FM通过chaire D&#39;IDEX卓越巴黎的索邦大学太阳城确认来自ANR资助下，INSU通过PNP赠款，研究所区大学法国以及在巴黎的索邦大学太阳城（ANR-10-的LabX-0023和ANR的Labex UniverEarth程序-11-IDEX-0005-02）。我们也感谢来自欧洲研究委员会的资助下，欧洲共同体的H2020框架方案/ ERC赠款协议＃637503（精粹）。

注:程序涉及动物已被批准的机构动物护理和使用委员会（IACUC）在巴黎大学狄德罗。 1.准备材料<ol><li>亚沸腾馏1升的酸（HNO 3，氢溴酸），以从杂质纯化它们。 </li><li>清洁烧杯和尖端适配器在热（〜100℃）浓HNO 3酸浴中至少两天。 </li><li>洗冷3 N HNO 3浴室的枪头数天，单独漂洗三次去离子水。 </li></ol> 2.样品制备<ol><li>麻醉小鼠腹腔注射氯胺酮和甲苯噻嗪。由脚趾捏法评估麻醉。 </li><li>收集的血液在肝素1.5 mL管存在一个心脏穿刺。 </li><li>分离的血细胞通过离心（10分钟，1500×g离心）等离子体和等离子体转移到性聚丙烯C使用聚丙烯提示ryogenic小瓶。 </li><li>通过在心脏切肝静脉和注射DPBS取下器官剩余的血液。颈椎脱位评估鼠标死亡。 </li><li>收获用无菌不锈钢器械的器官，释放周围脂肪，如果任何的他们，在聚丙烯瓶低温管理单元冻结他们。 </li></ol> 3.化学提纯<ol><li>首先，溶解样品中的约1毫升浓（30％） 的 H 2 O 2和〜1ml浓（〜15 M），HNO 3的混合。做一个通风橱内的所有这些步骤。 <ol><li>放置整个感兴趣的器官入15ml特氟隆烧杯中。然后，将H 2 O 2 / HNO 3添加到烧杯5。保持烧杯公开了几分钟，以避免由于有机物的氧化，反应的 CO 2的释放飞溅。 </li><li>最后，把烧杯在热板上在ABO血型UT 100℃几个小时或直到溶液是十分清楚的。 </li></ol></li><li>打开烧杯和干燥的热板上将溶液在约100℃。 </li><li>一旦样品是干，加入1毫升1.5N的HBr的到样本;关闭烧杯并让它溶解在热板上在100℃下进行几个小时。 </li><li>同时准备500微升列。 <ol><li>加入500μl的AG1X8 200-400目树脂为列，并把它与它下面的一个垃圾桶烧杯中柱架。通过交替洗涤树脂:5毫升MΩ⋅厘米水，5毫升0.5N的HNO 3，第 5毫升水，5毫升0.5N的HNO 3 18.2，然后加入5毫升水。调节所述树脂用5ml的1.5N的HBr。 </li></ol></li><li>从热板上取下烧杯中，并把它们在超声波浴中约30分钟，然后让烧杯冷却至RT。 </li><li>一旦烧杯冷却，洗涤树脂，打开烧杯。把末端适配器到t他注射器，加枪头;吸取1ml的样品，并将其加载到树脂（非常缓慢，为了不搅动树脂）。 </li><li>一旦所有的液体通过柱，加入5毫升的1.5N的HBr。 </li><li>一旦5毫升的1.5N的HBr通过柱子，用干净的15毫升烧杯更换垃圾桶烧杯中。 </li><li>加入5毫升的0.5N的HNO 3 2.5混合物在一个时间。在此阶段，锌从树脂洗脱。 </li><li>一旦5毫升HNO 3的穿过柱，取出烧杯，并放置在热板上，在100℃，直至干燥。 </li><li>取下柱夹列;垃圾桶树脂（用于每个样品的新树脂）。 </li><li>一旦样品是干的，重复的协议与在一个较小的塔的酸（100微升）相同的体积，然后将其放置在热板上，直至干燥。该样本是现在准备质谱。 </li></ol> 4.质谱测量<ol><li>分析锌同位素康波习得在多集电极 - 电感耦合等离子体质谱仪（MC-ICP-MS）。 <ol><li>使用表1中总结的机器参数。 </li></ol></li><li>放置法拉第筒收集在62镍，铜63，64锌，铜65，66锌，锌67和68的Zn质量（m / z）。 </li><li>制备含有500ppb的锌在0.1M HNO 3为同位素分析的溶液。 </li><li>通过使用喷雾室结合100μl/ min的聚四氟乙烯喷雾器分析Zn中500ppb的溶液。对于每个样品，测量30扫描（1块30个循环），其中每一个扫描的积分时间是8.389秒。 </li><li>通过从空白溶液（用于重新溶解的样品的0.1M HNO 3溶液）中减去上峰零强度校正的背景。 </li><li>控制和纠正可能64镍通过测量62镍峰的强度等压干扰。假设镍64/62 Ni比自然（0.2548），从正确的工具质量偏差此值，然后取出64镍的质量为64: 64 真正的锌= 64 测定锌-镍64 = 64锌测定 - （64镍/镍62）， 自然 x 62镍测量 。 </li><li>通过与江铃里昂锌标准的500 ppb的标准溶液（或其他可用的标准，例如IRMM-3702）包围每个样品更正仪器质量偏差。通过将样品的66锌/ 64 Zn的比率由之前和样品减去1之后测定并乘以1000这两个标准的66的Zn / 64 Zn的比率的平均值执行标准的包围（见公式1）。在江铃里昂锌标准典型的外部精度为0.05 permil / AMU（2个标准差，2 SD）。 </li></ol>

