我们感谢狼Frommer教授（卡内基科学研究所，斯坦福大学）的好心为我们提供的葡萄糖FRET质粒和乌韦Ludewig博士（霍恩海姆大学）的亲切供应谷氨酰胺FRET构建在pUTKan植物表达载体。 AB公司是由BBSRC针对性的优先级奖学金计划提供资金。既CK和KP是由英国研究理事会奖学金的生物制药工艺的支持。 CK还要感谢龙沙生物提供财政支持。该中心为合成生物学与创新是慷慨由EPSRC支持。

1。细胞系的复兴和维护<ol><li>恢复在补充有8 2mM L-谷氨酰胺和10ml / L的100倍的次黄嘌呤/胸苷补充物（完全培养基）9毫升的CD-CHO培养基中的CHO细胞。 </li><li>离心机在100×g离心5分钟。 </li><li>重悬在10毫升新鲜的完全生长培养基中的细胞。 </li><li>取出1ml样品，用光学显微镜测定的活菌浓度使用台盼蓝染料排除法用血细胞计数。 </li><li>启动培养以3×10 5细胞/在125毫升毫升的接种密度摇瓶。 </li><li>维持培养在培养箱中在37℃，5％CO 2的潮湿气氛中，在轨道摇动平台旋转在120rpm上。 </li><li>继代培养，每3-4天在完全生长培养基中以2×10 5细胞/ ml的接种密度。 </li></ol> 2。该细胞株与质粒的C转染ontaining的基因生物传感器<ol><li>使用大规模的质粒纯化试剂盒制备的质粒DNA。 </li><li>维持细胞在适当的条件下（37℃，5％CO 2）的12-24小时转染前，以确保细胞活跃分裂在转染时。 </li><li>使用台盼蓝染料排除方法计数细胞，然后制备20 ml的细胞培养物中加入125ml工作体积摇瓶中以1×10 6个细胞/ ml的浓度。 </li><li>使用合适的转染试剂盒中使用的细胞系。质粒DNA转染试剂的比例将依赖于细胞系和载体。值得推荐，这是通过测试的范围由制造商进行的最终转染前规定的范围内的比率的最优化。还维持含有细胞，但没有DNA的至少一个阴性对照的文化。 </li><li>孵育在静态模式下4天，然后转移到一个夏奇纳克平台转速为125转每分钟。 </li><li>添加适当的抗生素进行质粒选择到合适的浓度为通过杀灭曲线来确定。在这项研究中，硫酸博来霉素加至400微克/毫升供选择转染的细胞的终浓度。 </li><li>在该细胞系的适当时间间隔改变介质，抗生素每次添加，直到细胞中控制以及已经死亡。 </li><li>用最汇合井进步到颤抖的文化。 </li><li>冻结的细胞中含有10 7活菌1毫升冷冻混合冻存管建立细胞库。在这项工作中，这是由92.5％的生长培养基和7.5％二甲亚砜。 </li></ol> 3。批次和补料分批细胞生长曲线<ol><li>遵循用于细胞维持上述说明，建立转染的CHO细胞在250毫升的一式三份细胞培养摇瓶中的50毫升的工作容积。 </li><li>保持丘尔清晰度，在湿润的细胞培养箱中，在37℃，5％的CO 2，在轨道摇动平台旋转125转，并从每个生长培养除去4.1毫升样品在24小时的时间间隔。 </li><li>用100微升的样品，以确定与台盼蓝染料排斥法的活细胞浓度和细胞活力。 </li><li>重复，直到活细胞的浓度降低至零。 </li><li>重复分批补料细胞培养与补充葡萄糖或谷氨酰胺在第6天的适量恢复其浓度为36毫米和4毫米它们的初始值，分别为。建议额外的控制培养物也保持，这是与纯水的相同体积（12毫升这种情况下）被输送。 </li></ol> 4。 FRET比率测量<ol><li>取2毫升每日样品除去在上面的步骤3.2和离心机在100×g离心5分钟，在4℃下</li><li> Resusp在2ml冰冷的磷酸盐缓冲盐水（PBS）中的止细胞沉淀。转移到这一个6孔板和不含细胞的空白样品加至一个阱。 </li><li>紧接在四百三十五分之四百三十nm，发射四百三十五分之四百六十五纳米（蓝色）和五百十分之五百二十零纳米（黄色）的波长的激发波长测量的蓝色和黄色的荧光水平。 </li><li>计算FRET比率（检测过检测蓝色荧光黄色荧光的比值）。 </li></ol> 5。代谢产物的测定<ol><li>取2毫升每日样品除去在上面的步骤3.2和离心机在100×g离心5分钟，在4℃下除去上清液。 </li><li>再次重悬细胞沉淀在3毫升冰冷的PBS和离心机在100×g离心5分钟。 </li><li>去除上清，重悬细胞沉淀在3毫升冰冷的PBS如上。超声处理的样品5倍的每个3分钟，在15秒的脉冲和15秒关闭。在这一点上，所述细胞额外如果需要的话仙可能会被冻结。 </li><li>使用适当的葡萄糖测定试剂盒按照制造商的关于细胞提取物的样品的指示，以确定细胞内的葡萄糖的浓度（见表材料）。 注意：使用适当的标准来建立标准曲线。在这项工作中，标准的浓度0-100毫米之间不等。 </li><li>使用读数为标准，构建标准曲线，并找到最佳拟合的线性方程（在这种情况为：y = 1310.51x - 723.43其中y为吸光度读数和x是葡萄糖浓度）。 </li><li>利用标准曲线，并采取稀释考虑在内计算的葡萄糖浓度。细胞内的葡萄糖浓度然后可以使用这个公式计算： <img alt="" fo:content-width="4.5in" src="/files/ftp_upload/51200/book_bg.png" width="500" /> 对于CHO细胞，单细胞的体积，用12μM12上的泡孔直径计算</SUP&gt;。 </li><li>使用适当的谷氨酰胺测定试剂盒按照制造商的关于细胞提取物的样品的指示，以确定细胞内的谷氨酰胺浓度（见表材料）。 注意：使用适当的标准来建立标准曲线。在这项工作中，标准的浓度为0-2毫米之间。 </li><li>如在5.5以上，构建校准曲线（在这种情况下，y = 203.1x + 17.1其中y是吸光度读数和x是葡萄糖浓度） </li><li>利用标准曲线，计算出样品的谷氨酰胺浓度，取稀释考虑在内。细胞内谷氨酰胺浓度可以使用公式1以上计算。 </li></ol>

