研究支持NSF奖断路器-1021384。

1。厌氧缓冲区的制备<ol><li>准备1升100 mM磷酸钾缓冲液，pH值7.5。倒入500毫升毕希纳用搅拌棒瓶250毫升的缓冲。 </li><li>用橡皮塞密封瓶和放在轰动板。瓶到舒伦克线的连接短管。 </li><li>德加的缓冲区在室温下搅拌5小时，在真空状态下。在这段时间内，进行真空脱气，氩气冲洗每隔一小时的连续两轮。 </li><li> 10秒刷新瓶氩和真空歧管断开。将烧瓶内的手套箱。 </li><li>离开烧瓶打开手套箱剧烈振荡过夜。 </li></ol> 2。氧气从停流系统中删除<ol><li>准备1 L 0.1 M醋酸钠，pH值5.0。倒入250毫升布氏瓶125毫升此缓冲区和执行步骤1.2-1.4。 </li><li>掂量出5从黑曲霉（Aspergillus niger）（181300 U / G）和0.9克葡萄糖，用1.5 mL的Eppendorf管和猎鹰锥形管50毫升，分别毫克的葡萄糖氧化酶。放入手套箱两个容器。 </li><li>葡萄糖氧化酶和葡萄糖溶于50毫升厌氧0.1 M醋酸钠，pH值5.0（终浓度为18.13 U / ml的100毫米，分别）。填补这一解决方案（ 图1）水库注射器。 </li><li>确保该驱动器阀“负荷”的位置和填写的驱动器，注射器（ 图1）。关闭的F阀的“驱动器”的位置。清空停止注射器点击空 ​​临数据控制软件。推流电路通过提高相应的驱动器RAM手动从驱动器注射器F缓冲区。重复此步骤，共10倍。执行相同的程序与其他三个驱动器注射器。等待一小时。 </li><li>更换与同一缓冲区contai的水库注射器的解决方案宁葡萄糖氧化酶和葡萄糖。重复步骤2.4。 </li><li>重复步骤2.5，让溶液在一夜之间站在流系统。 </li><li>过夜培养后，重复步骤2.5。 </li></ol> 3。制备的氧饱和缓冲<ol><li>准备100 mM磷酸钾缓冲液（pH值7.5），倒入50毫升50毫升的小瓶，用搅拌棒。例与惠顿塞和惠顿铝密封的小瓶。 </li><li>放在冰小瓶。沉浸长针连接到到溶液中含有100％的氧气罐。作为一个发泄的小瓶瓶塞通过插入另一个短针。 </li><li> 100％，搅拌1小时的氧气气泡的解决方案。 </li><li>首先从小瓶短针，并等待10秒后，取出长针。放置在手套箱的封闭小瓶。 </li></ol> 4。 NADPH的溶液的制备<ol><li>在1.5 mL的Eppendorf管中掂量出1毫克和NADPH的地方，我T成手套箱。 </li><li>溶解在300μL厌氧100毫米钾磷酸盐缓冲液，pH值7.5的NADPH。从手套箱中取出30μL此解决方案，以确定与NADPH的浓度分光光度计（ε340纳米 = 6,270 M-1厘米-1）。 </li></ol> 5。除氧酶液<ol><li>在手套箱的冷冻和解冻400μL酶原液。酵素原液（360μM）事先准备好的100毫米磷酸钾缓冲液含有100 mM氯化钠（pH值7.5），并在液氮冷冻。 </li><li>酶液转移到一个小的轰动酒吧25毫升的小瓶，瓶盖与惠顿塞和惠顿铝密封的小瓶，从手套箱。 </li><li>放在冰瓶和小瓶的塞子通过插入一根针，它连接到一个舒伦克线。 </li><li>德加的表演，连续5轮的酶液真空脱气，氩气冲洗，每20分钟1小时。 </li><li> 10秒刷新瓶氩和断开真空多方面。小瓶放在手套箱内的冰。 </li></ol> 6。还原半反应：减少监测黄素<ol><li>准备停流仪器和单一的混合模式，按照制造商的协议支持数据的控制软件。 </li><li>打开循环水浴（15℃）。冒泡通过20分钟的氮去除水中的氧气。这可以防止通过流电路中的氧污染。 </li><li>保持连接停止流动的水洗澡住房循环浴驱动器注射器的温度和观察细胞15°C间。 </li><li>在控制面板窗口临数据控制软件选择光电二极管阵列，外部触发（激活停流操作），LogaritHMIC规模。 </li><li>启动临数据浏览器软件，并确定将存储的数据文件的目录。 </li><li>更换水库注射器C和F与厌氧100 mM磷酸钾缓冲液（pH值7.5）的解决方案。打开C和F阀的“驱动器”的位置。空的空临数据控制软件点击停止注射器。推流电路通过两个驱动器注射器手动提高相应的驱动器在总RAM五倍（点击之前，每个运动的RAM 空 ）缓冲区。 </li><li>为了确保流电路完全，葡萄糖氧化酶已被删除，执行步骤总额6.6四次。 </li><li>点击基线临数据控制软件。 </li><li>混合1100μL无氧100 mM磷酸钾缓冲液（终浓度混合后，在15微米停止流动）的，100μL酶原液。 </li><li>取代水库锡尔河的解决方案英格F与的酶液。关闭的F阀的“驱动器”的位置。空的空临数据控制软件点击停止注射器。推流电路通过提高相应的驱动器RAM手动从驱动器注射器F酶液。共执行了三次。在临数据的控制软件，设置采集时间为60秒。 </li><li>确保这两个驱动器注射器填补其相应的解决方案和驱动器公羊接触的驱动器注射器活塞。两个阀门打开的“驱动器”的位置，请在收购临数据控制软件来执行驱动器。重复此步骤2倍多，获得的数据，一式三份。这种氧化酶的形式产生的光谱。 </li><li>使用一个452纳米的从驱动器获得的价值，确定混合（ε452纳米 = 13700 M-1 cm-1处）之前，酶浓度，并准备了4毫升的核苷酸solutioñ1.5倍，更集中。 </li><li>更换水库注射器C的解决方案与NADPH的解决方案，空和填充停止注射器在6.10倍。 </li><li>重复步骤6.11。这期间减少酶的产量光谱。 </li></ol> 7。氧化半反应：监测黄素氧化<ol><li>准备停流仪器和混合双模式，按照制造商的协议支持数据的控制软件。 </li><li>重复步骤6.2-6.5。 </li><li>更换厌氧100 mM磷酸钾缓冲液（pH值7.5）中的四个水库注射器的解决方案。关闭的F阀的“驱动器”的位置，通过点击在临数据控制软件的空空站注射器。推动通过流电路的驱动器注射器手动提高相应的驱动器RAM F缓冲区。执行此步骤，在每个驱动器注射器总额的5倍。 </li><li>执行步骤730的四倍，在TOT人完全删除的内容，从流电路。 </li><li>点击在临数据控制软件的基线 。 </li><li>混合1000μL无氧100 mM磷酸钾缓冲液（终浓度混合后，在15微米停止流动）的，200μL酶原液。 </li><li>更换解决水库与酶液的注射器。关闭一个阀门的“驱动器”的位置。空的空临数据控制软件点击停止注射器。酶液，通过提高相应的驱动器RAM手动推入一个驱动器注射器通过流电路。执行此步骤总额的3倍。在临数据的控制软件，设置延迟时间和0.5秒至60秒的采​​集时间。 </li><li>确保所有驱动器注射器填补其相应的解决方案和驱动器公羊接触的驱动器注射器活塞。关闭所有阀门的“驱动器”的位置，然后单击“在Pro-DAT 收购执行驱动器控制软件。重复此步骤两次获得的数据，一式三份。这种氧化酶的形式产生的光谱。 </li><li>与NADPH的解决方案的1.5倍以上集中在注射器A.酶液的B阀打开的“驱动器”的位置，空和储值停止注射器如7.7中所述的三次更换注射器乙水库的解决方案。 </li><li>替换解决水库注射器C与含氧缓冲区的。的C阀打开“驱动器”的位置和空停止注射器和笔芯7.7倍。在临数据的控制软件，设置在15和900秒的时间延迟和收购，分别。 </li><li>重复步骤7.8。这期间再氧化的酶充分减少产量光谱。 </li></ol> 8。数据分析<ol><li>启动临数据转换软件。