研究提出支持的查尔斯河。

1。的MFIA程序的说明（图1） <ol><li>在的MFIA需要Charles River的抗原包被聚苯乙烯微球（珠），测试血清标记试剂（BAG，SPE）和缓冲区（小学稀释液，测定缓冲液）。 </li><li>试剂逐步加入到96孔过滤底微量滴定板的孔中。 </li><li>该MFIA是异构测试含义孵育随后由过滤器的洗涤步骤，以除去未结合的血清成分，或标记试剂进行。洗涤溶液添加到板的孔，以及过滤器的底部，保留珠粒通过抽吸除去。 </li><li> MFIA检测在室温下（27°C + / -2°C）。 </li><li>检测到抗原 - 抗体复合物在测试过程中血清孵育步骤形成与生物素化的山羊抗物种结合物（袋），其次，由R-藻红蛋白标记的链霉亲和素（SPE）的孵育。 </li><li>以测定读取器，珠粒通过一个在同一时间通过。检测器，在那里，它们暴露于两个激光器。一个激光激励内部识别磁珠的颜色集合，这对应于一个试验的染料;其他激发的藻红蛋白的测定期间捕获的报告染料。预定数目的珠，每次测定读取，作为中位数荧光指数（MFI）和藻红蛋白的荧光强度的报告。 <img alt="图1。" src="/files/ftp_upload/3715/3715fig1.jpg" /> 图1。MFIA程序。的xMAP基于MFIA是悬浮芯片采用彩色编码的聚苯乙烯5.6微米的珠抗原（或控制）的共价连接。由于珠粒在100个不同的颜色集，多达100个不同的检测可以在单井含量步骤执行，在过滤器的底微量滴定板中进行洗涤，使珠可以通过抽吸的真空歧管上。反应是阅读与Luminex公司的xMAP 100荧光。的强度phycoerythrin荧光报告为平均荧光指数（MFI） </li></ol> 2。在开始前，请注意以下几点： <ol><li> MFIA珠是光敏感<ol><li>直接光线照射量的限制是至关重要的。例行试验过程中发生的正常曝光量是可以接受的，但长期接触可导致漂白的珠子，这使他们无法读取的含量读者。 </li><li>的MFIA珠总是涡旋成悬浮，然后超声处理（10-30秒）在使用前，这是至关重要的成功的测定。 </li><li>从单珠含量读者收集信息。聚合珠群会导致更长的读取时间。 </li></ol></li></ol><ol start="2"><li>一定要保持测试板的板盖覆盖在所有孵化的步骤，以避免蒸发的分析解决方案。 </li><li>个人防护装备：实验室外套，GL在任何时候，在实验室环境中工作时，应佩戴oves和保护眼睛的。 </li></ol> 3。试剂<ol><li>表1列出了大部分所需的材料，以建立一个内部MFIA的实验室。附加的或重复的项目可能需要根据您的具体需求。请注意，这个清单不包括从查尔斯河（MFIA珠，控制和补充试剂）试剂购买。生物素化的结合物与链霉亲和标签的藻红蛋白的几个商业来源是可用的。但是从Charles River试剂滴定，以产生最佳的信号噪声的得分与我们的试剂，使用替代供应商或试剂等，不推荐。 表1中 。 CR-RADS使用一个BioPlex的的的的悬浮阵列从BioRad公司的阅读器和自动洗板机从BioTek的。等效的仪器，从不同的供应商提供。 <table border="1"><tboDY&gt; <tr><td> 项目 </td><td> 供应商 </td><td> 目录编号 </td></tr><tr><td> 设备 </td><td></td><td></td></tr><tr><td>悬浮芯片阅读器</td><td> * BioRad公司</td><td> 171-000205 </td></tr><tr><td> 96孔微孔板垫圈</td><td>百特</td><td> ELX50/8FMW </td></tr><tr><td>超声波清洗机/浴</td><td>科尔帕尔默</td><td> EW0884900 </td></tr><tr><td>模拟旋涡混合器</td><td> VWR </td><td> 58816-121 </td></tr><tr><td> -20°C冰箱</td><td>各个</td><td></td></tr><tr><td> 4°C冰箱</td><td>各个</td><td></td></tr><tr><td> 12道移液器20-200μl的</td><td> VWR </td><td> 83009-718 </td></tr><tr><td>轨道板振动筛</td><td> VWR /实验室线</td><td> 57019-600 </td></tr><tr><td>单道移液器，20-200μl的搭配秘诀</td><td> VWR </td><td> 83009-732 </td></tr><tr><td>单道移液器，2-20μL，搭配秘诀</td><td> VWR </td><td> 83009-726 </td></tr><tr><td>单道移液器，1001000μL，搭配秘诀</td><td> VWR </td><td> 83009-736 </td></tr><tr><td> Vacushield通风装置</td><td> VWR </td><td> 55095-006 </td></tr><tr><td>真空压力泵</td><td> VWR </td><td> 54908-037 </td></tr><tr><td>真空系统废液池</td><td> VWR </td><td> 80200-640 </td></tr><tr><td> 耗材 </td><td></td><td></td></tr><tr><td> 96 polystryrene板</td><td> Fisher Scientific则</td><td> 14-245-145 </td></tr><tr><td> MultiScreen的HTS-BV板，1.2微米过滤器，苯乙烯</td><td>密理博</td><td> MSBV N12 50 </td></tr><tr><td> 15ml锥形管（聚丙烯） </td><td>萨尔斯塔特</td><td> 62554.002 </td></tr><tr><td> 50ml锥形管（聚丙烯） </td><td>萨尔斯塔特</td><td> 62547.004 </td></tr><tr><td>血清瓶</td><td>萨尔斯塔特</td><td> 72694.007 </td></tr><tr><td>铝箔</td><td> VWR </td><td> 89079-068 </td></tr><tr><td> 1L瓶，无菌</td><td> VWR </td><td> 28199-246 </td></tr><tr><td> 0.22μm的瓶顶过滤器</td><td> VWR </td><td> 28199-307 </td></tr><tr><td>试剂水库（100毫升） </td><td> VWR </td><td> 82026-356 </td></tr><tr><td> 5ML，10ML，25ML移液管分配液体试剂</td><td> VWR </td><td></td></tr><tr><td> 试剂 </td><td></td><td></td></tr><tr><td> PBS，pH值7.4 1 BSA，粉（袋） </td><td>西格玛</td><td> P3813 </td></tr><tr><td>的Proclin 300 </td><td> SIGMA / Supelco公司</td><td> 48912-U </td></tr><tr><td>胶版纸球</td><td> Luminex公司</td><td>不同类型</td></tr></tbody></table></li></ol> 4。试剂准备<ol><li>需要两个缓冲区为MFIA程序。主要稀释剂是由Charles River提供，并包含一种特有的阻断剂，以减少非特异性的蛋白质的相互作用。在您的设备与市售试剂检测洗涤液。 </li><li>含量洗涤缓冲液：PBS / 1％BSA的pH为7.4作为从Sigma-Aldrich公司的粉末是可用的。 <ol><li>通过过滤的微粒的去除是至关重要的，因为这些颗粒可导致木屐在测定阅读器。一个0.2micron到无菌的，贴有标签的容器瓶顶过滤器，过滤器的缓冲。 </li><li> BAG和SPE稀释至含量浓度的洗涤液，2X工作。注：SPE是光敏感和sh·乌尔德·有限的光照射。 </li></ol></li></ol> 5。样品制备<ol><li>装配试验材料，试剂，一次性的。 <ol><li>多和单通道微量移液器和技巧</li><li> 96 - 孔低蛋白结合的微量滴定板（血清稀释）和过滤器的底板（MFIA测定） </li><li>根据标准程序采集血液样本。允许的血液标本血块完全由它们在室温下保持至少30分钟，然后离心和血清的去除。应振荡Undilute血清，简单地混合血清成分采样前。 </li><li>最终测试为MFIA稀释为1/50。这是必要的，使测定了2倍（1/25）样品的测试血清。未稀释的试验血清加1份血清，24份MFIA主要稀释剂稀释。测试血清中的96孔微量滴定板中的组织极大地降低了传输时间对样品的工商业污水附加费T形钢板，强烈推荐。 </li></ol></li></ol> 6。试板准备<ol><li>预润湿的96孔测试板，是必需的，以保证正确，即使以及疏散。即使整个板块将不会被使用需要这一步最初，你不会有，添加后续试剂或洗涤缓冲液井空。 <ol><li>一些实验室运行不到一个完整的96孔板的密封关闭未使用的测试板，并计划在以后的日子重新使用这些井的井。