作者想感谢克劳斯Kaestner博士Ngn3 - EGFP的小鼠胚胎干细胞系，布朗夫人露西和分析协助细胞分选流式细胞仪核心的礼物。这项工作是由NIH资助R21DK069997授予HTK的R01DK081587和加州再生医学研究所（摆围）授予兆瓦和LS的培训补助资金部分

1。胰腺细胞样细胞在体外的小鼠ES细胞分化生成的殖民地检测条件培养基（CM ） 小鼠胚胎干细胞的维护程序，胚体（EB）的悬浮培养和附件文化的形成，为进一步分化已先前所描述的1-3和本报告的重点。图1显示了一个在我们的分化协议所涉及的步骤示意图介绍。 为了获得高品质的空调媒体，重要的是首先产生6日龄胚，其中至少有30-40％的直径至少150微米。更大的分化应该包含在光学显微镜下的暗斑，表明增强分化（图2）。选择高品质的小牛血清（FCS）是至关重要的生成等胚。高品质的FCS地段的选择是基于自己的能力，以支持differentiatEBS的离子对胰岛素，胰高血糖素和淀粉酶2A基因表达的后期阶段（18-20天），由RT - PCR分析检测。此外，当一天6胚悬浮培养附件文化（约80 - 100胚在6孔板，每孔），重要的是，轻轻地旋转板收集到的井中心6日胚。对于原因不明，纵横捭阖，加强细胞与细胞接触EBS胰样细胞，在随后的附件文化阶段更好地发展的结果。 <ol><li>在培养第13天，取代新鲜附件文化的依恋文化传媒（DMEM/F-12（1:1），15％的血清替代淘汰赛，2毫米L -谷氨酰胺，50 U / mL青霉素，50μg/ mL链霉素）媒体containing10 MM烟酰胺，0.1纳米exendin - 4，和10毫微克/毫升的重组人激活素SSB（最终浓度）。 </li><li>在培养第16天，收集从步骤1.1媒体和过滤0.2微米聚醚ulfone膜。从这点出发，将收集媒体称为条件培养基（CM）。分装成15毫升猎鹰管的CM，并存放于-80 ° C直到前半固体培养（见下文第3节）。 </li></ol> 2。获得胰祖细胞在单细胞悬液，使用荧光激活细胞分选一阵敲门声在小鼠胚胎干细胞系，4个指定为Ngn3 - EGFP，EGFP报告基因取代一个Ngn3位点的等位基因是以前2 Ngn3是一个基本的螺旋-环-螺旋（BHLH）含有转录因子分化表示5天16文化发展过程中胰腺内分泌祖通常排序，获得差异Ngn3 - EGFP +和Ngn3 - EGFP -细胞1,2 <ol><li>分化的细胞分解成单细胞悬液。 <ol><li>孵育16天，文化的Wi第0.25％胰蛋白酶- EDTA（3分钟，37℃）逐出文化井的细胞。 </li><li>添加10％FCS停止胰蛋白酶的活性。 </li><li>用镁和钙的细胞，磷酸盐缓冲液（PBS）含有0.1％的牛血清白蛋白（BSA）和离心5分钟在300 XG。 </li><li>去除上清液，4 mg / mL胶原酶B和2000 U / mL的脱氧核糖核酸（DNA酶）我（30分钟，37℃）培养的细胞团块，以生产单细胞悬液。使用1000μL移液器破坏细胞颗粒每10分钟，以加快解离过程。 </li><li>洗涤细胞，用PBS含0.1％BSA和300 XG离心5分钟。 </li><li>去除上清液和含0.1％BSA的PBS重悬细胞。 </li></ol></li><li>制备细胞进行排序。 <ol><li>每毫升细胞悬液，以防止在排序细胞reaggregation添加2μLDNA酶我（1亩/毫升）。 </li><li>通过一个40至70微米米的单细胞悬液ESH除去大细胞聚集。 </li><li>添加的DAPI（1μg/ mL的终浓度）的单细胞悬液。 </li><li>在细胞分选获得的流式细胞仪检测数据。一个MoFlo MLS（Beckman Coulter公司）细胞分选，用于本报告。 </li><li>浇注的细胞群进行排序。 Ngn3 - EGFP +和Ngn3 - EGFP -细胞门控前向散射对侧向散射（图3A），其次的DAPI与侧散射（图3B），脉冲宽度与侧散射（图3C），最后FL1与自体荧光（图3D）。