来自以前发表的研究报告，这是财政支持雀巢作为说明示例中使用的所有NMR谱。

1。定植无菌动物和样品采集<ol><li>从隔离和房子他们在传统畜牧业室配备带过滤器在传统的动物将作为对照组（图1）面前笼中取出无菌动物。 </li><li>混合垃圾的一半（3天），从常规笼无菌动物的垃圾。始终保持1 / 3脏传统的枯枝落叶每次它是必要的更新，以保持水平（至少要保持3天）的细菌。 </li><li>收集在1.5毫升的微管的尿液处理管鼠标，帮助轻轻按摩排便排尿。管理单元立即在液氮冻结。至少在-40 ° C储存，直到NMR分析。一个20μL体积最小的是需要一个5毫米的NMR探头的收购，但建议使用30μL，以改善代谢分析质量。 </li><li>动物应bË安乐死不使用任何麻醉剂，以避免混淆核磁共振，由于肝代谢麻醉化合物（例如，使用颈椎脱位确认死亡放血其次） </li></ol> 2。收集肝活检的建议<ol><li>不要使用任何含有酒精，以避免污染的产品。工具使用仅用水或生理盐水溶液洗净。 </li><li>不要穿孔胆。胆漏的情况下，立即用清水或生理盐水溶液冲洗组织。 </li><li>收集从图2所示的左叶肝活检（约15-50毫克）。对于重复性的活组织切片检查，收集一直在避免周边地区组织较薄的左叶的中心。 </li><li>立即扣在液氮冻结活检，并存放于-80 °直到NMR分析彗星。 </li></ol> （3）1 H核磁共振收购尿微量</P&gt; <ol><li>准备在D2O（99.8％），pH值7.4，含有1毫米3 0.2M磷酸钠缓冲液- （三甲基硅基）丙酸- D 4（TSP）的。 </li><li>混合磷酸钠缓冲液30μL30μL尿。 </li><li>转移到1.7毫米核磁共振毛细管使用配备一个金属针（外径0.5毫米）50μL玻璃注射器（图3（2））混合溶液50μL。小心，以避免气泡。 </li><li>适合的毛细血管含尿液样本上的毛细管适配器（图3（3））和5毫米核磁共振探头（图3（1））放入一个2.5毫米的核磁共振微管。作为一个普通的5毫米核磁共振光谱采集管这管的组合。 </li><li>使用提取杆（图3（4））删除从2.5毫米NMR管的毛细管拧毛细管适配器，轻轻将其拉出。 </li></ol> 4 的1 H人力资源的MAS核磁共振肝组织活检：样品制备<ol><li> MAS转子组件和工具是在图4描述。 </li><li>插入活检（约15-50毫克）进入锆转子（图4（1）），并填写该卷的其余纯D 2 O核磁共振锁。小心不使任何气泡，因为这会改变随后垫补和数据采集的质量品质。 </li><li>将50μL特氟隆垫片（图4（2））使用圆柱形的螺丝（图4（5））。拧下它和校准使用深度计短边（图4（8））。在这一步，重要的是要付出特别注意样品的一部分，因为它可以通过间隔孔泄漏。如果是这样的话，那么活检的一部分被破坏和样品重量不再是可靠的。因此，有必要再次启动从开始的样品制备。 </li><li>将THEAD针（图4（3））轻轻螺丝，用螺丝刀（图4（6））。干出任何一块组织的残余水。 </li><li>放置在第（图4（4））顶部的转子和在转子打包机（图4（6））插入。坚决按，直到第到位。此外，不应该留下任何空间之间的转子和第。 </li><li>马克用黑色记号笔转子底部的一半，让光学旋转速率检测。 </li><li>广场内的核磁共振波谱仪的转子，并开始在5 kHz纺。收购1 H核磁共振谱使用CPMG脉冲序列13，根据制造商的指引。 </li><li>使用5.22 PPM（双峰）α异头葡萄糖共振核磁共振光谱校准。 </li><li>要解压缩的转子，继续使用第卸妆（图4（9））删除第。拧开THEAD针和删除聚四氟乙烯垫片使用的圆柱螺杆。使用水和洗涤剂彻底清洗。 </li></ol> 5。代表性的成果肠道微生物的活性，可使用尿代谢分析监测。大量的尿微生物已在文献中描述7,14-17合作的代谢产物通过1 H NMR识别。这些微生物的代谢产物是特别有用的监测殖民化进程，为他们提供了一个快速和无创的方法来估计时，新成立的生态系统是稳定的。图5A清楚地说明了肠道微生物代谢产物在殖民化过程中的外观。