我们不建议使用此方法，因为它可能会影响测试板的适当的过滤。 </li></ol></li></ol><ol start="2"><li>正确的测试板愿望的MFIA试验的成功是至关重要的。疏散样品井约5-10秒。吸气阱内容太快可导致胎圈聚集和缓慢的读取时间结束时的测定。 </li><li>后，每瓦特的试验板用纸巾吸干底面火山灰的步骤，以确保液体的排水已停止。如果涂抹试验板，可以导致液体试剂，毛细作用的井孵化过程和结果，在失败的山羊抗物种和/或种属的IgG珠分数。这些珠被设计得分MFI内的预先确定的范围内时，适量的主血清和标记试剂被加入到各孔。 </li></ol> 7。 MFIA珠混悬剂<ol><li>这是至关重要珠粒总是涡旋和超声处理，在使用前的成功的测定。收集信息，分析读者从单珠，珠聚集体或团块会导致更长的读取时间。 </li><li>涡的股票加上珠悬浮液（通常为10±5秒） </li><li>重悬珠其次超声波仪，浴中通过超声处理10-20秒。 </li><li>稀释20倍的股票停牌珠悬浮在主稀释2倍的工作。免除的2倍珠悬浮液50μL每个检测的预湿试验板。 </li></ol> 8。此外，试验和对照血清的测试板<ol><li>移取50μl的每一个2X测试和控制血清测试根据预定义的板图板。安全的盖子，板与弹性带或铝箔孵育轨道摇床，在黑暗中，在室温下，持续60分钟的试验板。 <ol><li>摇床转速应大于400，但低于700RPM。这是至关重要的，珠粒被保持在悬浮液中，以允许访问所有表面耦合抗原血清抗体。否则保持暂停珠粒将导致低IgG珠控制分数，并测定将需要被重复。 </li></ol></li></ol> 9。洗衣机的测试板<ol><li>使用真空歧管吸出很好的内容。疏散样品井约5-10秒。 </li><li>吸干佩普试验板的底面与ŕ后，每毛巾，以确保排液已停止。 </li><li>洗涤液的含量在50μl重悬珠。珠粒不能干出在测定过程中，这一点很重要。 </li></ol> 10。 BAG和SPE的测试板<ol><li>分液50μL2X工作液袋的所有MFIA珠孔中。的盖子固定到板与弹性带或铝箔。如上述，在黑暗中，在室温下振荡孵育，持续30分钟的试验板。 <ol><li>保温后洗的测试板和血清此外如前所述悬浮含量洗涤液珠。 </li></ol></li></ol><ol start="2"><li>分配50μL的2倍稀释至所有的孔中MFIA珠的SPE。的盖子固定到板与弹性带或铝箔。如上述，在黑暗中，在室温下振荡孵育，持续30分钟的试验板。 <ol><li>在此之后INCU的cDNA克隆洗的测试板和悬浮磁珠125μl洗涤液的含量和摇板〜两分钟之前到检测器悬浮珠。 </li></ol></li></ol> 11。阅读测试板<ol><li>试验板放置到测定读取器，在10分钟内再悬浮珠粒。在电源故障或其他事件的事件，测试板可以被存储在黑暗中在室温下，时间长达12小时，直到准备好读。 <ol><li>观察第一个样本，以验证所选择的珠面板相匹配的配置文件中的测试井珠。如果有是属于协议显示他们的指定的“胎圈区域&#39;珠，选择一个不正确的更新已经取得进展。 </li><li>如果小珠已经正确地悬浮，应填写在其指定的“区域”在分析阅读软件表示。 </li><li>如果小珠聚集在他们的地区，他们将填补一个缓慢的率。您可以删除测试版的读者和手动悬浮井。捣碎各孔与多通道移液器的3-4倍，以协助在分手胎圈粒料。更换测试板插入读卡器，然后继续。 </li></ol></li></ol> 12。代表性的成果<ol><li>最低数量的珠子，读法和藻红蛋白荧光强度的报道为平均荧光指数（MFI），范围从0到32,667。在比较计数25，50或100，每孔珠，我们看到无显着性差异，并定期计算每剂25珠，每个样品。检查结果报告的错误，如的珠数量不足或未能抗体/抗IgG珠分数。这些珠被设计为“得分”内的预先确定的MFI范围时，适量的主血清和标记试剂被加入到各孔中。样品井的错误，（珠计数低），或者未IgG​​对照珠分数重复进行，以获得有效的结果。 </li><li>结果导出到Excel工作表中。对于我们的数据解释分析分配： <ol><li>甲组织控制（TC）测试：对于大多数的啮齿动物MFIA检测，组织控制是野生型杆状病毒感染的昆虫细胞的提取物。 </li><li>一种含量托夫：此值是最小荧光预期调整为每个检测，以最大限度地提高诊断的准确性。对于大多数检测，荧光截止为3000。 </li></ol></li></ol><ol start="3"><li>净的中位数荧光指数（MFI）来计算由下面的公式为每个测定（组织和IgG对照测试除外）。