最终排序门Ngn3 - EGFP +细胞是故意转移到右侧（图3E;箭头），以限制污染Ngn3 - EGFP -细胞在排序。对于阴性对照闸，无论是每天6 Ngn3 - EGFP胚胎干线或相同的阶段，每天16文化，从非转基因株系，如TL1 ES细胞（图3D，左侧面板），可用于EBS。 </li></ol></li><li>排序Ngn3 - EGFP +和Ngn3 - EGFP <suP&gt; -细胞。 </li></ol> 3。电镀排序细胞半固体培养<ol><li>基质胶的准备。 <ol><li>在基质胶分装至1 mL，每管使用前在-20 ° C存储。 </li><li>冷冻分装应置于4 ° C过夜或冰刚刚解冻，使用前3个小时。解冻分装必须保持在半固态的文化准备，以避免resolidification冰。 </li></ol></li><li>甲基纤维素溶液（3.3％）的准备。 甲基纤维素粉末不能高压灭菌。为了获得细菌甲基解决方案在编制过程中，使用清洁的称量船和蒸压搅拌棒，烧杯，烧瓶和其他硬件。 <ol><li>前一天的甲基纤维素溶液的制备，存储200毫升的无菌双蒸水在4 ° C此外，准备的DMEM/F12媒体（由溶解粉状DMEM / F - 12在做准备了一倍蒸馏水）含100 U / mL青霉素和500毫升2X100μg/ mL链霉素，储存于4 ° C。 </li><li>在一天的甲基准备，加无菌双蒸水500毫升到1000毫升烧杯，在上面的铝箔片。煮沸至少30分钟，以去除水中的气泡。 </li><li>一个2000毫升的玻璃瓶中放入一个大型搅拌棒（至少3英寸长）。 </li><li>测量300毫升的开水，并转移到容量瓶中。 </li><li>烧瓶放在小的轰动板（不加热）。慢慢搅拌的热水，以避免产生气泡。 </li><li>称取甲基纤维素粉33克，非常缓慢地把它添加到热水。热水是必要的湿甲基粉效率和援助在寒冷的温度随后解散。 </li><li>继续搅拌粉末，直到温度降低到约40-45 ° C。 </li><li>加入200毫升冷无菌双蒸水冷却甲基混合物。紧接后，你应该开始看到的解决方案变成透明粘稠，表明甲基是水相溶解。手摇动烧瓶，以确保解决方案是彻底混合。 </li><li>加入500毫升冷2X的DMEM/F12媒体。继续漩涡手工混合。 </li><li>在寒冷的房间的轰动板放在烧瓶中，慢慢搅拌24-48小时甲基解决方案。 </li><li>分装成125毫升，每储存瓶，并储存在-20 ° C的解决方案一个样品应分装到无菌petridish的，并置于37 ° C培养箱无菌测试。解冻甲基纤维素溶液可存放在冰箱中长达2个月。 </li></ol></li><li>空调媒体准备。 解冻CM（见第1条），只是在使用前。保持整个冰半固体培养制备的CM。 </li><li>准备以下的生长因子：1.0 M，0.1微米exendin - 4，10μg/ mL的重组人激活素βB烟和10＆MU; g / mL的VEGF - A的。储存在-20烟酰胺和exendin - 4 ° C和激活素βB和VEGF - A在-80℃，解冻前立即使用。所有半固体培养媒体组件应保持在冰上，在电镀过程。应当指出，除了血管内皮生长因子- A，后来发现没有必要的集落形成。 </li><li>小牛血清的制备。 FCS应在使用前灭活的热量。孵育30分钟的FCS在56 ° C水浴中灭活补体蛋白。如果最初的FCS股票瓶拥有超过500毫升分装成125毫升小瓶。表面积增加将确保整个卷和彻底加热。允许瓶在室温下冷却至少1小时前贮存于-20 ° C解冻的FCS是0.2μm的过滤后方可使用。 </li><li>排序细胞混合文化的组成部分。 期间随时准备在任何时候，以防止凝固的冰细胞和所有半固态媒体组件基质胶。与基质胶包括接触的所有物资，枪头​​，注射器和针头，应冷。放置在-20℃至少一个半小时前使用这些用品。 <ol><li>确定接种于24孔板，每孔细胞数量。可镀多达25000细胞每24孔。一个可以执行在最初的实验中的细胞密度滴定，以确定最佳的种子细胞数量。 </li><li>添加到5毫升的聚苯乙烯管文化的组成部分，在表1中所​​述的顺序单元盖（请注意，最后添加，以避免过早刺激细胞）。添加3.3％的甲基聚苯乙烯管第一次和最后排序细胞。注意，3.3％甲基纤维素溶液，需要制定并添加到16 ½号针头和适当的标记的毕业注射器聚苯乙烯管。在一般情况下，为了正确测量粘性的解决方案的体积，如3.3％的甲基纤维素溶胶ution和最终混合文化，重要的是先在注射器吸一些解决方案，然后立即推的解决方案，以排出空气。 3.3％甲基纤维素溶液可被加热到室温，如果实在是太难了画出来。但是，请确保它是在冰上冷却后配发到聚苯乙烯管和前基质胶此外的。 </li></ol></li><li>板的半固体培养基含有排序细胞。 <ol><li>在确保聚苯乙烯管帽坚决落实到位，大力管，彻底动摇手。在此过程中会产生气泡。 </li><li>管放在冰上5分钟或直到小气泡表面。 </li><li>一个1ml注射器使用16半或18个半针头吸出内容。按照3.6.2的意见，没有空气在注射器绘制准确的体积粘性溶液。 </li><li>到每个24孔，慢慢地免除500 UL媒体。使用低附件24孔板。直接添加媒体以及中心。半固态媒体会自动传播，在井。对于每个实验样品，quadruplicated井经常使用，以获得统计学意义的结果。只有最里面的一个水平方向的24孔板4列用于文化。用无菌水填充剩下的水井，以帮助细胞含有文化的加湿。 </li><li>在37 ° C和5％CO 2空气培养的细胞。 </li><li>延迟增长的因素除了。启动后的文化，它可以添加生长因子。这种演习可能被用来解决某些生长因子的生存影响。可通过1 mL注射器滴生长因子的解决方案，以每孔100μL用26克3 / 8针，并允许扩散到半固体培养基。 </li></ol></li></ol> 4。光显微镜下观察菌落形成应付应观察细胞电镀后立即以确保单个细胞随机均匀地分布整个出井。如果大多数的细胞或造成菌落分布在井的边缘，这将表明，配药入井的半固体培养基太用力（见3.7.4节）。由于半月板效应在殖民地的文化井的边缘密度相比，那些居住在该中心可能会更高。 <ol><li>菌落总数的量化。由于媒体的3维特征，殖民地将出现在不同的重点。因此，可能需要通过光镜观察期间的重点调整为每个殖民地。附加下24孔一格，当计数，以避免两次注册相同的殖民地。网格可能会获得通过切割一个NUNC petridish墙（货号No.169558）。一个10倍的物镜，光镜下应使用OBServe和计数的殖民地。 </li></ol> 5。代表性的成果： 以前的实验表明，2.5 × 10 5小鼠R1 ES细胞（50毫升媒体总）开始，人们可以预期产生约5000适当大小的6天的胚。 Ngn3 - EGFP胚胎干细胞的效率较低; 4倍以上的细胞产生类似大小分化第6天。由于25日6胚包含平均为1.88 ± 0.67 × 10 5细胞，可从5000 EBS 1约37.6 × 10 6细胞。在附件的文化细胞有望扩大2.72 ± 0.32倍， 因此 ，5000一天6 EBS将产生约100 × 10 6天16个单元，前排序。浇注后，Ngn3 - EGFP +细胞会弥补约10％的总人口（议定书案文第2.2.5节和图3）日16细胞。 示例的photomicrogr胰岛素对小鼠ES细胞的菌落aphs如图4所示。这些殖民地有一个独特的形态;小（60-100微米）的单个细胞的全面菌落小，皮肤黝黑，不反光的殖民地。在我们以前的出版物，实时RT - PCR和双单独钦点菌落免疫荧光染色显示表达胰岛素，C肽和胰高血糖素。这些结果表明，单Ngn3 - EGFP +细胞能够分化成至少包含两个胰岛激素同时细胞，类似如内分泌细胞的原始第一波。 