此图显示了以下步骤2中所述的程序为动物得到一个尿代谢轮廓殖民统治20天，使用在步骤1中描述的过程。这种动物没有排出任何indoxyl phenylacetylglycine（PAG），在无菌状态下的硫酸对甲酚（0天蓝色）的硫酸和数量很少。由于殖民化的进展，这些蛋白质代谢标记的肠道菌群大大增加，达到一个平衡点，在第20天（红色）。这是一个组所示，图5B使用PAG的动物特别容易监控共鸣。此图是由下图5A灰色（δ7.40-7.43）中强调的共振，对应于一个特定的共振PAG的一组7只动物（三重）相结合的面积。 1 H高解像度魔角旋转（人力资源的MAS）核磁共振光谱是一种非破坏性的技术，允许任何一种活检 18代谢概况快速和可重复性收购。在这个协议中，我们使用这个强大的技术获得前（蓝色）和（红色）（图6）定植后的2小鼠肝代谢轮廓。这个数字说明以及可以从基于核磁共振的代谢轮廓MAS派生的信息。大量的氨基酸，以及衍生代谢物，如能量代谢葡萄糖，糖原，乳酸，甘油三酸脂（D）- 3 -羟丁酸和​​nicotinurate，可以可视化。这些配置文件中还含有氧化应激相关的信息（即抗坏血酸一个CID，谷胱甘肽），核苷酸代谢（即肌苷，尿苷）和甲胺代谢（即胆碱，三甲胺- N -氧化物）。在这个例子中，这是很清楚，无菌鼠标几乎没有显示糖原和非常低血糖和甘油三酯察看7数额。 <img src="/files/ftp_upload/3642/3642fig1.jpg" alt="图1。"/> 图1。定植协议概述。无菌和常规动物都装在过滤器端配备由方交换了自己的窝，以便逐步从传统的肠道菌群（1）定植的笼子。使用1 H核磁共振的代谢分析（2-3），肠道微生物的活动都受到监控。肝脏代谢评估的1 H HR MAS基于核磁共振的代谢分析（4-5） 。 <img src="/files/ftp_upload/3642/3642fig2.jpg" alt="图2。"/> 图2鼠标生活ř解剖。肝脏是显示，比如平坦的侧面表的器官。可重复活检，这是应始终从虚线矩形的左叶中心收集的样本。 <img src="/files/ftp_upload/3642/3642fig3.jpg" alt="图3。"/> 图3 1.7毫米核磁共振毛细管套件与microvolumes工作重点 ：（1）：2.5毫米核磁共振微管，2：1.7毫米核磁共振毛细管，3：毛细管适配器，4：提取杆。 <img src="/files/ftp_upload/3642/3642fig4.jpg" alt="图4。"/> 图4 MAS的转子设备的关键 ：1：MAS的转子，2：50μL特氟隆垫片，3：THEAD针，4：第5：圆柱螺杆，6：螺丝刀，7：包装机转子，8：深度计。 <img src="/files/ftp_upload/3642/3642fig5.jpg" alt="图5。"/> 图5 colonizatio尿代谢概况演化。N. <ol class="ualpha"><li>放大6.8-7.8 ppm的微生物共同代谢产物，可以可视化。1 H NMR谱之间的光谱芳香地区均来源于单个人在第0天（蓝色），4（绿色），15（橙色）和20 （红色）后定植。灰色地带对应的是综合图B.主要领域：1，MeHistamine：1 methylhistamine; Indoxyl - S：Indoxyl硫酸;他：组氨酸，P -甲酚 - S：P -甲酚硫酸; PAG ：Phenylacetylglycine。 </li><li>殖民统治期间的平均PAG的浓度（N = 7）。学生t -检验用来比较PAG浓度的差异，在不同的时间点：A：0天相比，P &lt;0.05; B：P &lt;0.01相比，第10天。 </li></ol><img src="/files/ftp_upload/3642/3642fig6.jpg" alt="图6。"/> 典型的600兆赫的1 H人力资源的MAS无菌（蓝色）和前无菌（重新产生的肝活检核磁共振光谱图6 。D）小鼠。粗体质子负责甘油三酯共振键 ：3 HB：3 -羟丁酸，谷胱甘肽：谷胱甘肽，TGS：三酸甘油脂，TMAO：三甲胺- N -氧化物。

