下在此温度下进行。 </li><li>调度所采集的血液成15 mL管（每管5mL）和离心机在250×g离心13分钟，得到PRP。 注意:此离心步骤导致产生的三个层的样品中:1）上层，血浆，血小板组成，和白血细胞的一小部分。 2）中间层，富含白细胞的部分。 3）底层，其基本上由红血细胞。 </li><li>通过吸取上层LA收集PRP揭掉仔细到一个新的15ml管中，以最大程度地防止污染与红和白血细胞，并在37℃下孵育10分钟。 </li><li>离心PRP在2200×g离心12分钟（5毫升PRP）。 注意:此离心步骤应与低速制动器或无制动来执行。 </li><li>除去上清液（贫血小板血浆），并使用塑料巴斯德吸管小心地重悬沉淀，用10毫升含2μL/ mL肝素的TA缓冲液（5000 U / mL）和25μMPGI 2的2.5μL/毫升。在37℃下孵育10分钟。 </li><li>添加2.5μL/ mL的25μMPGI 2和离心机在1900×g离心8分钟（与低速制动器或无制动）。 </li><li>除去上清，重悬与使用塑料巴斯德吸管含有2.5μL/ mL的25μMPGI 2的5毫升TA缓冲液中的沉淀物。在37℃下孵育10分钟。 </li><li>在温育期间，吸管的血小板悬浮液的150μL在到1.5mL管中并计数血小板，使用自动化的细胞计数器（即通过测量直流电阻的变化来检测血细胞的尺寸）。 </li><li> 10分钟温育后，加入2.5μL/ mL的25μMPGI 2与血小板悬浮液，并立即在1900×g下离心8分钟。 </li><li>去除上清，重悬沉淀至250,000血小板/μL的浓度与TA缓冲区的适当体积（ 即 ，如果细胞数为50万每μL，在10毫升TA缓冲液重悬）含有腺苷三磷酸双的32μL/ mL，在为0.01U /毫升（最终浓度0.32 U / mL）中。 注:腺苷三磷酸双高浓度使用，以避免在不存在激动剂的ATP 10,11自发分泌诱导P2X1受体的脱敏作用。这是重要的，因为胶原蛋白诱导的反应是通过快速旁分泌诱导/三磷酸腺苷P2X1的分泌激活发布FROM活化血小板。如果血小板信号通路不严重要求P2X1功能的保存，使用0.02 U / mL的腺苷三磷酸双。一些研究（在Mahaut-Smith 等人的综述10）证明了0.02 U / ml的腺苷三磷酸双避免了与可忽略的响应P2X1 ADP受体脱敏P2Y1。 </li><li>执行aggregometric测量之前孵育细胞悬浮液在37℃下至少30分钟。制剂是5至8小时是稳定的。 </li></ol> 4.集合度<ol><li>准备在蒂罗德缓冲液的纤维蛋白原（56毫克/毫升）。 </li><li>吸管260μL的血小板悬浮液在玻璃试管（ 图1A;左试管）含有10微升的纤维蛋白原（56毫克/毫升）和磁力搅拌棒，然后孵育2的悬浮液- 3分钟，在37℃在温育的孔本在凝集（ 图1B和1C）。 </li><li>与Panx1预孵化通过在37℃下加入2.8 mM或28 mM储备溶液（最终浓度为100μM和1mM，分别地）进行7分钟的10μL抑制剂亮蓝FCF。 </li><li> 通过测量含有10微升血纤维蛋白原（56毫克/毫升），10微升亮蓝FCF（2.8 mM或28毫摩尔）和TA缓冲无血小板的比色皿的OD校准凝集到一个假设100％的聚集值。 <ol><li>放置反应杯在聚合以及下自动搅拌和按键盘链接到凝集的计算机上的相应按钮（ 即按F1如果聚合孔1时）。 注意:下面描述的该实验包括亮蓝FCF。用于校准的化合物必须被调整到实验条件。 </li></ol></li><li> 通过使用将被用于下自动stirrin实验相同的血小板样品校准凝集到一个假设0％的聚集值G。 <ol><li>放置在聚集比色皿井和按键盘连接到凝集计算机上的相应按钮。继续之前等待约20-30秒。这种延迟供应，以确保没有聚集加入激动剂之前发生。 注意:如血小板数的任何差异可能对测量的OD的效果，0％校准步骤需要重复为每个单独的测量。 </li></ol></li><li>加入20μL所需激动剂，如15微克/毫升的胶原蛋白（1微克/毫升终浓度）或1.125毫花生四烯酸（75μM终浓度），放入试管中。立即通过按下键盘链接到凝集的计算机上的相应按钮来启动下连续搅拌下自动记录。 注:除了激动剂诱导血小板激活。血小板聚集体可清楚地在实验（ 图1A的端部的玻璃比色皿来区分;右击牛逼比色皿）。 </li><li>记录6分钟后自动停止。在这一点上，通过点击计算机的保存图标保存数据。 注:聚集速率的计算是由计算机，它体现了聚集过程以百分比的最终结果进行。 </li><li> 分析数据。 <ol><li>有关为洗涤血小板悬浮液和血小板聚集的浊测量的制备方案附加的广泛信息，指的是由该领域的专家12,13撰写其他论文。 </li></ol></li></ol>