分散的单个细胞集落检测，使那些有能力开始在体外（图5）的增殖和分化的祖细胞的研究。因此，这是一个实验，可以测量个别祖细胞的内在功能。的祖先有capability成为胰岛素表达殖民地被称为胰岛素表达的菌落形成单位（ICFUs）（图5）。一致的想法，Ngn3 - EGFP Ngn3标志着在发展胰腺内分泌祖细胞，5 +，但不Ngn3 - EGFP -从mES细胞衍生的人口的细胞丰富的 ICFUs 2然而，这些祖细胞的频率仍然是低;总Ngn3 - EGFP +从第16天的文化孤立人口约0.1％至0.6％ICFUs 2不管，预计Ngn3 - EGFP +细胞集落形成效率的一个明显的区别是最高的，其次由。预排序的细胞，然后Ngn3 - EGFP -细胞。 <img alt="图1" src="/files/ftp_upload/3148/3148fig1.jpg" /> 图1。时间轴在小鼠ES细胞体外分化为胰腺细胞样细胞。对于条件培养基（CM）生成，小鼠胚胎干CELLS是增长15％FCS第6天的monothioglycerol减少剂量（6.0 × 10 -3米以下四个天的前两天和6.0 × 10 -4中号）产生分化处于悬浮状态。日6 EBS（80-100每口井），然后转移到附件6以及含有15％的淘汰赛血清替代的菜。可能会增加5％FCS之间文化的6-10天，以加快EBS的附件。烟酰胺，exendin - 4和重组人激活素βB将被添加到媒体文化的第13天（见1.1节）。媒体上收集培养16天，在这一点已成为空调的媒体。 16天的细胞，然后进行排序，进入半固体培养基菌落测定镀。 <img alt="图2" src="/files/ftp_upload/3148/3148fig2.jpg" /> 图2，大天，6胚从小鼠胚胎干细胞衍生的机构代表的显微照片。理想的一天，6胚至少150微米直径度计，有黑暗的中心（箭头）。早期胰腺祖细胞标记（如PDX - 1和Sox9）的表达水平在这些EBS（数据未显示）。 <img alt="图3" src="/files/ftp_upload/3148/3148fig3.jpg" /> 图3。排序mES细胞派生日16 Ngn3 - EGFP表达的大门。 （一）正向和侧向散射门，细胞的大小和粒度分别指示，消除非细胞碎片。 （二）的DAPI是用于识别死亡细胞，将染色阳性，在405-413纳米和排放检测与四十〇分之四百五十○过滤器激发。 （三）脉冲宽度，前向散射的电子脉冲的宽度，揭示双峰，应通过荧光激活细胞分选前通过淘汰。 （四）浇注EGFP（通道FL1）与自体荧光使用16日从父母的控制，如TL1（左侧面板）细胞株细胞照亮真实的荧光，从而Ngn3 - EGFP的&lt;SUP&gt; +细胞群（右图）。 （五）红色箭头显示的EGFP +门手动排序，以增加假阳性细胞的分离之前右移。 （D）和（E）的门控基于对地区活动（R1 - R3）（AC）。 <img alt="图4" src="/files/ftp_upload/3148/3148fig4.jpg" /> 图4。代表显微照片胰岛素表达在半固体培养的殖民地。殖民地是随机分布，出现小暗，非反光集群。单个菌落，以后可以检测基因和蛋白表达的RT - PCR和免疫组化分析，分别为（未显示;见视频）。 <img alt="图5" src="/files/ftp_upload/3148/3148fig5.jpg" /> 图5。殖民地的实验，让单系祖细胞的功能和定量评估。造成菌落记数，用来计算频率uency之间总镀细胞的祖细胞。在每个殖民地组成的细胞发起祖的谱系潜力的指标。使用这个实验中，我们发现，丰富Ngn3 - EGFP +从小鼠胚胎干细胞衍生的细胞表达胰岛素的菌落形成单位（ICFUs），祖细胞有能力分化为胰岛素表达殖民地。 <img alt="表1" src="/files/ftp_upload/3148/3148table1.jpg" /> 表1。半固体培养媒体组件和添加顺序。