<ol>
	<li>Cani, P. D., Delzenne, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microflora+as+a+target+for+energy+and+metabolic.">Gut microflora as a target for energy and metabolic.</a> Curr. Opin. Clin. Nutr. Metab. Care. 10, 729-734 (2007).</li><li>Ley, R. E., Turnbaugh, P. J., Klein, S., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+ecology:+human+gut+microbes+associated+with+obesity.">Microbial ecology: human gut microbes associated with obesity.</a> Nature. 444, 1022-1023 (2006).</li><li>Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+pandemics+and+the+modification+of+digestive+bacterial+flora.">Obesity pandemics and the modification of digestive bacterial flora.</a> Eur. J. Clin. Microbiol. Infect. Dis. 27, 631-634 (2008).</li><li>Turnbaugh, P. J., Backhed, F., Fulton, L., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet-induced+obesity+is+linked+to+marked+but+reversible+alterations+in+the+mouse+distal+gut+microbiome.">Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome.</a> Cell. Host. Microbe. 3, 213-223 (2008).</li><li>Balcombe, J. P., Barnard, N. D., Sandusky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+routines+cause+animal+stress.">Laboratory routines cause animal stress.</a> Contemp. Top. Lab. Anim. Sci. 43, 42-51 (2004).</li><li>Muyzer, G., Smalla, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+denaturing+gradient+gel+electrophoresis+(DGGE)+and+temperature+gradient+gel+electrophoresis+(TGGE)+in+microbial+ecology.">Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology.</a> Antonie van Leeuwenhoek. 73, 127-141 (1998).</li><li>Claus, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization-induced+host-gut+microbial+metabolic+interaction.">Colonization-induced host-gut microbial metabolic interaction.</a> MBio. 2, (2011).</li><li>Waters, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+magic+angle+spinning+1H+NMR+spectroscopy+of+intact+liver+and+kidney:+optimization+of+sample+preparation+procedures+and+biochemical+stability+of+tissue+during+spectral+acquisition.">High-resolution magic angle spinning 1H NMR spectroscopy of intact liver and kidney: optimization of sample preparation procedures and biochemical stability of tissue during spectral acquisition.</a> Anal. Biochem. 282, 16-23 (2000).</li><li>Bollard, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+1H+and+1H-13C+magic+angle+spinning+NMR+spectroscopy+of+rat+liver.">High-resolution 1H and 1H-13C magic angle spinning NMR spectroscopy of rat liver.</a> Magnetic resonance in medicine. 44, 201-207 (2000).</li><li>Lindon, J. C., Holmes, E., Nicholson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+methods+and+applications+in+biomedical+magnetic+resonance.">Pattern recognition methods and applications in biomedical magnetic resonance.</a> Progress in Nuclear Magnetic Resonance Spectroscopy. 39, 1-40 (2001).</li><li>Tate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinction+between+normal+and+renal+cell+carcinoma+kidney+cortical+biopsy+samples+using+pattern+recognition+of+(1)H+magic+angle+spinning+(MAS)+NMR+spectra.">Distinction between normal and renal cell carcinoma kidney cortical biopsy samples using pattern recognition of (1)H magic angle spinning (MAS) NMR spectra.</a> NMR. Biomed. 13, 64-71 (2000).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographical+variation+in+metabolic+signatures+of+human+gastrointestinal+biopsies+revealed+by+high-resolution+magic-angle+spinning+1H+NMR+spectroscopy.">Topographical variation in metabolic signatures of human gastrointestinal biopsies revealed by high-resolution magic-angle spinning 1H NMR spectroscopy.</a> Journal of Proteome Research. 6, 3944-3951 (2007).