<ol>
	<li>Patel, S. R., Hartwig, J. H., Italiano, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biogenesis+of+platelets+from+megakaryocyte+proplatelets.">The biogenesis of platelets from megakaryocyte proplatelets.</a> J Clin Invest. 115 (12), 3348-3354 (2005).</li><li>Andrews, R. K., Berndt, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+physiology+and+thrombosis.">Platelet physiology and thrombosis.</a> Thromb Res. 114 (5-6), 447-453 (2004).</li><li>Go, A. 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=Heart+disease+and+stroke+statistics--2014+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2014 update: a report from the American Heart Association.</a> Circulation. 129 (3), e28-e292 (2014).</li><li>Creager, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+the+CAPRIE+trial:+efficacy+and+safety+of+clopidogrel.+Clopidogrel+versus+aspirin+in+patients+at+risk+of+ischaemic+events.">Results of the CAPRIE trial: efficacy and safety of clopidogrel. Clopidogrel versus aspirin in patients at risk of ischaemic events.</a> Vasc Med. 3 (3), 257-260 (1998).</li><li>Born, G. V., Cross, M. 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+Aggregation+of+Blood+Platelets.">The Aggregation of Blood Platelets.</a> J Physiol. 168, 178-195 (1963).</li><li>Cazenave, J. P., Hemmendinger, S., Beretz, A., Sutter-Bay, A., Launay, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Platelet+aggregation:+a+tool+for+clinical+investigation+and+pharmacological+study+Methodology].">[Platelet aggregation: a tool for clinical investigation and pharmacological study Methodology].</a> Ann Biol Clin (Paris). 41 (3), 167-179 (1983).</li><li>Molica, F., 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=Functional+role+of+a+polymorphism+in+the+Pannexin1+gene+in+collagen-induced+platelet+aggregation.">Functional role of a polymorphism in the Pannexin1 gene in collagen-induced platelet aggregation.</a> Thromb Haemost. 114 (2), 325-336 (2015).</li><li>Taylor, K. A., Wright, J. R., Vial, C., Evans, R. J., Mahaut-Smith, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+human+platelet+activation+by+surface+pannexin-1+channels.">Amplification of human platelet activation by surface pannexin-1 channels.</a> J Thromb Haemost. 12 (6), 987-998 (2014).</li><li>Wang, J., Jackson, D. G., Dahl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+food+dye+FD&amp;C+Blue+No.+1+is+a+selective+inhibitor+of+the+ATP+release+channel+Panx1.">The food dye FD&amp;C Blue No. 1 is a selective inhibitor of the ATP release channel Panx1.</a> J Gen Physiol. 141 (5), 649-656 (2013).</li><li>Mahaut-Smith, M. P., Jones, S., Evans, R. 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+P2X1+receptor+and+platelet+function.">The P2X1 receptor and platelet function.</a> Purinergic Signal. 7 (3), 341-356 (2011).</li><li>Hechler, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+platelet+functions+and+thrombosis+through+selective+or+nonselective+inhibition+of+the+platelet+P2+receptors+with+increasing+doses+of+NF449+[4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis+-benzene-1,3-disulfonic+acid+octasodium+salt].">Inhibition of platelet functions and thrombosis through selective or nonselective inhibition of the platelet P2 receptors with increasing doses of NF449 [4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis -benzene-1,3-disulfonic acid octasodium salt].</a> J Pharmacol Exp Ther. 314 (1), 232-243 (2005).</li><li>Cazenave, J. 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=Preparation+of+washed+platelet+suspensions+from+human+and+rodent+blood.">Preparation of washed platelet suspensions from human and rodent blood.</a> Methods Mol Biol. 272, 13-28 (2004).</li><li>Jarvis, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation:+turbidimetric+measurements.">Platelet aggregation: turbidimetric measurements.</a> Methods Mol Biol. 272, 65-76 (2004).