换句话说，具体的测定信号的总测定的MFI减去这个值再除以1000的TC（背景）的MFI 0-32代替0-32,667之间简单地设置的范围内的净MFI。 净MFI =（MFI 抗体检测 - MFI 组织控制 ）/ 1000 氖吨MFI和TC MFI被转换成分数相比，针对每种抗原的含量截止。 <img alt="图2。" src="/files/ftp_upload/3715/3715fig2.jpg" /> 图2。的代表MFIA数据抗体测试血清与所有控制传递。 </li></ol><ol start="4"><li> 12.4 MFIA解释<ol><li>试验板的结果只应被解释，如果系统的适宜控制和血清控制（IgG抗体/抗-IgG珠）的结果满足认可准则，表2中列出。这两个IgG对照珠失败可能表明了一些程序上的错误，如不适当稀释或不足量的共轭或SPE，不正确的测试血清稀释或使用的血清从一个immunocomprimised的动物。 表2 <table border="1"><tbody><tr><td colspan="3"> 验收标准含量控制 </td></tr><tr><td rowspan="2"> 控制 </td&gt; <td colspan="2"> 可以接受的结果 </td></tr><tr><td> 得分 </td><td> 分类 </td></tr><tr><td>高范围免疫血清</td><td> ≥4.5 </td><td>积极</td></tr><tr><td>低范围免疫血清</td><td> ≥1.5 </td><td> ≥边缘</td></tr><tr><td>非免疫动物血清</td><td> &lt;2.5 </td><td> ≤边缘</td></tr><tr><td>稀释液</td><td> &lt;2.5 </td><td> ≤边缘</td></tr><tr><td>免疫球蛋白订珠* </td><td> ≥Cutoff/1000 </td><td>通过</td></tr></tbody></table> *珠涂与种属特异性抗检测血清免疫球蛋白（Ig）设置：此示例适宜控制分数没有增加样本量不足，可能会导致过高的样品稀释，不正确的物种或检测血清的免疫浩ST。 </li></ol><ol start="2"><li>如果有任何的测试板含量控制除了高频范围内的血清免疫失败，结果是不能接受的，必须重复进行。通过低范围血清控​​制分数线是至关重要的，因为它演示了检测的灵敏度水平测试板。 </li><li>的山羊抗-IgG抗体珠种得分是基于主要的测试血清的量加入到测定。如果这个控制胎圈失败，但种的IgG抗体通过（井B1，图3），则它表明足够的检测试剂（BAG，SPE）以及被添加到测试，它是一​​种血清相关的问题。如果测试试剂的量是不足的，在一个特定的井的IgG和抗-IgG控制珠将失败（E1，图3）。 <img alt="图3。" src="/files/ftp_upload/3715/3715fig3.jpg" /> 图3。的代表MFIA测试结果与故障（橙色）。井A1，C1和F1表明，TC（组织无功）失败TC得分表示在括号中。韦尔斯B1和E1示出了两种类型的IgG抗体的内部控制，故障，受试抗体不足，（B1）或测试试剂（E1）袋/ SPE分别。 </li></ol><ol start="4"><li>如果测试板含量控制的效果是令人满意的，单独的检测分数归类为示于表3。这是非常重要的替代测试方法，以确认任何边缘或积极的发现。 表3 <table border="1"><tbody><tr><td colspan="4"> MFIA分数分类 </td></tr><tr><td colspan="3"> 得分 </td><td rowspan="2"> 分类 </td></tr><tr><td> TC </td><td> 净 </td><td> TC +网* </td></tr><tr><td rowspan="2"> ≥2 </td><td> &lt;0.5 </td><td> &lt;2.5 </td><td>负极（ - ） </td></tr><tr><td&gt;≥0.5 </td><td> ≥2.5 </td><td> TC反应（T） </td></tr><tr><td rowspan="3"> &lt;2 </td><td> &lt;1.5 </td><td></td><td> - </td></tr><tr><td> 1.5≤X &lt;2.5 </td><td></td><td>边缘（B） </td></tr><tr><td> ≥2.5 </td><td></td><td>正（+） </td></tr></tbody></table> *分类负仍然可以被确定的一个非零的TC得分提供的TC +净得分（=得分）银是负</li></ol></li></ol>