<ol>
	<li>Moser, I., Jobst, G., Urban, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensor+arrays+for+simultaneous+measurement+of+glucose,+lactate,+glutamate,+and+glutamine.">Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine.</a> Biosens. Bioelectron. 17 (4), 297-302 (2002).</li><li>Billingsley, K., 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=Fluorescent+nano-optodes+for+glucose+detection.">Fluorescent nano-optodes for glucose detection.</a> Anal. Chem. 82 (9), 3707-3713 (2010).</li><li>Dattelbaum, J. D., Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Determination+of+Glutamine+Using+a+Genetically+Engineered+Protein.">Optical Determination of Glutamine Using a Genetically Engineered Protein.</a> Anal. Biochem. 291 (1), 88-95 (2001).</li><li>Kfouri, 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=Toward+a+miniaturized+wireless+fluorescence-based+diagnostic+imaging+system.">Toward a miniaturized wireless fluorescence-based diagnostic imaging system.</a> IEEE J. Sel. Top. Quantum Electron. 14 (1), 226-234 (2008).</li><li>Dittrich, P. S., Manz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+detection+in+microfluidic+channels&#8212;the+Holy+Grail+in+muTAS.">Single-molecule fluorescence detection in microfluidic channels&#8212;the Holy Grail in muTAS.</a> Analytical and Bioanalytical Chemistry. 382 (8), 1771-1782 (2005).</li><li>Hou, B. H., Codamo, J., Pilbrough, W., Hughes, B., Gray, P. P., Munro, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sensors+for+monitoring+dynamic+changes+of+intracellular+metabolite+levels+in+mammalian+cells.">Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells.</a> Nat. Protoc. 6 (7), 1818-1833 (2011).</li><li>Bermejo, C., Haerizadeh, F., Takanaga, H., Chermak, D., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+cytosolic+glucose+and+ATP+levels+in+yeast+using+optical+sensors.">Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.</a> Biochem. J. 432, 399-406 (2010).</li><li>Yang, H. Y., Bogner, M., Stierhof, Y. D., Ludewig, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H(+)-independent+glutamine+transport+in+plant+root+tips.">H(+)-independent glutamine transport in plant root tips.</a> Plos One. 5, (2010).</li><li>Fehr, M., Takanaga, H., Ehrhardt, D. W., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+high-cavacity+bidirectional+glucose+transport+across+the+endoplasmic+reticulum+membrane+by+genetically+encoded+fluorescence+resonance+energy+transfer+nanosensors.">Evidence for high-cavacity bidirectional glucose transport across the endoplasmic reticulum membrane by genetically encoded fluorescence resonance energy transfer nanosensors.</a> Mol. Cell. Biol. 25 (24), 11102-11112 (2005).</li><li>Ai, H. W., Hazelwood, K. L., Davidson, M. W., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+FRET+pairs+for+ratiometric+imaging+of+dual+biosensors.">Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors.</a> Nat. Methods. 5 (5), 401-403 (2008).</li><li>Ouyang, M. X., Sun, J., Chien, S., Wang, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+hierarchical+relationship+of+Src+and+Rac+at+subcellular+locations+with+FRET+biosensors.">Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (38), 14353-14358 (2008).</li><li>Han, Y., 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=Cultivation+of+recombinant+Chinese+hamster+ovary+cells+grown+as+suspended+aggregates+in+stirred+vessels.">Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.</a> J. Biosci. Bioeng. 102 (5), 430-435 (2006).</li><li>Deuschle, K., Okumoto, S., Fehr, M., Looger, L. L., Kozhukh, L., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+optimization+of+a+family+of+genetically+encoded+metabolite+sensors+by+semirational+protein+engineering.">Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering.</a> Protein Sci. 14 (9), 2304-2314 (2005).</li><li>Wong, D. C. F., Wong, N. S. C., Goh, J. S. Y., May, L. M., Yap, M. G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+N-Glycosylation+gene+expression+in+CHO+cell+Fed-batch+cultures.">Profiling of N-Glycosylation gene expression in CHO cell Fed-batch cultures.</a> Biotechnol. Bioeng. 107 (2), 516-528 (2010).</li><li>Behjousiar, A., Kontoravdi, C., Polizzi, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Monitoring+of+Intracellular+Glucose+and+Glutamine+in+CHO+Cell+Culture.">In Situ Monitoring of Intracellular Glucose and Glutamine in CHO Cell Culture.</a> PLoS One. 7, (2012).</li><li>Dwyer, M. A., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periplasmic+binding+proteins:+a+versatile+superfamily+for+protein+engineering.">Periplasmic binding proteins: a versatile superfamily for protein engineering.</a> Curr. Opin. Struct. Biol. 14 (4), 495-504 (2004).</li></ol>