</li><li>Thompson, N. T., Scrutton, M. C., Wallis, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+volume+changes+associated+with+light+transmittance+changes+in+the+platelet+aggregometer:+dependence+upon+aggregating+agent+and+effectiveness+of+stimulus.">Particle volume changes associated with light transmittance changes in the platelet aggregometer: dependence upon aggregating agent and effectiveness of stimulus.</a> Thromb Res. 41 (5), 615-626 (1986).</li><li>Malinski, J. A., Nelsestuen, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+turbidity+to+the+stages+of+platelet+aggregation.">Relationship of turbidity to the stages of platelet aggregation.</a> Biochim Biophys Acta. 882 (2), 177-182 (1986).</li><li>Riess, H., Braun, G., Brehm, G., Hiller, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+evaluation+of+platelet+aggregation+in+whole+human+blood.">Critical evaluation of platelet aggregation in whole human blood.</a> Am J Clin Pathol. 85 (1), 50-56 (1986).</li><li>Hayward, C. 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=An+evaluation+of+methods+for+determining+reference+intervals+for+light+transmission+platelet+aggregation+tests+on+samples+with+normal+or+reduced+platelet+counts.">An evaluation of methods for determining reference intervals for light transmission platelet aggregation tests on samples with normal or reduced platelet counts.</a> Thromb Haemost. 100 (1), 134-145 (2008).</li><li>Frelinger, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+function+tests,+independent+of+platelet+count,+are+associated+with+bleeding+severity+in.">Platelet function tests, independent of platelet count, are associated with bleeding severity in.</a> ITP. Blood. 126 (7), 873-879 (2015).</li><li>Fusegawa, Y., Goto, S., Handa, S., Kawada, T., Ando, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+spontaneous+aggregation+in+platelet-rich+plasma+is+increased+in+habitual+smokers.">Platelet spontaneous aggregation in platelet-rich plasma is increased in habitual smokers.</a> Thromb Res. 93 (6), 271-278 (1999).</li><li>Davis, R. B., Boyd, D. G., McKinney, M. E., Jones, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+exercise+and+exercise+conditioning+on+blood+platelet+function.">Effects of exercise and exercise conditioning on blood platelet function.</a> Med Sci Sports Exerc. 22 (1), 49-53 (1990).</li><li>Yee, D. L., Sun, C. W., Bergeron, A. L., Dong, J. F., Bray, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregometry+detects+platelet+hyperreactivity+in+healthy+individuals.">Aggregometry detects platelet hyperreactivity in healthy individuals.</a> Blood. 106 (8), 2723-2729 (2005).</li><li>Cattaneo, M., 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=Recommendations+for+the+Standardization+of+Light+Transmission+Aggregometry:+A+Consensus+of+the+Working+Party+from+the+Platelet+Physiology+Subcommittee+of+SSC/ISTH.">Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.</a> J Thromb Haemost. , (2013).</li><li>Patel, D., Zhang, X., Veenstra, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+hemichannel+and+pannexin+channel+electrophysiology:+how+do+they+differ?.">Connexin hemichannel and pannexin channel electrophysiology: how do they differ?.</a> FEBS Lett. 588 (8), 1372-1378 (2014).</li><li>European Food Safety Authority. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientific+Opinion+on+the+re-evaluation+of+Brilliant+Blue+FCF+(E+133)+as+a+food+additive.">Scientific Opinion on the re-evaluation of Brilliant Blue FCF (E 133) as a food additive.</a> EFSA Journal. 8 (11), 1853-1889 (2010).</li><li>Lages, B., Scrutton, M. C., Holmsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+gel-filtered+human+platelets:+isolation+and+characterization+in+a+medium+containing+no+added+Ca2+,+Mg2+,+or+K+.">Studies on gel-filtered human platelets: isolation and characterization in a medium containing no added Ca2+, Mg2+, or K+.</a> J Lab Clin Med. 85 (5), 811-825 (1975).</li></ol>

