Name: high precision zinc isotopic measurements applied to mouse organs
Uploaded: 2015-05-22T13:00:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about High Precision Zinc Isotopic Measurements Applied to Mouse Organs at JoVE.com

FM Chaire d&#39;Excellence IDEX Sorbonne Paris Cité aracılığıyla ANR fon kabul, INSU PNP hibe yoluyla, Institut Universitaire de France yanı sıra Sorbonne Paris Cité (ANR-10-LabX-0023 ve ANR at LABEX UniverEarth programı -11-IDEX-0005-02). Biz de Avrupa Topluluğu&#39;nun H2020 çerçeve programı / ERC hibe anlaşması # 637503 (Bozulmamış) kapsamında Avrupa Araştırma Konseyi fon teşekkür ederim.

NOT: hayvanlarla ilgili Prosedürler Université Paris Diderot Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. 1. Malzemenin Hazırlanması <ol><li> Alt kaynatma kirlilikten arındırmak için asit 1 L (HNO 3, HBr) distile olur. </li><li> Sıcak (~ 100 ° C) &#39;de beher ve ucu adaptörü temizleyin en az iki gün HNO 3 asidi banyosu yoğunlaştı. </li><li> Birkaç gün boyunca soğuk 3 N HNO 3 banyosu içinde pipet uçları yıkayın ve de-iyonize su ile tek tek üç kez yıkayın. </li></ol> 2. Numune Hazırlama <ol><li> Ketamin ve ksilazin intraperitoneal enjeksiyonu ile farelere anestezi. Ayak tutam yöntemi ile anestezi değerlendirin. </li><li> 1.5 ml tüpler içinde heparin mevcudiyetinde bir kardiyak delme ile kan toplanır. </li><li> Santrifüjleme (10 dakika, 1500 x g) kan hücrelerinden plazma ayrılır ve polipropilen c plazma aktarmakPolipropilen ipuçlarını kullanarak ryogenic şişeler. </li><li> Kalbinden DPBS hepatik ven kesme ve enjekte ederek organlara kalan kanı çıkarın. Servikal dislokasyon ile fare ölümü değerlendirin. </li><li> Steril paslanmaz çelik aletlerle organlarını varsa yağ çevreleyen onları serbest ve polipropilen kriyojenik şişeleri onları-dondurma ani olarak. </li></ol> 3. Kimyasal saflaştırma <ol><li> İlk olarak, konsantre (% 30) ~ 1 ml H2O 2 ve konsantre edilerek (~ 15 M) HNO 3 ~ 1 ml &#39;lik bir karışımı içinde örnekleri çözülür. Davlumbaz içindeki tüm adımları yapın. <ol><li> 15 ml teflon beher içerisine ilgi bütün organı yerleştirin. Daha sonra, beher 5 H2O 2 / HNO 3 ekleyin. Organik maddenin oksidasyon reaksiyonu ve CO 2 salınımı nedeniyle sıçramalarını önlemek için bir kaç dakika için açık bir beher tutun. </li><li> Son olarak, Abo sıcak bir plaka üzerinde beheri koydusolüsyon saat kadar veya bir kaç ut 100 ° C çok açıktır. </li></ol></li><li> Beher açın ve yaklaşık 100 ° C&#39;de bir sıcak plaka üzerinde çözeltisi kurutun. </li><li> Örnek kuruduktan sonra, numunelere 1.5 N HBr 1 ml ekleyin; beher kapatın ve birkaç saat boyunca 100 ° C&#39;de sıcak bir plaka üzerinde çözülür edelim. </li><li> Bu arada 500 ul sütunları hazırlar. <ol><li> Sütun AG1X8 200-400 meş reçine 500 ul ekleyin ve altında bir çöp beher sütun rafa koydu. 5 mi M? ⋅ cm su, 0.5 N HNO 3 5 ml, 5 ml su, 0.5 N HNO 3 5 ml 18.2 ve su daha sonra 5 mi: sırayla değişen reçineyi yıkayın. 