点击“ 选项 ”图标，并选择保存在ProDataCSV。 </li><li>打开其中存储的数据文件的目录。 APL的专业数据转换器窗口拖动文件。数据将被自动保存为CSV格式的文件在同一目录中。 </li><li>打开CSV格式的文件，使用Microsoft Excel（微软雷德蒙，华盛顿，美国）。正常化减去相应的吸收在700 nm处的基线停流的痕迹。 </li><li>使用KaleidaGraph（Synergy软件，读，PA）与时间，以适当的指数函数拟合在相应的最大吸收的情节分析修正的痕迹。所示，在452 nm处的吸光度，以确定混合后停止流量仪表的NADPH酶和黄素减少率作为时间的函数绘制图2B。在这种情况下，获得最适合的数据，采用双指数方程和0.65和0.23 S -1率分别为第一和第二阶段的减少，水库计算的职衔。要确定的C4A-hydroperoxyflavin中间的和减少与氧酶反应后，再氧化形成率，我们分析了在380和452 nm处，分别为（ 图3B）停流的痕迹。使用一个单一的指数方程，我们计算的第一步和第二步的氧化半反应速率为 1.4和0.006，分别。 </li></ol> 9。代表结果从前面的章节中所描述的实验结果显示， 一个F思达还原半反应如何，可以通过测量在452 nm处的吸光度的变化，对应黄素的氧化还原状态的变化监测。这一步的速度，可以由数据拟合到相应的方程（ 图2步骤8.4）。减幅获得（0.65秒-1）是类似猫的K值稳态实验，表明减少是速率决定步骤的反应。趁着双混合停流模式，氧化半反应中间体率可确定（ 图3;步骤8.4）。在一个F思达催化反应，C4A-hydroperoxyflavin的明确检测（380 nm的最大 λ），并形成和衰变率，可以计算。所获得的再氧化（0.006秒-1）的速度缓慢表示，C4A-hydroperoxyflavin是在没有非常稳定的鸟。 <img alt="计划1" src="/files/ftp_upload/3803/3803scheme1.jpg" /> 计划1。 一个F思达机制。的基金会辅isoallozaxine的环。氧化黄素（一）结合的NADPH（二）发生反应，形成减少黄素和NADP +（c）。经过与氧分子和有约束力的反应C4A-hydroperoxyflavin的鸟氨酸，形成（四）。这是羟基化的物种。鸟氨酸羟化后，的hydroxyflavin（五）必须脱水形成氧化酶。 NADP +仍然是整个催化循环的约束，是最后被释放（F）的产品。 <img alt="图1" src="/files/ftp_upload/3803/3803fig1.jpg" /> 图1。停流仪器。一）图片应用光物理SX20元件停流分光光度计。二）图片的样品处理单元。双混合模式的流电路）计划。 <img alt="图2" src="/files/ftp_upload/3803/3803fig2.jpg" /> 图2。厌氧与NADPH减少思达在停流仪器。思达30微米和45微米的NADPH等量混合后录制的）的光谱变化。第一谱（氧化SIDA）和最后频谱（充分减少希德A）是蓝色和红色，分别突出。 B）在吸收452 nm的记录作为时间的函数跟踪。 <img alt="图3" src="/files/ftp_upload/3803/3803fig3.jpg" /> 图3。氧化思达在停流仪器。一个记录后，充分减少思达和含氧缓冲区的混合等量）的光谱变化。终浓度为15μM的拔毒，22.5μM的核苷酸，0.95毫米的氧气。频谱记录在0.034，4.268和727.494小号对应充分的酶减少，C4A-hydroperoxyflavin中间的（λ 最大的380纳米），氧化酶（λ 最大的450纳米），分别。 b）在382和452 nm处的吸光度跟踪记录作为时间的函数。