作者什么都没有透露。

该FIBS使在体内和关键分子原位定量，在这种情况下生长限制性营养物质，除去从淬火和提取方法所产生的不确定性。调查结果表明，有在FIBS信号和细胞内浓度在1-5毫米的范围内葡萄糖和0.3-2mm的谷氨酰胺之间的良好关系。在批量的CHO细胞培养中，这些浓度中的指数，固定的，并且早下降阶段遇到的问题。该指数相和固定相是最关键的适宜喂养策略的设计方面，因此使用这些生物传感?...

代谢物监测在生物加工各种应用，包括工艺开发，媒体和饲料的设计，以及代谢工程。各种方法可用于浓度测量通过使用酶1，化学2，或结合试验3。一个有趣的选项是使用荧光，蛋白质为基础的体内生物传感器作为关键代谢产物的细胞内浓度的记者。在超低量测定法，荧光是一种方便的工具，如小型化实际上是改善了信号-噪声比4,5和蛋白为基础的传感器可遗传编码的意义，没有外源试剂是必需的代谢物的分析。福斯特共振能量转移（FRET）生物传感器包括一个配体结合域连接的两个荧光蛋白。 FRET是能量从光激发捐助者的一个非辐射转移到受体荧光分子要价较高，<html>编接近（&lt;100）。配体的裂解或结合引起的构象变化的传感器，其中，反过来，诱导的荧光基团的接近而变化，导致通过在发光光谱的变化测定FRET效率的变化。 FRET集成生物传感器（FIBS）的细胞系中是组成型产生的和它们的光谱特性改变的基础上代谢物在细胞内的浓度。 FIBS具有快速响应时间使它们非常适合进行测量的动态模型6。以前的应用包括监测单7-9和10多个代谢物，以及提供有关时空分布11个数据。载脂蛋白最大，其中配体结合打乱降低能量传递和载脂蛋白民，其中配体结合所带来的两个荧光基团成更紧密的接触（ 图1）的荧光团的接近：FIBS可以在两种配置中创建。 _content“&gt;在这项工作中，一个协议，提出构建中国仓鼠卵巢（CHO）细胞系中是组成型表达一个FIBS为代谢物，葡萄糖或谷氨酰胺，一种方法是建立在传感器体 ​​内的校准，以使将来的定量胞内代谢物浓度的测量，如示于图2。基于此，葡萄糖或谷氨酰胺的细胞内浓度可在补料分批的CHO细胞培养物测定中，向其中两种养分中加入高浓度的过程，其结果证明由FIBS和先前构建的校准曲线使用的荧光信号，准确预测细胞内浓度是可能的，并经一个独立的酶法测定，该方法提供了超过当前的分析技术显着的优点，因为它是无创的，低成本的快，给，可在星期一FIBS的实时信号itored整个文化。

<table border="0" cellpadding="0" cellspacing="0" width="936">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">CHO-S Cells</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;">11619-012</td>
			<td style="width:459px;">Cell line will vary depending on the goals of the study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">pCDNA4/TO vector&#160;</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">TransIT-PRO Transfection Reagent</td>
			<td style="width:120px;">Mirus Bio</td>
			<td style="width:120px;">MIR 5700</td>
			<td style="width:459px;">Transfections can be accomplished using any method suitable for the cell line under study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Zeocin</td>
			<td style="width:120px;">Invivogen</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">CD-CHO medium</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">10743-011</td>
			<td style="width:459px;">Cell growth medium is dependent upon the cells under study</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">100X HT Supplement</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">11067-030</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">L-Glutamine 200 mM (100X), Liquid</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">25030032</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">InfinitePRO 200 plate reader</td>
			<td style="width:120px;">Tecan</td>
			<td style="width:120px;">FLx800TBI</td>
			<td style="width:459px;">Any 96-well fluorescence plate reader that can access the required wavelengths can be substituted</td>
		</tr>
		<tr height="21">
			<td height="190" rowspan="6" style="height:190px;width:239px;">Filters for plate reader</td>
			<td rowspan="6" style="width:120px;">Tecan</td>
			<td align="right" style="width:120px;">30000463</td>
			<td rowspan="6" style="width:459px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(520NM BW 10NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(430NM BW 35NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022787</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">(465NM BW 35NM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Maxiprep Plasmid Purification Kit</td>
			<td style="width:120px;">Qiagen</td>
			<td style="width:120px;">12163</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Amplex Red glucose/glucose oxidase assay kit</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:120px;">A22189</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">EnzyChrom&#160; Glutamine Assay Kit</td>
			<td style="width:120px;">BioAssay systems</td>
			<td style="width:120px;">EOAC-100</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Improved Neubauer haemocytometer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:120px;">MNK-420-010N</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Incubator&#160;</td>
			<td style="width:120px;">Nuaire</td>
			<td style="width:120px;">NU-5510E</td>
			<td style="width:459px;"></td>
		</tr>
	</tbody>
</table>