<ol>
	<li>Marechal, C. N., Telouk, P., Albarede, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+analysis+of+copper+and+zinc+isotopic+compositions+by+plasma-source+mass+spectrometry.">Precise analysis of copper and zinc isotopic compositions by plasma-source mass spectrometry.</a> Chemical Geology. 156 (1), 251-273 (1999).</li><li>Marechal, C. N., Albarede, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-exchange+fractionation+of+copper+and+zinc+isotopes.">Ion-exchange fractionation of copper and zinc isotopes.</a> Geochim. Cosmochim. Acta. 66 (9), 1499-1509 (2001).</li><li>Weiss, D. J., Mason, T. F. D., Zhao, F. J., Kirk, G. J. D., Coles, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+discrimination+of+zinc+in+higher+plants.">Isotopic discrimination of zinc in higher plants.</a> New Phytologist. 165 (3), 703-710 (2005).</li><li>Jouvin, D., Louvat, P., N, M. F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+isotopic+fractionation:+why+organic+matters.">Zinc isotopic fractionation: why organic matters.</a> Environ Sci Technol. 43 (15), 5747-5754 (2009).</li><li>Moynier, F., 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=Isotopic+fractionation+and+transport+mechanisms+of+Zn+in+plants.">Isotopic fractionation and transport mechanisms of Zn in plants.</a> Chemical Geology. 267 (3-4), 125-130 (2009).</li><li>Moynier, F., Herzog, G., Albarede, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+composition+of+zinc,+copper,+and+iron+in+lunar+samples.">Isotopic composition of zinc, copper, and iron in lunar samples.</a> Geochim. Cosmochim. Acta. 70 (24), 6103-6117 (2006).</li><li>Moynier, F., 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=Isotopic+fractionation+of+zinc+in+tektites.">Isotopic fractionation of zinc in tektites.</a> Earth Planet. Sci. Lett. 277 (3-4), 482-489 (2009).</li><li>Chen, H., Savage, P., Teng, F. Z., Helz, R., Moynier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+isotope+fractionation+during+magmatic+differentiation+and+the+isotopic+composition+of+the+bulk+Earth.">Zinc isotope fractionation during magmatic differentiation and the isotopic composition of the bulk Earth.</a> Earth Planet. Sci. Lett. 369-370, 34-42 (2013).</li><li>Moeller, 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=Calibration+of+the+new+certified+materials+ERM-AE633+and+ERM-AE6447+for+copper+and+IRMM+3702+for+zinc+isotope+amount+ratio+determination.">Calibration of the new certified materials ERM-AE633 and ERM-AE6447 for copper and IRMM 3702 for zinc isotope amount ratio determination.</a> Geostd. Geoan. Res. 36 (2), 177-199 (2012).</li><li>Bigeleisen, J., Mayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+equilibrium+constants+for+isotopic+exchange+reactions.">Calculation of equilibrium constants for isotopic exchange reactions.</a> J. Chem. Phys. 15, 261-267 (1947).</li><li>Luck, J. M., Ben Othman, D., Albarede, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zn+and+Cu+isotopic+variations+in+chondrites+and+iron+meteorites:+Early+solar+nebula+reservoirs+and+parent-body+processes.">Zn and Cu isotopic variations in chondrites and iron meteorites: Early solar nebula reservoirs and parent-body processes.</a> Geochim. Cosmochim. Acta. 69 (22), 5351-5363 (2005).</li><li>Moynier, F., Dauphas, N., Podosek, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Search+for+70Zn+Anomalies+in+Meteorites.">A Search for 70Zn Anomalies in Meteorites.</a> Astrophys. J. 700 (2), L92-L95 (2009).</li><li>Paniello, R., Day, J., Moynier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zn+isotope+evidence+for+the+origin+of+the+Moon.">Zn isotope evidence for the origin of the Moon.</a> Nature. 490 (7420), 376-380 (2012).</li><li>Pichat, S., Douchet, C., Albarede, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+isotope+variations+in+deep-sea+carbonates+from+the+eastern+equatorial+Pacific+over+the+last+175+ka.">Zinc isotope variations in deep-sea carbonates from the eastern equatorial Pacific over the last 175 ka.</a> Earth and Planetary Science Letters. 210 (1-2), 167-178 (2003).</li><li>Moynier, F., Fujii, T., Shaw, A., Le Borgne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+of+natural+Zn+isotopes+in+mice.">Heterogeneous of natural Zn isotopes in mice.</a> Metallomics. 5 (6), 693-699 (2013).</li><li>Balter, V., 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=Bodily+variability+of+zinc+natural+isotope+abundance+in+sheep.">Bodily variability of zinc natural isotope abundance in sheep.</a> Rapid Com. Mass. Spec. 24, 605-612 (2010).</li><li>Balter, V., 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=Contrasting+Cu,+Fe,+and+Zn+isotopic+patterns+in+organs+and+body+fluids+of+mice+and+sheep,+with+emphasis+on+cellular+fractionation.">Contrasting Cu, Fe, and Zn isotopic patterns in organs and body fluids of mice and sheep, with emphasis on cellular fractionation.</a> Metallomics. 5 (11), 1470-1482 (2010).</li><li>Urgast, D. S., 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=Zinc+isotope+ratio+imaging+of+rat+brain+thin+sections+from+stable+isotope+tracer+by+LA-MC-ICP-MS.">Zinc isotope ratio imaging of rat brain thin sections from stable isotope tracer by LA-MC-ICP-MS.</a> Metallomics. 4, 1057-1063 (2012).</li><li>Marin-Carbonne, J., Rollion-Bard, C., Luais, B. <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+measurements+of+iron+isotopes+by+SIMS:+MC-ICP-MS+intercalibration+and+application+to+a+magnetite+crystal+from+the+Gunflint+chert.">In-situ measurements of iron isotopes by SIMS: MC-ICP-MS intercalibration and application to a magnetite crystal from the Gunflint chert.</a> Chem. Geol. 285 (1-4), 50-61 (2011).</li><li>Fietzke, J., 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=Boron+isotope+ratio+determination+in+carbonates+via+LA-MC-ICP-MS+using+soda-lime+glass+standards+as+reference+material.">Boron isotope ratio determination in carbonates via LA-MC-ICP-MS using soda-lime glass standards as reference material.</a> J. Anal. Atom. Spec. 25, 1953-1957 (2010).</li></ol>