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V., Allou, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+protocatechuate+dioxygenase+for+maintaining+anaerobic+conditions+in+biochemical+experiments.">The use of protocatechuate dioxygenase for maintaining anaerobic conditions in biochemical experiments.</a> Analytical Biochemistry. 286 (2), 187-192 (2000).</li></ol>

我们什么都没有透露。

酶催化氧化还原反应通常含有如血红素和黄素显着的吸光度的变化发生在催化循环的辅助因子。黄素氧化的形式呈现在〜360和450 nm的吸收最大值，它的减少通常在7 450纳米以下的吸收减少监测。在一般情况下，一些短暂的中间体存在，但形式和衰变太快，要定期分光光度计测量。使用应用光物理SX20的停流分光光度计（或类似的工具），它是可以在毫秒时间尺度来衡量的吸光度变化（死区时间，2毫秒）。在这里，我们研究的黄素依赖型单加氧酶的F思达和氧化还原半反应，作为一个模型。氢转移速率决定通过测量与NADPH酶在厌氧条件下混合后在452 nm处的吸光度变化。随后，采取advantagE，酶的双模式混合停流仪器与NADPH反应，直到实现全面减少，减少的酶，辅酶+复杂与氧气混合。在此过程中，它是可以检测瞬态含氧的黄素中间体的形成和衰变率来衡量。这些中间体的鉴定提供了在催化反应物种的性质有关的实验数据。在一个F思达，C4A-hydroperoxyflavin（通常在370-380 nm的监测），这是羟基化物种的形成。此外，每一步的速率常数测量可以获取有关速率决定步骤的反应信息，并有助于阐明酶的动力学和化学机制。 在一般情况下，类似的方法可用于的其他flavoenzymes，或变化发生其中吸收的蛋白质，如蛋白质，是续AIN血红素，哆磷酸盐，非血红素铁8-10。对这种方法的局限性，需要大量纯化的酶，但是这可以通过使用高产量表达系统来克服。一个决定最佳录音谱蛋白浓度，用足够的蛋白质，所以可以观察到一个足够强的信号，但不是太多，使酶不被浪费。通常情况下，停流实验中使用的含黄素酶的酶浓度最低的是6-10微米（混合后），并确定使用相应的酶的摩尔消光系数。在一个F思达的情况下，酶的约束基金会的比例是50-65％2。 APO-蛋白被视为在这些实验中无效，因为绑定的FAD辅基催化是必要的。这种方法的另一个可能的限制是如果在酶的过程发生的速度比2毫秒（停止流动的死区时间），他们不会被观察到，但日ERE报告利率可以下降到解决这一问题的策略。这种情况的一个例子，包括使用高NaCl浓度在1铁氧还蛋白-NADP +还原酶11反应。停止流流电路的氧气擦洗往往是一个棘手的一步，在这个实验中，需要特别注意。这里描述的葡萄糖氧化酶，葡萄糖系统在大多数实验室中成功地使用，因为它是一个有效和廉价的方法。然而，也有一些缺点，其中包括对H 2 O 2的生产，并作为protocatechuate-protocatechuate双加氧酶系统的其他替代品，对于某些应用，应考虑12。利用厌氧手套箱，使得它更容易确保厌氧条件下，但并非是必不可少的。我们希望酶在缺氧的情况下减少或与氧气发生反应，在浓度，氧气必须从停止流流电路那就是我们指定。虽然停流是在手套箱，有流电路中的氧气，如果我们在以前的实验中使用的有氧缓冲区。吸光度测量此外，荧光和圆二色检测可以在应用光物理SX20的停流分光光度计进行相应的配件。

<table border="1"><tbody><tr><td> 通用实验室设备 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>真空泵</td><td>韦尔奇</td><td> - </td></tr><tr><td>布氏烧瓶</td><td>费舍尔</td><td> 70340-500 </td></tr><tr><td>搅拌棒</td><td>费舍尔</td><td> 14-512-129 </td></tr><tr><td>搅拌板</td><td>费舍尔</td><td> 11-100-49S </td></tr><tr><td>舒伦克线</td><td> kontes玻璃</td><td> - </td></tr><tr><td>氩罐</td><td>公司Airgas </td><td>受体UPC300 </td></tr><tr><td>液氮罐</td><td>公司Airgas </td><td> NI200 </td></tr><tr><td>氮气罐，超高纯度等级</td><td>公司Airgas </td><td>倪UHP200 </td></tr><tr><td>氧气罐</td><td>公司Airgas </td><td>牛年40 </td></tr><tr><td> 5％氢平衡氮气罐</td><td>公司Airgas </td><td> X02NI95B200H998 </td></tr><tr><td> SX20圣opped，流分光光度计</td><td> AppliedPhotophysics </td><td> - </td></tr><tr><td>手套箱</td><td>怕羞</td><td> - </td></tr><tr><td>水浴</td><td>布林克曼劳达</td><td> - </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 耗材 </td><td></td><td></td></tr><tr><td> 50毫升BD猎鹰管</td><td>费舍尔</td><td> 14-432-23 </td></tr><tr><td> 15毫升BD猎鹰锥形管</td><td>费舍尔</td><td> 05-527-90 </td></tr><tr><td> 1.5毫升的Eppendorf离心管</td><td>费舍尔</td><td> 05-402-18 </td></tr><tr><td> 50和25毫升玻璃瓶</td><td>费舍尔</td><td> 06-402 </td></tr><tr><td>橡胶瓶塞</td><td>费舍尔</td><td> 06-447H </td></tr><tr><td>铝封条</td><td>费舍尔</td><td> 06-406-15 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> reagenTS </td><td></td><td></td></tr><tr><td>磷酸钾，二氢</td><td>费舍尔</td><td> AC2714080025 </td></tr><tr><td>磷酸钾，二元</td><td>费舍尔</td><td> P288-500 </td></tr><tr><td>醋酸钠</td><td>西格玛</td><td>的S-2889 </td></tr><tr><td> 答：从葡萄糖氧化酶尼日尔 </td><td>西格玛</td><td> G7141-250KU </td></tr><tr><td> D-葡萄糖</td><td>费舍尔</td><td> D16-500 </td></tr><tr><td> β-核苷酸</td><td>费舍尔</td><td> ICN10116783 </td></tr><tr><td> L（+）-鸟氨酸盐酸盐</td><td>费舍尔</td><td> ICN10116783 </td></tr></tbody></table>

monitoring the reductive and oxidative half-reactions of a flavin-dependent monooxygenase using stopped-flow spectrophotometry