<ol>
	<li>Jacobson, R. H., 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=Principles+of+validation+of+diagnostic+assays+for+infectious+diseases.">Principles of validation of diagnostic assays for infectious diseases.</a> Manual of Standards for Diagnostic Tests and Vaccines. , 8-15 (1996).</li><li>Wright, 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=International+standards+for+test+methods+and+reference+sera+for+diagnostic+tests+for+antibody+detection.">International standards for test methods and reference sera for diagnostic tests for antibody detection.</a> Rev. sci. tech. Off. int. Epiz. 17 (2), 527-533 (1998).</li><li>Pepe, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Statistical Evaluation of Medical Tests for Classification and Prediction. , (2003).</li><li>Barr, M. C., 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=Comparison+and+interpretation+of+diagnostic+tests+for+feline+immunodeficiency+virus+infection.">Comparison and interpretation of diagnostic tests for feline immunodeficiency virus infection.</a> J. Am. Vet. Med. Assoc. 199, 1377-1381 (1991).</li></ol>

所有的作者查尔斯河RADS工作，动物血清学检测服务和试剂的主要供应商。

MFIA测试过程中是高效率，需要更少的设备和更小的体积比传统的单重测试样品和试剂。的多路传输系统的功能，为用户提供了灵活性，屏幕上同时多株或血清型的实验室啮齿动物（例如冠状病毒，细小病毒等）的共同代理。这也使得我们，感兴趣的区域（即特定的病毒家族）的基础上设计定制的胎圈板，是适应其他类型的生物分子，包括细胞因子和其他生物标志物筛选。此外，它允许我们将内部控制的实验来验证样品与系统适用性，从而保证结果的准确性。这些措施包括组织控制和IgG抗体检验血清种免疫球蛋白（αIg）的镀膜珠集，以评估样品的适用性。一个组织控制磁珠检测的非特异性结合的血清免疫球蛋白和αIg珠控制确认该血清已被添加并含有足够的免疫球蛋白浓度。种免疫球蛋白血清，另一个控制珠，涂表明，标记的试剂和检测器的正常工作。其他市售的多重格式的血清学检测（即芯片，ImmunoComb）可能无法提供同一级别的结果确认。 的能力，缩小的可能来源分析失败之前重新测试可以帮助研究者节省时间和材料。关键方面的MFIA的，应确认之前重复不合格的样本。由于在环境温度下进行测定，应验证，实验室的温度是约27℃±2℃，较高的温度可导致比对照和样品的预期分数降低。洗衣机也许是最关键的一步检测。保险试验板正确洗涤，吸干，并再悬浮可以消除由于低胎圈计数（由于凝集珠）或添加试剂不足（丢失通过排汗满分试验板过滤器底部）的抽样误差绝大多数。我们经常在数法（代理人）25珠，并没有发现任何统计学上的差异，结果计数较高的珠子，这也将导致一个较长的读取时间为板。 我们已经进行了全面的验证研究MFIA数种，常用的实验动物（小鼠，大鼠，仓鼠，豚鼠和兔）证明诊断的准确度，重复性和耐用性测试大量已知阳性和阴性血清样品，并比较其ELISA，IFA和MFIA结果的。的检出限（即，标准血清免疫滴定终点）MFIA相媲美，而且在某些情况下，超越，那些相应的ELISA。测量SPF啮齿类动物血清，诊断的特异性，超过99％的整体通信betweeŇELISA和MFIA已知的阳性和已知的阴性血清进行大于95％。总之，这些结果证明，多路MFIA是一个很好的替代单一ELISA，是适合其用途，即在常规的血清学监测实验室动物的殖民地。