<ol>
	<li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Committing+embryonic+stem+cells+to+early+endocrine+pancreas+in+vitro.">Committing embryonic stem cells to early endocrine pancreas in vitro.</a> Stem cells. 22, 1205-1217 (2004).</li><li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-expressing+colonies+developed+from+murine+embryonic+stem+cell-derived+progenitors.">Insulin-expressing colonies developed from murine embryonic stem cell-derived progenitors.</a> Diabetes. 56, 921-929 (2007).</li><li>Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+in+vitro+differentiation+assay+for+pancreatic-like+cell+development+from+murine+embryonic+stem+cells:+detailed+gene+expression+analysis.">Characterization of an in vitro differentiation assay for pancreatic-like cell development from murine embryonic stem cells: detailed gene expression analysis.</a> Assay. Drug. Dev. Technol. 9, 403-419 (2011).</li><li>Lee, C. S., Perreault, N., Brestelli, J. E., Kaestner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin+3+is+essential+for+the+proper+specification+of+gastric+enteroendocrine+cells+and+the+maintenance+of+gastric+epithelial+cell+identity.">Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity.</a> Genes. 16, 1488-1497 (2002).</li><li>Gu, G., Dubauskaite, J., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+the+pancreatic+lineage:+NGN3++cells+are+islet+progenitors+and+are+distinct+from+duct+progenitors.">Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.</a> Development. 129, 2447-2457 (2002).</li><li>Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+proliferation+of+hematopoietic+stem+cells.">Differentiation and proliferation of hematopoietic stem cells.</a> Blood. 81, 2844-2853 (1993).</li><li>Gittes, G. K., Galante, P. E., Hanahan, D., Rutter, W. J., Debase, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-specific+morphogenesis+in+the+developing+pancreas:+role+of+mesenchymal+factors.">Lineage-specific morphogenesis in the developing pancreas: role of mesenchymal factors.</a> Development. 122, 439-447 (1996).</li><li>Miralles, F., Battelino, T., Czernichow, P., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+plays+a+key+role+in+morphogenesis+of+the+pancreatic+islets+of+Langerhans+by+controlling+the+activity+of+the+matrix+metalloproteinase+MMP-2.">TGF-beta plays a key role in morphogenesis of the pancreatic islets of Langerhans by controlling the activity of the matrix metalloproteinase MMP-2.</a> J. Cell. Biol. 143, 827-836 (1998).</li><li>Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cultivation+of+human+islets+from+expanded+ductal+tissue.">In vitro cultivation of human islets from expanded ductal tissue.</a> Proc. Natl. Acad. Sci. U. S. A. 97, 7999-8004 (2000).</li><li>Gao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+endocrine+progenitor+cells+and+critical+factors+for+their+differentiation+in+human+adult+pancreatic+cell+culture.">Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture.</a> Diabetes. 52, 2007-2015 (2003).</li><li>Gao, R., Ustinov, J., Korsgren, O., Otonkoski, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+neogenesis+of+human+islets+reflects+the+plasticity+of+differentiated+human+pancreatic+cells.">In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells.</a> Diabetologia. 48, 2296-2304 (2005).</li><li>Suarez-Pinzon, W. L., Lakey, J. R., Brand, S. J., Rabinovitch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+with+epidermal+growth+factor+and+gastrin+induces+neogenesis+of+human+islet+beta-cells+from+pancreatic+duct+cells+and+an+increase+in+functional+beta-cell+mass.">Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet beta-cells from pancreatic duct cells and an increase in functional beta-cell mass.</a> J. Clin. Endocrinol. Metab. 90, 3401-3409 (2005).</li><li>Hao, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cell+differentiation+from+nonendocrine+epithelial+cells+of+the+adult+human+pancreas.">Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas.</a> Nature Medicine. 12, 310-316 (2006).</li><li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+pancreatic+progenitor+cells--recent+studies.">Minireview: pancreatic progenitor cells--recent studies.</a> Endocrinology. 149, 4312-4316 (2008).</li><li>Seaberg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+identification+of+multipotent+precursors+from+adult+mouse+pancreas+that+generate+neural+and+pancreatic+lineages.">Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.</a> Nature Biotechnology. 22, 1115-1124 (2004).</li><li>Rovira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+centroacinar/terminal+ductal+progenitor+cells+in+adult+mouse+pancreas.">Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 75-80 (2010).</li></ol>