作者什么都没有透露。

测量的再现性，通过在不同的分析会话进行同一样品重复分析评价。例如如图6所示 ，我们已复制同一陆地岩石7倍，我们获得了记录在表2中的结果。 正如预期的那样从同位素分馏10的理论和测量任何太阳系材料至今（ 如陨石11-13，3-5植物，深海沉积物中14，动物15-17），结果遵循质量-依赖法（参见图1）。</stro...

高精度的测量（较好100ppm以下/原子质量单位）锌稳定同位素组合物只可能为大约15年由于多集电极等离子体源质谱仪的发展，至今在地球被主要应用于与行星科学。应用到医疗领域是新颖的，并具有很强的潜力作为生物标志物用于修改锌的代谢（ 例如，阿尔茨海默病）的疾病。本文报道一种测量高精度锌的天然稳定同位素比率在各脏器鼠标的方法。同样的情况也适用于人类样本。该方法包括器官，锌从原子的其余部分的化学提纯，然后在一个质谱仪同位素比率分析的溶解。 锌同位素测量的质量取决于化学纯化的质量（锌的纯度，低空白排版ARED到的锌存在于样品中的量，该过程的高化学产率）和在仪器偏差的控制。最终锌馏分的高纯度是需要删除这两个同量异序的干扰和非等压干扰创建矩阵的效果。等压核素创建直接干扰（ 例如，64镍）。非等压干扰产生的所谓"基质"的效果，并通过改变电离相比，纯锌标准到的样本进行比较，以1的条件改变的测量值的分析精度。低空白（&lt;10毫微克）表明不存在由外部锌污染的样品是将偏压测量同位素组成。以Zn同位素可以在离子交换层析2被分馏，所有的锌原子的集合确保没有同位素分馏发生时，这意味着该化学过程应当有充分的产率。最后，质谱测量时的器乐同位素分馏的校正是通过"标准包围"的方法进行。 因此，存在的主要困难，以获得精确的测量结果控制外部污染（ 即低空白），生产全收率化学纯化的洁净的任何其他原子或分子，并校正在质谱仪器乐同位素分馏。在本文中，我们将介绍我们的分析协议锌从小鼠器官以及在质谱测量分开。 提取是使用稀释的酸的低量（的HBr / HNO 3介质）上的阴离子交换树脂的微柱（0.5微升和0.1微升）。它有一个完整的产量和测量有一个外部的可重复性优于50 ppm的在66锌/锌64的比例。冰毒的另一个优点外径是，它是非常快的。该方法因此很好地适应于医学，其中一个需要分析大量的样品相比，地球科学，在那里被开发这些分析方法。