1.5 N HBr 5 ml reçine hale getirin. </li></ol></li><li> Bardak oda sıcaklığına soğumasını bekleyin, sonra sıcak plaka beher çıkarın ve yaklaşık 30 dakika boyunca bir ultrason banyosunda koydu ve. </li><li> Beher soğutulur ve reçine yıkandı sonra, beher açın. T ucu adaptörü koyO, şırınga, bir pipet ekleyin; numune 1 ml pipet ve (reçine tahrik etmemek için çok yavaş) reçine üzerine yük. </li><li> Tüm sıvı sütunu içinden geçtikten sonra, 1.5 N HBr 5 ml ekleyin. </li><li> 1.5 N HBr 5 mi kolonu boyunca geçtikten sonra, temiz bir 15 ml&#39;lik bir cam kaba olan çöp kabı değiştirin. </li><li> Aynı anda, 0.5 N HNO 3 2.5 ml 5 ml. Bu aşamada çinko reçineden elüe edilir. </li><li> HNO 3 5 ml sütunu içinden geçtikten sonra, beher çıkarın ve kuruyana kadar 100 ° C&#39;de bir sıcak plaka üzerine yerleştirin. </li><li> Sütun tutucudan sütun çıkarın; reçine çöp (her bir örnek için, yeni bir reçine kullanmak). </li><li> Örnek kuruduktan sonra, daha küçük bir sütun üzerinde asidi (100 ul), aynı hacimde protokolü tekrar ve daha sonra kuruyana kadar bir sıcak plaka üzerine yerleştirin. Örnek kitle spektrometresi için hazırdır. </li></ol> 4. Kütle-spektrometresi Ölçümü <ol><li> Zn izotop alçı AnalizÇok toplayıcı-indüktif eşleşmiş plazma-kütle spektrometresi (MC-ICP-MS) lan de. <ol><li> Tablo 1&#39;de özetlenmiştir makine parametrelerini kullanın. </li></ol></li><li> 62 Ni, 63 Cu, Zn 64, 65 Cu, Zn 66, 67 Zn ve 68 Zn KÜTLE (m / z) toplamak için Faraday bardak yerleştirin. </li><li> Izotop analizi için 0.1 M HNO 3 500 ppb Zn içeren bir solüsyon hazırlayın. </li><li> 100 ul / dakika teflon nebulizer ile birlikte bir püskürtme odası kullanarak Zn 500 ppb çözeltisi analiz eder. Her bir örnek için, her bir tarama entegrasyon süresi 8,389 sn olduğu 30 taramalar (30 döngü, 1 blok) ölçer. </li><li> Boş solüsyonu (örnekleri yeniden çözünmesi için kullanılan 0.1 M HNO 3 çözeltisi) on-tepe sıfır yoğunlukları çıkarılarak arka plan düzeltin. </li><li> Kontrol ve 62 Ni doruk şiddeti ölçülerek doğru mümkün olan 64 Ni izobarik girişim.Doğru, 64 Ni / 62 Ni oranı doğal olduğunu enstrümantal kitle önyargı bu değeri (0,2548) varsayalım, ve daha sonra kütle 64 64 Ni kaldırın: Ölçülen 64 Zn gerçek = 64 Zn - ölçülen 64 Ni = 64 Zn - (64 Ni / 62 Ni) 62 x doğal Ni ölçüldü. </li><li> JMC Lyon Zn standardının 500 ppb, standart solüsyonu (ya da IRMM-3702 gibi başka mevcut standart) numunelerin her bracketing ile enstrümantal kitle önyargı düzeltin. Önce ve örnek eksi 1 sonra ölçüldü ve 1.000 ile çarpılması iki standart 66 Zn / 64 Zn oranının ortalama numunenin 66 Zn / 64 Zn oranı bölünmesi standart braketi gerçekleştir (Denklem 1 e bakınız). JMC Lyon Zn standart Tipik dış hassasiyet 0,05 permil / amu (2 standart sapma, 2 sd) &#39;dir. </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>

Yazarlar ifşa hiçbir şey yok.