作者什么都没有透露。

有一个在寻找能够以防止血栓形成，而不增加出血的风险调控血小板功能的新药极大的兴趣。为了这个目的， 体外实验室试验能可靠和可重复地监测人体血小板凝集反应是绝对必要的。比浊集合度​​是执行一个简单的技术。然而，一些注意事项需要牢记。测量需要在连续搅拌下将要执行的聚集过程在很大程度上取决于搅拌。同样重要的是，保持在血小板的37℃的生理温度，以避免任何类?...

血小板是血液，其主要有凝血功能在一起的重要组成部分的因素，是阻止血管损伤后出血。血小板是从骨髓1的巨核细胞衍生的小（2-3微米）的无核片段。血小板循环在非激活状态，在此期间，它们显示为透镜形结构。在内皮的中断，血小板聚集到血管损伤部位，以堵塞漏洞，一个称为原发性止血过程。最初，血小板附着到内皮下分子，如胶原蛋白和血管性血友病因子，被暴露的损伤粘附步骤2的结果。然后，他们改变形状和分泌的化学信使，激活步骤。最后，它们通过桥接受体聚集步骤相互连接。初期止血之后是涉及凝血级联的活化与纤维蛋白沉积的二次处理，其稳定S中的初始血栓2。 急性局部缺血事件如心肌梗死3通常由血栓形成，因为动脉粥样硬化斑块的物理破坏（破裂）的结果。目前的抗血小板药物是这种普遍的疾病的治疗的基石，但它们的临床益处被出血风险增加的限制。在心血管患者中，阿司匹林和抗P2Y12化合物最处方药，靶向血栓烷A2和分别ADP通路4，这是导致血小板活化的主要途径。不过，迈向新的目标，将最大限度地平衡血栓形成及出血性风险创新性的研究仍然是必要的。 从20世纪60年代5至今天，浊度凝集起到研究了至关重要的作用，增强了我们的血小板反应性和我的知识N的人类抗血栓剂的效力的监督。比浊法最初应用于从血液样品中提取PRP。事实上，血液收集在含有柠檬酸允许快速和大生产的PRP，而无需对血小板的完整性和功能的任何影响的试管中进行。然而，PRP的短期稳定性（约3小时），并且剩余的血浆酶如凝血酶，和具有潜在假象聚合简档相关联的低钙浓度是使用PRP的主要不便。一个重要的进步已经的方法的发展为血小板隔离用另外的离心和洗涤步骤6。总之，PRP从收集在酸 - 柠檬酸盐 - 葡萄糖（ACD）全血中分离和血小板被串行离心步骤之后，分离被再悬浮于等渗磷酸缓冲液（蒂罗德缓冲液）含有葡萄糖，人血清白蛋白和的二价C前ations（CA 2+和Mg 2+）。为了避免血小板反应性的变化，台氏缓冲液的pH值仔细地保持在7.35-7.4。另外，血小板的不希望的激活是由之前的一些离心步骤加入前列环素（PGI 2）防止。最后，加入腺苷三磷酸双可防止洗涤血小板变得对ATP / ADP的作用有抗性。将所得的血小板悬浮液缺乏凝血因子和血小板的稳定性增加至少两倍相比PRP的解决方案。此外，事实上，血小板活性，但是完整权证浊度测量的再现性，并提供研究激动剂或血小板聚集的拮抗剂的作用以最佳的方式的能力。 使用这种方法，我们在最近的研究已经表明，通过遗传方法抑制Panx1通道的形成（敲除小鼠）或由药理减小Panx1通道活性接近降低胶原诱导的血小板聚集7。 Panx1形成ATP释放信道，其在多种细胞类型，包括人血小板7,8遍在表达。事实上，我们证实通过比浊法对人洗涤的血小板，一个7分钟预温育与更多或更少的特定化学阻断剂（丙磺舒，甲氟喹和10种 Panx1肽）之前加入各种激动剂的一个面板，抑制特异性胶原诱导血小板聚集而以AA和ADP的血小板反应并没有受到影响。我们表明，ATP释放通过Panx1通道在GPVI信号传导途径，导致胶原诱导的聚集特异性干扰。有趣的是，与其他疾病（丙磺舒，甲氟喹）应用的多个FDA批准的化合物影响血小板Panx1通道的活性。一方面，这会打开新的治疗前景选择结构延续修改血小板反应性。在另一方面，应该考虑这些化合物的潜在的辅助疗效。在这种背景下，安全的食品染料亮蓝FCF在多个糖果和能量饮料中使用已描述为Panx1 9的选择性抑制剂。在这里我们描述用于人洗涤的血小板和血小板聚集的浊度测量的隔离的协议适合于调查亮蓝FCF染料的血小板聚集的拮抗剂的作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>citric acid monohydrate (C6H8O7*H2O)</td>
			<td>Roth</td>
			<td>5949-29-1</td>
			<td>danger of eye damage/irritation</td>
		</tr>
		<tr>
			<td>trisodium citrate dihydrate (Na3C6H5O7*2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S1804</td>
			<td>-</td>
		</tr>
		<tr>
			<td>D(+)-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium dihydrogenophosphate monohydrate (NaH2PO4*H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2*6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium chloride hexahydrate (CaCl2*6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>442909</td>