我们什么都没有透露。

到目前为止一直局限于殖民地检测，允许个人胰腺祖细胞的数量和功能评估，这极大地妨碍了在胰腺干细胞和祖细胞生物学的研究进展。殖民地检测的精髓在于它措施祖细胞的内在能力，而不是仅仅其表型，使祖细胞的功能和宗族潜力待观察。这也是一个定量的工具，可以决定在一个特定人群的祖细胞数量。在造血干细胞领域，菌落检测鉴定起到了重要的角色，以及破译生长因子的要求，相互作用，以及这些细胞的谱系承诺机制，在胰腺祖细胞生物学，胰腺器官 7领域6 8或散装9-13已制定了文化，然而，这样的分析没有解决生物questi在单细胞水平上的附加。没有单细胞分析，多潜在的胰腺干/祖细胞的存在不能被明确证明14。 在这里，我们表明，胰岛素表达细胞集落形成祖细胞，可使用一个立体的，含基质胶，半固态的文化系统检测。虽然已经开发了允许单一的成人胰腺祖细胞的增殖和分化成“pancreatospheres”一个悬浮培养系统 15,16，我们的殖民地检测提供了额外的优势。首先，我们的半固体培养媒体系统允许电镀高达25000排序，每500μL培养基细胞每24而产生的集落形成的线性范围。相比之下，暂停pancreatosphere试验需要使用单从细胞分选的细胞沉积的方法，让单细胞分析，因而体积较大的文化传媒和孵化空间是必需的。因此，我们的殖民地检测，是比较容易和成本效益进行大量不同的细胞时需要同时为祖活动相比。其次，我们的半固体培养基，使细胞外基质成分和生长因子，它允许的增殖和分化发生在同一井均匀分布。相反，它是比较困难的，提供这样一个环境，尤其是矩阵，在悬浮培养细胞。我们都知道，它与我们的方法不能明确证明，每一个殖民地是从一个单细胞产生，两个祖先可能是随机定位彼此形成一个殖民地附近。不过，这个问题是可以克服的二次分析使用单细胞操纵。排序的单细胞直接手工采摘可以明确证明每个殖民地的单细胞来源。 总之，胰岛素表达细胞集落形成亲活动在半固体培养基genitors本文所述在单细胞水平胰腺祖细胞的机理研究提供了独特的机会。例如，在以前的报告中，我们发现除了基质胶，细胞外基质蛋白的原油混合物，成文化，媒体为胰岛素表达 Ngn3 - EGFP +细胞，2个殖民地，提示细胞外基质的形成需要重要的祖。使用这个殖民地检测，现在将有可能进一步剖析，并定义为这种活动的具体矩阵要求。除了 ​​研究从小鼠胚胎干细胞衍生的祖细胞，我们目前正在调查中，这在体外检测系统是否也支持形成围产期和成年小鼠的胰腺和肝脏中产生的其他独特的殖民地，以及从人类尸体胰腺组织缺乏胰岛。

<table border="1"><tbody><tr><td> 材料/试剂 </td><td> 公司 </td><td> 目录 数 </td><td> 评论 </td></tr><tr><td> 1X的DMEM / F - 12（1:1） </td><td> Mediatech </td><td> 15 - 090 - CV </td><td></td></tr><tr><td>胎牛血清</td><td>组织培养 生物制品</td><td> 201 </td><td>请参阅第 1.1 </td></tr><tr><td> L -谷氨酰胺</td><td> Gibco公司</td><td> 25030-081 </td><td></td></tr><tr><td> 1X贝科的PBS </td><td> Mediatech </td><td> 21 - 031 - CV </td><td></td></tr><tr><td>牛血清 白蛋白</td><td>西格玛</td><td> A8412 </td><td> 7.5％ </td></tr><tr><td>基质胶</td><td>流式细胞 迪金森</td><td> 356231 </td><td>生长减缓 因子</td></tr><tr><td>甲基</td><td> Sinetsu 化工， 日本东京</td><td></td><td> 1500厘泊 （高- VIscosity） </td></tr><tr><td>烟</td><td>西格玛</td><td> N0636 </td><td></td></tr><tr><td>人类 重组 激活素βB </td><td> R＆D系统</td><td> 659 - AB - 025 </td><td></td></tr><tr><td> Exendin - 4 </td><td>西格玛</td><td> E7144 </td><td></td></tr><tr><td>人类 重组 VEGF - A </td><td> R＆D系统</td><td> 293 - VE - 010 </td><td></td></tr><tr><td> 24孔板</td><td>配角</td><td> 3473 </td><td>超低 附件</td></tr><tr><td> 60毫米的Petri盘 网格线</td><td> NUNC </td><td> 169558 </td><td></td></tr></tbody></table>