</li><li>Meiboom, S., Gill, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+spin-echo+method+for+measuring+nuclear+relaxation+times.">Modified spin-echo method for measuring nuclear relaxation times.</a> The review of scientific instruments. 29, 688-691 (1958).</li><li>Nicholson, J. K., Holmes, E., Wilson, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microorganisms,+mammalian+metabolism+and+personalized+health+care.">Gut microorganisms, mammalian metabolism and personalized health care.</a> Nat. Rev. Microbiol. 3, 431-438 (2005).</li><li>Martin, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+probiotic+Lactobacillus+paracasei+treatment+on+the+host+gut+tissue+metabolic+profiles+probed+via+magic-angle-spinning+NMR+spectroscopy.">Effects of probiotic Lactobacillus paracasei treatment on the host gut tissue metabolic profiles probed via magic-angle-spinning NMR spectroscopy.</a> Journal of Proteome Research. 6, 1471-1481 (2007).</li><li>Swann, 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=Variation+in+Antibiotic-Induced+Microbial+Recolonization+Impacts+on+the+Host+Metabolic+Phenotypes+of+Rats.">Variation in Antibiotic-Induced Microbial Recolonization Impacts on the Host Metabolic Phenotypes of Rats.</a> J. Proteome. Res. , (2011).</li><li>Jacobs, D. M., Gaudier, E., van Duynhoven, J., Vaughan, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-digestible+food+ingredients,+colonic+microbiota+and+the+impact+on+gut+health+and+immunity:+a+role+for+metabolomics.">Non-digestible food ingredients, colonic microbiota and the impact on gut health and immunity: a role for metabolomics.</a> Curr. Drug. Metab. 10, 41-54 (2009).</li><li>Beckonert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+magic-angle-spinning+NMR+spectroscopy+for+metabolic+profiling+of+intact+tissues.">High-resolution magic-angle-spinning NMR spectroscopy for metabolic profiling of intact tissues.</a> Nat. Protoc. 5, 1019-1032 (2010).</li><li>Hooper, L. V., Sansonetti, P., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in microbiology. 31, 559-589 (2002).</li><li>Rahija, R. J., Fox, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+7.">Ch. 7.</a> The mouse in biomedical research. , 217-234 (2007).</li><li>Goodwin, B. L., Ruthven, C. R., Sandler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+flora+and+the+origin+of+some+urinary+aromatic+phenolic+compounds.">Gut flora and the origin of some urinary aromatic phenolic compounds.</a> Biochemical Pharmacology. 47, 2294-2297 (1994).</li><li>Koopman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Normalization'+of+germfree+mice+after+direct+and+indirect+contact+with+mice+having+a+'normal'+intestinal+microflora.">'Normalization' of germfree mice after direct and indirect contact with mice having a 'normal' intestinal microflora.</a> Lab Anim. 20, 286-290 (1986).</li><li>Nishikata, N., Shikata, N., Kimura, Y., Noguchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+lipid-dependent+regulation+of+de+novo+lipogenesis+and+lipid+partitioning+by+ketogenic+essential+amino+acids+in+mice.">Dietary lipid-dependent regulation of de novo lipogenesis and lipid partitioning by ketogenic essential amino acids in mice.</a> Nutrition and Diabetes. 1, 1-12 (2011).</li><li>Spagou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+GC-MS+metabolic+profiling+study+of+plasma+samples+from+mice+on+low-+and+high-fat+diets.">A GC-MS metabolic profiling study of plasma samples from mice on low- and high-fat diets.</a> J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 879, 1467-1475 (2011).</li><li>Sanchez-Patan, F., Monagas, M., Moreno-Arribas, M. V., Bartolome, B. <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+microbial+phenolic+acids+in+human+faeces+by+UPLC-ESI-TQ+MS.">Determination of microbial phenolic acids in human faeces by UPLC-ESI-TQ MS.