			<td>danger of eye damage/irritation</td>
		</tr>
		<tr>
			<td>N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (Hepes)</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>15630</td>
			<td>-</td>
		</tr>
		<tr>
			<td>human serum albumin</td>
			<td>CSL Behring</td>
			<td>00257/374</td>
			<td>-</td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Corrosive and irritative for the respiratory system. Can cause severe skin and eye damages.</td>
		</tr>
		<tr>
			<td>Eppendorf 5810 R</td>
			<td>Fisher Scientific</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>heparin</td>
			<td>Drosspharm AG/SA</td>
			<td>20810</td>
			<td></td>
		</tr>
		<tr>
			<td>prostacyclin I2 (PGI2)</td>
			<td>Cayman</td>
			<td>18220</td>
			<td>-</td>
		</tr>
		<tr>
			<td>apyrase from potatoes</td>
			<td>Sigma-Aldrich</td>
			<td>A6535</td>
			<td>-</td>
		</tr>
		<tr>
			<td>fibrinogen (Haemocomplettan)</td>
			<td>CSL Behring</td>
			<td>HS 73466011</td>
			<td>-</td>
		</tr>
		<tr>
			<td>thrombo-aggragometer SD-Medical</td>
			<td>SD-Innovation</td>
			<td>TA8V</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Brilliant blue FCF (Erioglaucine disodium salt)</td>
			<td>Sigma-Aldrich</td>
			<td>80717</td>
			<td>Harmful to aquatic life with long lasting effects (Avoid release to the environnement)</td>
		</tr>
		<tr>
			<td>collagen</td>
			<td>Horm, Nycomed</td>
			<td></td>
			<td>-</td>
		</tr>
		<tr>
			<td>arachidonic acid</td>
			<td>Bio/Data corporation</td>
			<td>C/N 101297</td>
			<td>-</td>
		</tr>
		<tr>
			<td>cell counter Sysmex KX-21N</td>
			<td>Sysmex Digitana&#160;</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>-</td>
		</tr>
		<tr>
			<td>glass cuvettes</td>
			<td>SD-Innovation</td>
			<td>THCV1000</td>
			<td>-</td>
		</tr>
		<tr>
			<td>magnetic stirrers</td>
			<td>SD-Innovation</td>
			<td>THA100</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

turbidimetry on human washed platelets: the effect of the pannexin1-inhibitor brilliant blue fcf on collagen-induced aggregation