a quantitative assay for insulin-expressing colony-forming progenitors

在体外的功能和定量分析，单细胞分析的缺乏已经阻碍了胰腺干细胞和祖细胞生物学领域。单祖细胞的分析是至关重要的，因为它们提供了明确的方式，明确地显示个别祖先的血统潜力。虽然已制定产生“pancreatospheres”从单细胞悬浮培养方法，存在一些局限性。首先，这是费时，执行单细胞的细胞大量沉积，其中大量的文化传媒和空间，反过来命令。二，造成pancreatospheres编号是劳动密集，尤其是当频率低pancreatosphere发起祖。第三，pancreatosphere检测是不是一种有效的方法，以使两个相同的文化胰腺祖细胞的增殖和分化以及，重新stricting检测的实用性。 为了克服这些限制，一个半固体培养基胰腺祖细胞的殖民地检测已开发和在本报告中提出。这种方法需要从现有的造血细胞集落检测，其中甲基纤维素是用来向媒体提供的粘度概念优势，使祖细胞在三维空间中停留，因为他们进行增殖以及分化。为了丰富胰岛素表达细胞集落形成祖从一个异构的人口，我们可以利用细胞表达神经元素（NGN）3，胰腺内分泌祖细胞标记。小鼠胚胎干细胞（ES）细胞衍生Ngn3表达细胞与增强型绿色荧光蛋白记者标签，排序和多达25,000个细胞被镀成低附件24的培养皿。满足每口井包含以下主要组件的半固体培养基500μL：hylcellulose，基质胶，烟酰胺，exendin - 4，激活素βB，空调媒体收集的小鼠胚胎干细胞源性的胰腺细胞样细胞。经过8至12天的文化，胰岛素表达菌落形态具有鲜明的形成，可以进一步使用定量RT - PCR和immunoflourescent染色，以确定每个殖民地的宗族组成的胰腺癌基因表达分析。 总之，我们的殖民地检测可以方便地检测和内细胞的异质人口的功能祖细胞定量。此外，半固态的媒体格式允许对细胞的细胞外基质成分和生长因子的统一演示，使祖细胞增殖和分化体外。这个殖民地检测胰腺祖细胞在单细胞水平的机理研究提供了独特的机会。

Watch this Scientific Journal Video about A Quantitative Assay for Insulin-expressing Colony-forming Progenitors at JoVE.com

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

描述一个立体的克隆形成实验，让胰祖细胞分化为胰岛素表达殖民地。此方法采用半固体甲基纤维素，基质胶和生长因子的媒体的优势，其中单祖细胞增殖和分化<em&gt;在体外</em&gt;，允许量化人口的功能祖。

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

a-quantitative-assay-for-insulin-expressing-colony-forming-progenitors

Research

JoVE Journal

Biology

13.4K 次观看.  California State University Channel Islands. 描述一个立体的克隆形成实验，让胰祖细胞分化为胰岛素表达殖民地。此方法采用半固体甲基纤维素，基质胶和生长因子的媒体的优势，其中单祖细胞增殖和分化在体外，允许量化人口的功能祖。

视频: A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

科研

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

使用CD71/TER119流流式细胞仪检测小鼠红系祖细胞的识别和分析

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

来自人类脐带血内皮细胞集落形成细胞的表型和功能特性

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

一个定量聚合酶重组酶扩增法的发展与内部阳性对照

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...