</a> J. Agric. Food. Chem. 59, 2241-2247 (2011).</li><li>Roux, A., Lison, D., Junot, C., Heilier, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+liquid+chromatography+coupled+to+mass+spectrometry-based+metabolomics+in+clinical+chemistry+and+toxicology:+A+review.">Applications of liquid chromatography coupled to mass spectrometry-based metabolomics in clinical chemistry and toxicology: A review.</a> Clin. Biochem. 44, 119-135 (2011).</li><li>Ryan, D., Robards, K., Prenzler, P. D., Kendall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+and+potential+developments+in+the+analysis+of+urine:+a+review.">Recent and potential developments in the analysis of urine: a review.</a> Anal. Chim. Acta. 684, 8-20 (2011).</li><li>Nagayama, K., Wuthrich, K., Bachmann, P., Ernst, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+J-resolved+1H+n.m.r.+spectroscopy+for+studies+of+biological+macromolecules.">Two-dimensional J-resolved 1H n.m.r. spectroscopy for studies of biological macromolecules.</a> Biochem. Biophys. Res. Commun. 78, 99-105 (1977).</li><li>Aue, W. P., Bartholdi, E., Ernst, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+spectroscopy.+Application+to+nuclear+magnetic+resonance.">Two-dimensional spectroscopy. Application to nuclear magnetic resonance.</a> J. Chem. Phys. 64, 2229-2246 (1975).</li><li>Bodenhausen, G., Ruben, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+abundance+15N+NMR+by+enhanced+heteronuclear+spectroscopy.">Natural abundance 15N NMR by enhanced heteronuclear spectroscopy.</a> Chemical. Physics. Letters. 69, 185-189 (1980).</li><li>Fan, T. W. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolite+profiling+by+one-+and+two-dimensional+NMR+analysis+of+complex+mixtures.">Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures.</a> Progress in nuclear magnetic resonance spectroscopy. 28, 161-219 (1996).</li><li>Fan, T., Lane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+profiling+of+metabolites+and+isotopomers+by+NMR.">Structure-based profiling of metabolites and isotopomers by NMR.</a> Progress in Nuclear Magnetic Resonance Spectroscopy. 52, 48-48 (2008).</li><li>Fonville, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+partial+least+squares+models+and+related+chemometric+approaches+in+metabonomics+and+metabolic+phenotyping.">The evolution of partial least squares models and related chemometric approaches in metabonomics and metabolic phenotyping.</a> Journal of Chemometrics. 24, 636-649 (2010).</li><li>Merrifield, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metabolic+system-wide+characterisation+of+the+pig:+a+model+for+human+physiology.">A metabolic system-wide characterisation of the pig: a model for human physiology.</a> Mol. Biosyst. , (2011).</li><li>Tugnoli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+human+gastric+mucosa+by+HR-MAS+magnetic+resonance+spectroscopy.">Molecular characterization of human gastric mucosa by HR-MAS magnetic resonance spectroscopy.</a> International Journal of Molecular Medicine. 14, 1065-1071 (2004).</li><li>Sitter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+HR+MAS+MR+spectroscopic+profiles+of+breast+cancer+tissue+with+clinical+parameters.">Comparison of HR MAS MR spectroscopic profiles of breast cancer tissue with clinical parameters.</a> NMR Biomed. 19, 30-40 (2006).</li><li>Beckonert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiling,+metabolomic+and+metabonomic+procedures+for+NMR+spectroscopy+of+urine,+plasma,+serum+and+tissue+extracts.">Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.</a> Nat. Protoc. 2, 2692-2703 (2007).</li></ol>