比浊法是用于测量悬浮在血浆（富含血小板的血浆，PRP）或在缓冲液（洗涤血小板）血小板聚集实验室技术，通过使用一个或激动剂的组合。使用从它们的等离子体环境和在没有抗凝血剂的分离洗涤血小板的允许研究固有血小板特性。间激动剂，花生四烯酸（AA），腺苷二磷酸（ADP）的大面板，凝血酶和胶原是最常使用的。聚合反应是由在连续搅拌下测量随血小板悬浮液的时间的相对光密度（OD）进行定量。在均匀的悬浮液血小板限制光的通过加入激动剂后，血小板形状改变发生在制造OD小的暂时性增加。以下该初始活化步骤，血小板凝块逐步形成，从而允许光通过穿过悬浮液作为水库的ULT OD降低。聚集过程最终以百分比表示，相对于贫血小板血浆或缓冲液的OD。严格校准因此在每次实验开始时是必不可少的。作为一般规则:校准到0％，通过测量非刺激的血小板悬浮液的OD同时测量不含血小板悬浮介质的OD表示的100％的值设定。血小板聚集通常显现为实时聚合曲线。比浊法是最常用的实验室技术用于血小板功能的调查之一，被认为是历史的金标准，并用于旨在抑制血小板聚集的新药物的开发。在这里，我们描述了详细方案1）制备人洗涤血小板和2）人的胶原诱导的聚集的浊分析与食品染料亮蓝FCF，这是最近的iDEN洗涤，预处理血小板tified如Pannexin1（Panx1）通道的抑制剂。

聚集仪软件自动生成聚集曲线，给人以百分比最大聚集值。这些值可以在为了进行统计分析，在条形图的形式可视化最大凝集值被复制到一个数据分析软件。任选地，聚合曲线的每个单独的点可被相继地以可视化的曲线导出到一个电子表格软件，然后统计软件（ 例如的GraphPad）。一些研究者使用聚合曲线的最大斜率计算曲线下的速度和面积，以评估血小板活性。滞?...

Watch this Scientific Journal Video about Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation at JoVE.com

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

我们描述了洗涤的血小板的人血液，然后通过比浊法激动剂诱导的血小板聚集的测量隔离的简单方法。作为例子，我们申请研究的预温育与Pannexin1通道抑制剂亮蓝FCF后的人血小板聚集响应于胶原此方法。

比浊法对人洗涤血小板:所述Pannexin1抑制剂的影响亮蓝FCF对胶原诱导的聚集

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

turbidimetry-on-human-washed-platelets-effect-pannexin1-inhibitor

Research

JoVE Journal

Medicine

11.9K 次观看.  University of Geneva. 我们描述了洗涤的血小板的人血液，然后通过比浊法激动剂诱导的血小板聚集的测量隔离的简单方法。作为例子，我们申请研究的预温育与Pannexin1通道抑制剂亮蓝FCF后的人血小板聚集响应于胶原此方法。 

视频: Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

单侧颈脊髓损伤的挫伤模型，利用无限的地平线冲击

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

科研

医学

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

B7-H1（PD-L1）免疫组化染色石蜡的嵌入式幻灯片的胰腺癌组织

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

在感染艾滋病毒的个体的测量弱者。体弱患者的识别是第一步改良与弱者拨回

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

基于短暂性脑缺氧缺血性血栓性中风模型

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

人体胎盘生理学综合研究的4血管取样方法<em&gt;体内</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

测定化学物质对线虫线虫生长和繁殖的影响

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

人体滋养层模型研究母体肥胖炎症对胎盘自噬的调节作用

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...