ölçümlerin tekrarlanabilirliği farklı analitik oturumları sırasında yürütülen aynı numunelerin çoğaltılmış analizlerle değerlendirilir. Örnek 6 için, aynı karasal kullanıcıların rock 7 defa tekrarlanır olan ve Tablo 2&#39;de rapor edilen sonuçları elde edilmiştir. Izotop fraksiyon 10 teorisinin beklenen ve bugüne kadar herhangi bir güneş sistemi malzeme ölçüldüğü gibi (örneğin, göktaşı 11-13, 3-5,<...

yüksek hassasiyetli ölçümü (daha iyi 100 ppm / atomik kütle birimi) çinko izotop bileşimi sadece çoklu-toplayıcı plazma kaynak kitle spektrometresi gelişimine yaklaşık 15 yıl sayesinde mümkün olmuştur ve o zamandan beri çoğunlukla Earth uygulanmış ve gezegen bilimleri. Tıp alanında uygulamaları yenidir ve çinko metabolizmasını (örneğin, Alzheimer hastalığı) modifiye hastalıklar için biyobelirteçler gibi güçlü bir potansiyele sahiptir. Bu makale çeşitli fare organlarda yüksek hassasiyetle çinkonun doğal izotop oranları ölçmek için bir yöntem bildirir. Aynı insan örnekleri için geçerli olacaktır. yöntem, organ, atomunu kalan çinkonun kimyasal saflaştırma ve daha sonra bir kitle spektrometresi üzerinde izotop oranı analiz çözünme meydana gelir. Zn izotop ölçümlerin kaliteli kimyasal arıtma kalitesi (Zn saflığı, düşük boş abone ol bağlıdırörnek Zn miktarına ared, yüksek kimyasal usul verim) ve enstrümantal önyargı kontrolü. Nihai Zn fraksiyonunun yüksek saflıkta bir matris efekti oluşturmak İzobarik müdahaleler ve non-izobarik girişim hem kaldırmak için gereklidir. Isobaric nuclides doğrudan müdahaleler (örneğin, 64 Ni) oluşturun. Sigara izobarik müdahaleler olarak adlandırılan &quot;matris&quot; etkisi oluşturabilir ve numuneler 1 göre olan saf çinko standardına göre iyonizasyon durumunu değiştirerek ölçümleri analitik hassasiyet değiştirebilir. Düşük boş (&lt;10 ng) dış Zn ile numunelerin kontaminasyon olduğunu gösterir önyargı ölçülen izotop bileşimini olur o. Zn izotopları, iyon-değiştirme kromatografisi 2 boyunca kısımlara gibi, bütün Zn atomları toplanması herhangi bir izotopik fraksiyonasyon kimyasal işlemi tam verim sahip olması gerektiği anlamına gelen oluşmasını temin eder. Son olarak, kütle spektrometresi ölçümü sırasında enstrümantal izotop fraksiyon düzeltilmesi &quot;standart dizeleme&quot; yöntemi ile yapılır. Bu nedenle, ana zorluklar elde etmek için hassas ölçümler başka atomlar veya moleküller temiz tam verim kimyasal arıtmaya üreten, (boş, yani düşük) dış kirlenmeyi kontrol ve kütle spektrometresi-enstrümantal izotop fraksiyonasyon düzeltiyorsanız. Bu yazıda, fare organları yanı sıra kitle spektrometresi ölçümlerinden Zn ayırmak için analitik protokol anlatacağız. ekstraksiyon, anyon-değişim reçine mikro sütununa (0.5 ul ve 0.1 ul) ile seyreltildi asitlerin düşük miktarda (HBr / HNO 3 ortam) kullanılarak yapılır. Bu tam bir verim vardır ve ölçümler harici tekrarlanabilirlik 66 Zn / 64 Zn oranı daha iyi 50 ppm var. Meth diğer bir avantajıod çok hızlı olmasıdır. Bu yüzden, yöntem, çok iyi bir bu analitik yöntemler geliştirilmiştir bilimleri ile karşılaştırıldığında numune çok sayıda analiz etmek için ihtiyacı olan, tıp şekilde uyarlanmıştır.