我们什么都没有透露。

在这个协议中，我们描述了一个渐进的殖民化过程，在一个开放的环境，进一步探讨肝脏代谢的影响肠道菌群的1 H HR MAS完整活检核磁共振分析评估。殖民化的各种方法在文献中被描述。最常用的方法来定义的菌群与殖民动物灌胃或污染的饮用水19,20。粪便接种也可以使用先前描述21。定植方法，这里介绍的是来自一个“正常化”无菌动物库普曼JP等描述的方法。在1986年...

特定的试剂和设备表： <table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> 2.5毫米微管</td><td>新时代</td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1.7毫米的毛细管</td><td> Sigma - Aldrich公司</td><td> NORS175001 </td><td></td></tr><tr><td>毛细管适配器</td><td>新时代</td><td> NE-325-5/1.7 </td><td></td></tr><tr><td>萃取棒</td><td>新时代</td><td> NE - 341 - 5 </td><td></td></tr><tr><td> HR - MAS的转子BL4 50μL 球面聚四氟乙烯垫片套件</td><td>布鲁克</td><td> HZ07213 </td><td></td></tr><tr><td> 50μL插入工具箱</td><td>布鲁克</td><td> B2950 </td><td></td></tr><tr><td>提前三600MHz核磁共振</td><td>布鲁克</td><td></td><td></td></tr><tr><td> 1 H HR MAS NMR固体探头</td><td>布鲁克</td><td></td><td></td></tr><tr><td> 99.9％的氧化氘</td><td> Sigma - Aldrich公司</td><td> 530867 - 1L </td><td></td></tr><tr><td> 3 - （三甲基硅基）丙酸 酸- D 4（TSP）， </td><td> Sigma - Aldrich公司</td><td> 269​​913 </td><td></td></tr></tbody></table>

assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1h nmr spectroscopy

这是众所周知的，肠道细菌有助于显著主机的动态平衡，提供一系列的好处，如免疫保护和维生素的合成。他们还提供主机与大量的营养物质，使这个生态系统的一个重要的代谢器官。在肠道菌群和代谢综合征之间的联系的理解之间的主机和其肠道菌群的代谢相互作用，越来越多的证据的范围内正在成为现代生物学的一个重要挑战。1-4 定植（也称为正常化的进程）指定建立在前者的无菌动物的微生物。虽然这是一个自然的过程，在出生时发生，它也用于成人无菌动物来控制肠道花卉的生态系统，并进一步确定其主机上的代谢的影响。控制殖民化进程的一个普通的程序是使用SINGL灌胃方法E或微生物的混合物。这种方法的结果，并提出在一个非常快的殖民统治的极端压力5的缺点。因此，它是有用的，以尽量减少压力和获得殖民化进程较慢，逐步观察细菌建立在主机上的新陈代谢的影响。 在这个手稿中，我们描述了一个过程，在一个渐进的殖民过程中，使用非破坏性的代谢分析技术，以评估肝脏代谢的修改。我们建议监测评估肠道微生物的代谢活性微生物的代谢产物 1 H核磁共振的代谢分析尿中排泄反映肠道微生物定植。这使得肠道微生物的活动超出了通常由粪便细菌DGGE（变性 ​​梯度凝胶电泳）监测评估的肠道微生物生态系统的稳定建立稳定的升值 。6定植在传统的开放式环境，是由传统的动物，这将作为控件沾脏的垃圾发起。正在coprophagous动物的啮齿动物，这确保了同质定植如前所述。 肝代谢分析是直接从一个完整的肝活检使用高分辨率的1 H魔角旋转核磁共振光谱测量。这半定量的技术提供一个快速的方法进行评估，不破坏细胞结构，如甘油三酯，葡萄糖和糖原的主要代谢产物，以进一步估计殖民化进程和肝脏代谢 7-10之间复杂的相互作用。这种方法也可以适用于任何组织活检 11,12 。

Watch this Scientific Journal Video about Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy at JoVE.com

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

一个渐进的殖民化过程进行了介绍，以进一步评估其主机上的肝脏代谢的影响。定植被监视的非侵入性的，而基于核磁共振的代谢分析评估微生物的代谢产物的尿中排泄肝脏代谢高分辨率魔角旋转（人力资源的MAS）核磁共振完好活检的分析评估。

在无菌无鼠标进定植评估肝代谢的变化 1 H核磁共振光谱

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

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Research

JoVE Journal

Immunology and Infection

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

13.0K 次观看.  The University of Reading. 一个渐进的殖民化过程进行了介绍，以进一步评估其主机上的肝脏代谢的影响。定植被监视的非侵入性的，而基于核磁共振的代谢分析评估微生物的代谢产物的尿中排泄肝脏代谢高分辨率魔角旋转（人力资源的MAS）核磁共振完好活检的分析评估。

视频: Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

科研

免疫与感染

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

定植 Euprymna scolopes鱿鱼由弧菌鲵</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating肝白细胞的选择性收获

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

由荧光相关光谱测量的一次淋巴细胞的细胞膜分子扩散

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...