曲霉一个铁载体（SIDA）是一个基金会含有单加氧酶催化合成氧肟酸铁载体，是重要的毒力（如ferricrocin或N&#39;，N“，N”&#39; - triacetylfusarinine丙类）1氨酸羟化。由思达催化反应可以分为还原和氧化半反应（图1），在还原半反应，氧化基金会绑定到一个F思达，减少NADPH的2,3。在氧化半反应，减少辅反应与氧分子，形成一个C4A-hydroperoxyflavin中间，其中一个氧原子转移到鸟。在这里，我们描述了一个程序来衡量利率和检测思达不同光谱形式安装在仪器使用停流厌氧手套箱。在停流仪器，小体积的反应物迅速混合，后日停止流通的ë停止注射器（ 图1），在观察细胞中的解决方案的光谱变化随着时间的推移记录。在实验的第一部分，我们将展示我们如何可以使用单一的模式，直接测量黄素NADPH的一个F思达厌氧减少停流仪器。然后，我们使用双混合设置一个F思达第一厌氧NADPH的减少，老龄化循环期间在指定的时间，然后在观察细胞（ 图1）氧分子的反应。为了执行这个实验中，厌氧缓冲区是必要的，因为只有还原半反应监测时，任何解决方案中的氧气反应会减少的黄素辅，并形成一个C4A-hydroperoxyflavin中间，最终会衰变成氧化黄素。这不会使用户能够准确地测量削减率自会有完成营业额的ENZyme。当氧化半反应正在研究的酶必须在缺氧的情况下减少之间的氧化还原的步骤，以便观察。在这个实验中所使用的缓冲区之一是饱和的氧气，使我们可以研究在高浓度的氧气氧化半反应。这些通常的程序进行了研究含黄素的单加氧酶或氧化还原半反应时。停止流进行预稳态实验的时间尺度是秒毫秒，这让内在的速率常数的测定和反应中间体的检测和鉴定。这里所描述的程序，可以应用于其他黄素依赖型单加氧酶5,6。

Watch this Scientific Journal Video about Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry at JoVE.com

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

我们描述了一停流仪器的使用调查还原和氧化半反应<em&gt;曲霉</em&gt;一个铁载体（SIDA），黄素依赖型单加氧酶。然后，我们显示相应的在思达反应的物种的光谱，并计算其形成的速率常数。

监视依赖黄素单加氧酶和氧化还原半反应，用停流分光光度法

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of ...

monitoring-reductive-oxidative-half-reactions-flavin-dependent

Research

JoVE Journal

Bioengineering

15.0K 次观看.  Virginia Polytechnic Institute and State University. 我们描述了一停流仪器的使用调查还原和氧化半反应曲霉一个铁载体（SIDA），黄素依赖型单加氧酶。然后，我们显示相应的在思达反应的物种的光谱，并计算其形成的速率常数。

视频: Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

科研

生物工程

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

在一个容器的支架部署在监测的墙力学

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental technique and image analysis protocol presented here can also be applied for imaging microscopic bubbles in other fields of research; therefore, it has a wide range of applications. We apply this to image cavitation around dental ultrasonic scalers. It is important to image cavitation to characterize it and to understand how it can be exploited for various applications. Cavitation occurring around dental ultrasonic scalers can be used as a novel method of dental plaque removal, which would be more effective and cause less damage than current periodontal therapy techniques. We present a method for imaging the cavitation bubble clouds occurring around dental ultrasonic scaler tips using a high-speed camera and a zoom lens. We also calculate the area of cavitation using machine learning image analysis. Open source software is used for image analysis. The image analysis presented is easy to replicate, does not require programming experience, and can be modified easily to suit the application of the user.

Cavitation microbubbles are imaged using a high-speed camera attached to a zoom lens. The experimental setup is explained, and image analysis is used to calculate the area of the cavitation. Image analysis is done using ImageJ.

使用高速摄像机和图像分析对快速移动微泡区域进行成像和定量

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.