fibs-enabled noninvasive metabolic profiling

在计算生物学的时代，新的高通量实验系统是必要的，以便填充和完善的模型，使他们能够进行预测进行验证。理想的情况是这样的系统将是低量，这就排除了采样和破坏性的分析时，时间过程的数据将被获取。所需要的是一种在原位监测工具，它可以在实时和非侵入性地报告必要的信息。一个有趣的选项是使用荧光，蛋白质为基础的体内生物传感器作为细胞内浓度的记者。已经发现应用中代谢物的定量一个特定类的体内生物传感器是基于共振能量转移（FRET）的配体结合结构域连接的两个荧光蛋白之间。 FRET集成生物传感器（FIBS）细胞系中都产生组成，他们有快速的响应时间和他们的茶谱根据代谢物在细胞内的浓度特性的研究改变。在本文中，该方法用于构建中国仓鼠卵巢（CHO）细胞系中是组成型表达一个FIBS为葡萄糖和谷氨酰胺和校准体内批次细胞培养物中FIBS以使胞内代谢物浓度的将来量化进行说明。从进料 - 分批的CHO细胞培养数据表明，FIBS能够在每种情况下，检测在细胞内的浓度所产生的变化。使用从FIBS和先前构建的校准曲线的荧光信号，细胞内浓度，精确地确定所确认的独立酶法测定。

该方法的概述示于图2。在这里提出的工作，CHO细胞用的FIBS载体和稳定细胞系，在400微克/毫升硫酸博来霉素的抗生素压力被选定。两个独立的稳定细胞系组成性表达的葡萄糖和谷氨酰胺的传感器因此被创建。 图1描述了在本研究中使用的生物传感器2的配置。葡萄糖传感器是基于载脂蛋白 - 最大原理和相应的质粒是PRSET-FLIPglu600μΔ11Aphrodite。这是一种融合增强型青色荧光蛋?...

Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

FIBS-enabled Noninvasive Metabolic Profiling

如何在原位代谢轮廓校正共振能量转移集成生物传感器（FIBS）用于描述呈现。该FIBS可用于测量非侵入性代谢物辅助的代谢模式和生物过程条件的高通量筛选的发展的细胞内水平。

FIBS功能的无创性代谢轮廓

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

fibs-enabled-noninvasive-metabolic-profiling

Research

JoVE Journal

Bioengineering

9.6K 次观看.  Imperial College London. 如何在原位代谢轮廓校正共振能量转移集成生物传感器（FIBS）用于描述呈现。该FIBS可用于测量非侵入性代谢物辅助的代谢模式和生物过程条件的高通量筛选的发展的细胞内水平。 

视频: FIBS-enabled Noninvasive Metabolic Profiling

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

科研

生物工程

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

原位压极限和骨牙周韧带齿纤维联合的相关性无创成像

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

定量和大小分析外囊泡的使用可调电阻脉冲检测

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

在Micropost阵列方便，准确机械力分析

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

利用N酰基修饰 Mannosamines 对唾液酸的代谢 Glycoengineering

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

磁共振显微镜下切除活体脑组织的代谢支持

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

纳米传感器检测体内蛋白酶活性的无创诊断

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...