multiplexed fluorometric immunoassay testing methodology and troubleshooting

为了确保质量，在生物医学研究中使用的动物模型，我们开发了大量的诊断测试策略和方法来确定，如果动物被暴露在偶然的传染性病原体（如病毒，支原体和其他挑剔的微生物）。感染免疫功能的动物通常是短暂的，但常常可以检测到数天至数周之内血清抗体反应，感染和坚持的整个生命周期的主机。血清学检查是主要的诊断方法，实验室动物进行监控。间接酶联免疫吸附试验（ELISA）在历史上一直是主要的筛选方法为血清学监测。作为单重进行的ELISA，其中微生物的抗原 - 抗体反应测定，每。比较MFIA进行作为一个复用的测定。由于微球有100个不同的颜色集，可以进行多达100种不同的检测simultaneously在一个单一的微孔板。这种创新降低血清，试剂和一次性用品的例行测试，同时增加的量，获得的信息以及从一个单一的测试所需的量。此外，我们还可以将多个内部控制珠，以确认样品与系统适用性，从而保证结果的准确性。这些措施包括组织控制和IgG抗体检验血清种免疫球蛋白（αIg）的镀膜珠集，以评估样品的适用性。在ELISA和IFA，组织控制检测到的非特异性结合的血清免疫球蛋白。 （血清对照）αIg控制证实，血清已被添加并包含足够的IgG控制系统适应性控制（珠），涂有血清种免疫球蛋白，免疫球蛋白浓度，同时表明，标记试剂和Luminex阅读器是否正常工作。

Watch this Scientific Journal Video about Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting at JoVE.com

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

使用Luminex公司的xMAP微球技术，我们开发了复用荧光免疫法（MFIA）为血清学监测各种实验室动物物种的。 MFIA是抗原，组织控制或免疫球蛋白共价连接到颜色编码的聚苯乙烯微球的悬浮液微阵列。解决MFIA测试方法以及各种疑难排解主题。

复用荧光免疫检测方法和故障排除

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

multiplexed-fluorometric-immunoassay-testing-methodology

Research

JoVE Journal

Biology

28.2K 次观看.  Charles River. 使用Luminex公司的xMAP微球技术，我们开发了复用荧光免疫法（MFIA）为血清学监测各种实验室动物物种的。 MFIA是抗原，组织控制或免疫球蛋白共价连接到颜色编码的聚苯乙烯微球的悬浮液微阵列。解决MFIA测试方法以及各种疑难排解主题。

视频: Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

科研

生物学

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

聚合酶链反应：基本协议加上故障排除和优化策略

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

Solid Phase Synthesis of a Functionalized Bis-Peptide Using &quot;Safety Catch&quot; Methodology

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

一个官能二肽的固相合成使用“安全锁扣”方法论

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

复用的恒化器阵列的设计与应用

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

全自动毛细 Electrophoretic-based 免疫分析方法的程序和关键优化策略

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...