<table><tbody><tr><td>Multi-collection inductively-coupled-plasma mass-spectromter</td><td>Thermo-Fisher</td><td></td><td></td></tr><tr><td>Anion-exchange resin AG1 X8 200-400</td><td>Bio-Rad</td><td>140-1443-MSDS</td><td></td></tr><tr><td>Teflon beakers</td><td>Savillex&amp;nbsp;</td><td>200-015-12</td><td></td></tr><tr><td>In-house-made teflon colunms made with shrinkable teflon</td><td></td><td></td><td></td></tr></tbody></table>

high precision zinc isotopic measurements applied to mouse organs

我们提出一个过程，在小鼠器官高精度锌同位素比值来衡量。锌是由5稳定同位素（64锌，66的Zn，67的Zn，68 Zn和70的Zn），这些小鼠器官之间自然分级。我们首先说明如何以溶解，以释放所述锌原子的不同器官;这个步骤是通过HNO 3和H 2 O 2的混合物中实现。然后，我们从所有其他元素净化锌原子，特别是从等压干扰（ 例如 ，镍），通过阴离子交换色谱法中的稀的HBr / HNO 3介质。前两个步骤都在一个干净的实验室使用高纯度的化学品进行。最后，同位素比率通过使用多集电极感应耦合等离子体质谱仪，在低分辨率测量。将样品使用喷雾室和同位素分馏诱导质谱仪是correc注入泰德通过样品的比与一个标准（标准包围技术）的比例。这充分典型的过程产生的同位素的比例与50ppm的（2 SD）的重现性。

在1.5N的HBr的，主要锌物种（ZnBr3-）形式非常强配合物与阴离子交换树脂，而大多数其他元素不与树脂相互作用。锌，然后，通过改变介质稀释的HNO 3，改变Zn构成的形态至Zn 2+这是从树脂6,7释放回收。 同位素比通常表示为每1000个的偏差相对于标准部分: <img alt="式（1）" fo:content-width="2.5in" src="/files/ftp_upload/52479/52479eq1.jpg" /><p class=...

Watch this Scientific Journal Video about High Precision Zinc Isotopic Measurements Applied to Mouse Organs at JoVE.com

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

适用于鼠标机关高精度锌同位素测量

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

high-precision-zinc-isotopic-measurements-applied-to-mouse-organs

Research

JoVE Journal

Chemistry

7.4K 次观看.  Université Paris Diderot. We present the technique to measure with high precision zinc isotope ratios in mouse organs.

视频: High Precision Zinc Isotopic Measurements Applied to Mouse Organs

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

综合程序，以评估<em&gt;在体内</em&gt;癌症纳米医学的性能

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

科研

癌症研究

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

制备和评价<sup&gt;99米</sup&gt;锝标记的三齿螯合物预靶向使用生物正交化学

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

化学

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

原子吸纳光谱测量哺乳动物细胞细胞内锌池

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

生物化学

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

啮齿动物组织的霰弹枪脂质组学

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

从小鼠多个器官分离和定量寨卡病毒

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

免疫与感染

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

高脂喂养小鼠体内成分及代谢锁定分析

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

医学

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

平滑肌细胞近系追踪小鼠晚期动脉粥样硬化病变细胞成分的定量分析

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

小鼠肝细胞核球状估计的高通量原位方法

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

使用正电子发射断层扫描对实验性自身免疫性脑脊髓炎小鼠模型中的 CD19+ B 细胞进行成像

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

适用于鼠标机关高精度锌同位素测量

摘要

探索更多视频

此视频中的章节

综合程序，以评估

制备和评价