<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

Biz fare organlarda yüksek hassasiyetli çinko izotop oranları ile ölçmek için bir prosedür mevcut. Çinko doğal fare organları arasında parçalandı olan 5 duraylı izotop (64 Zn, 66 Zn, 67 Zn, 68 Zn ve 70 Zn) oluşmaktadır. Biz ilk Zn atomları boşaltmak için farklı organlara çözmek için nasıl gösterir; Bu adım, HNO 3 ve H2O 2 bir karışımı ile gerçekleştirilir. Daha sonra, bir seyreltik HBr / HNO 3 ortam içinde anyon değişim kromatografisi ile izobarik etkileşimler (örneğin, Ni), özellikle de, diğer elemanları çinko atomu saflaştırılması. Bu, ilk iki adım yüksek saflıkta kimyasallar kullanılarak temiz bir laboratuarda gerçekleştirilmiştir. Son olarak, izotop oranları düşük çözünürlükte, çoklu toplayıcı indüktif çiftli-plazma kütle spektrometre kullanılarak-ölçülür. Numuneler düzeltmesi olan bir püskürtme odası ve kütle-spektrometresi ile indüklenen izotopik parçalaması kullanılarak enjekte edilirBir standart (standart dizeleme tekniği) oranına örneklerin oranının karşılaştırarak ted. Bu, tam tipik prosedür 50 ppm (2 sd) tekrarlanabilirlik ile bir izotop oranı üretir.

1.5 N HBr, ana çinko türleri (ZnBr3-) anyon değiştirme reçinesi ile form güçlü kompleksleri, çoğu diğer elemanlar reçine ile etkileşime yok iken. Çinko, daha sonra seyreltilmiş HNO 3 orta değiştirme reçinesi 6,7 salınır Zn Zn 2+ türleşmesini değiştirerek elde edilir. Izotop oranı tipik olarak, standart göre 1000 sapmalar başına kısımlar olarak ifade edilir: <img alt="Denklem 1" fo:content-width="2.5in"...

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.

Fare Organlar Uygulanan Yüksek Hassasiyetli Çinko İzotopik Ölçümler

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

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

Video: 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.

Kapsamlı Usulü değerlendirin<em&gt; İn Vivo</em&gt; Kanser Nanomedicines performans

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

Kanser Araştırmaları

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.

Hazırlama ve Değerlendirilmesi<sup&gt; 99m</sup&gt; Bioorthogonal Kimya Kullanarak Öncesi hedeflemek için üç dişli Şelatlar Tc-etiketli

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

Kimya

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.

Mammalin hücrelerinde hücre Içi çinko havuzlarını ölçmek için atomik absorbance spektroskopisi

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

Biyokimya

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.

Kemirgen Dokularının Av Tüfeği Lipidomikleri

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.

Farede Birden Fazla Organdan Zika Virüsünün İzolasyon ve Nicelikselleştirilmesi

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

İmmünoloji ve Enfeksiyon

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.

Vücut kompozisyonu ve metabolik Caging Analizi yüksek yağ Fed farelerde

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

Tıp

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.

Kantitatif analiz düz kas hücre soy izleme farelerin gelişmiş aterosklerotik lezyonları içinde hücresel kompozisyon

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.

Farelerde Hepatosit Nükleer Ploidi Tahmini için Yerinde Yüksek Throughput Yöntemi

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.

Pozitron Emisyon Tomografisi Kullanılarak Deneysel Otoimmün Ensefalomiyelit Fare Modelinde CD19+ B Hücrelerinin Görüntülenmesi

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

Fare Organlar Uygulanan Yüksek Hassasiyetli Çinko İzotopik Ölçümler

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Kapsamlı Usulü değerlendirin

Hazırlama ve Değerlendirilmesi