Biz minnetle Wayne State Üniversitesi (MJA), Amerikan Yaşlanma Araştırma (SMV) Vakfı hibe ve Kariyer Bağımsızlık Geçiş Biyomedikal Görüntüleme National Institute Ödülü (R00EB007129) ve Ulusal Sağlık Enstitüleri Biyomühendislik Pathway başlangıç ​​fonları kabul Sağlık (MJA).

1. LnCl 3 tuzları kullanarak Metalation <ol><li> 30-265 mM bir çözüm üretmek için ligand su içinde eritin. Bu video 73 mM konsantrasyonda ligand 2 - (4-isothiocyanatobenzyl) diethylenetriamine pentaacetic asit (DTPA-p-SCN-Bn) kullanılmıştır. </li><li> 1 M NH 4 OH ekleyerek ligand çözüm pH 5.5 ve 7.0 arasında ayarlayın. Bu video, 0.2 mL 1 M NH 4 OH çözüm kullanılmıştır. </li><li> 5-1000 mM bir konsantrasyon ile bir çözüm üretmek için su LnCl 3 1-2 benzerleri eritin. Bu video, EuCl 3 ve GdCl 3 111 mm konsantrasyonlarda kullanılır. Metal aşırı bir metalation genellikle tamamlanması için sürücü ve dolayısıyla arıtma basitleştirmek için kullanılır. </li><li> Karıştırılırken LnCl 3 ligand çözüm çözümü ekleyin. </li><li> LnCl 3 eklenmesinden sonra, 0.2 M NH 4 OH ekleyerek ortaya çıkan reaksiyon karışımının pH 5.5 ve 7.0 arasında ayarlayın. Bu video toplam 0.5 mL 0.2 M NH 4 OH çözüm kullanılmıştır. Ligand aside duyarlı fonksiyonel gruplar içeriyorsa, bu adımı sırasında pH birden çok kez ayarlayın. DİKKAT - çözüm çok basit olursa, herhangi bir temel duyarlı fonksiyonel gruplar, izotiyosiyanat gibi, konjugasyon için kullanılamaz hale olacaktır. </li><li> PH ölçümleri ile reaksiyon izleyin. PH sabit kalır reaksiyon tamamlandı. </li></ol> 2. Raising pH tetkikleri (bu video dahil, ancak baz duyarlı fonksiyonel grupları olmaksızın ligandlar için iyi değil) <ol><li> ≥ 11 pH ayarlamak için reaksiyon karışımının NH 4 OH konsantre ekleyin. Bu adım, çözünmez hidroksit uncomplexed gibi herhangi bir metal hızlandırabilir. </li><li> Filtre 0.2 mikron filtre ile süpernatantı. Reaksiyon karışımı filtre takunya, santrifüj ve filtreleme önce şişeden tavsiye edilir. </li><li> Diyaliz gerçekleştirilmez ise, düşük basınç (döner buharlaştırma ya da dondurarak kurutma tavsiye edilir) altında çözücü kaldırmak. </li><li> Serbest lantanide kalırsa Adımlar 2,1-2,3 tekrar edilebilir. </li></ol> 3. Diyaliz workup <ol><li> Ekstra uzunluğu (örnek hacmi yaklaşık% 10) bırakarak numune hacmi tutmak için uygun bir uzunluğu (üreticinin yönergeleri izleyin) diyaliz tüp kesin. Bu video, 100-500 dalton moleküler ağırlığı cut-off (MWCO) membran kullanılan, ancak büyük MWCO boru konjugasyon metalation önce yapılır uygun olarak kullanılabilir. Ayrıca, diyaliz kasetleri istenirse diyaliz tüp alternatif olarak kullanılabilir. </li><li> Eğer üretici kurallarına göre uygun ortam sıcaklığında 15 dakika süreyle su kesme diyaliz tüp bekletin. </li><li> Su (diyalizat) ile bir diyaliz rezervuar (1 L beher Bu video) doldurun. Diyalizat hacmi yaklaşık 100x örnek olmalıdır. </li><li> Katlama bir iki kez tüp sonu ve bir diyaliz kapatma kelepçe ile boru katlanmış kısmı güvenli. Diyaliz sırasında kapalı kalmasını sağlamak için lastik bir bant ile kapatılması sonuna sarın. </li><li> 0.2 mikron filtre ile reaksiyon karışımı Filtresi ve boru gözyaşı dikkatli olmak boru açık ucunu süzüntü yük. Tüp kapatmak için yeterli baş boşluk bıraktığınızdan emin olun. </li><li> Iki kez, bir kapatma ile güvenli ve kapatılması adım 3.4 &#39;deki gibi bir lastik bant ile sarın boru kalan açık ucunu katlayın. </li><li> Bir lastik bant kullanarak diyaliz hortumun bir ucunu kelepçe hava içeren bir cam flakon takın. Diğer kelepçe kum içeren bir şişe takın. Bu şişeleri boru diyalizat dalmış kalmasını sağlar. </li><li> Diyalizat içeren diyaliz rezervuar dolu tüp yerleştirin. </li><li> Ortam sıcaklığında yavaş bir hızda (vorteks) manyetik bir heyecan plaka kullanarak diyalizat karıştırın. </li><li> Bir gün ders (diyalizat Bu video, 2.5, 6.5 ve 11.5 saat olarak değiştirildi) üzerinde 3x diyalizat değiştirin ve sonra (20-28 saat diyaliz toplam) diyaliz gecede devam etmesine izin. </li><li> Diyalizat diyaliz tüpünü çıkarın ve dikkatli bir örnek kaldırmak için bir kapatma açık. 3x diyaliz tüp su ile yıkayın ve numune yıkamalar birleştirmek. </li><li> Düşük basınç altında su çıkarın. Bu video Dondurarak kurutma kullanılır. </li></ol> 4. Serbest metal varlığının değerlendirilmesi <ol><li> Metal kompleks çözülür asetat tamponu (buffer hazırlanması: 400 ml suya 1.4 ml asetik asit çözülür, 1 M NH 4 OH ile 5.8 pH ayarlamak ve toplam hacmi 500 ml su ekleyin) ve xylenol turuncu bir gösterge (16 mcM pH 5.8 tamponu). Bu video, 0.3 mg kompleks 0.3 mL tampon dağıldı ve 3 mL indikatör çözeltisi eklendi. </li><li>, Menekşe sarı gösterge bir renk değişimi gözlem yoluyla serbest metal varlığını tespit. </li><li> İsterseniz bir kalibrasyon eğrisi 1 oluşturarak, serbest metal miktarı belirlenebilir. Alternatif olarak, boya arsenazo III xylenol turuncu 2 yerine kullanılan olabilir. Serbest metal kalırsa, daha önce karakterizasyonu, diyaliz, bir tuzsuzlaştırma sütun veya yüksek performanslı sıvı kromatografi (HPLC) kullanarak saflaştırılmış örnek olmalıdır. </li></ol> 5. Su koordinasyon sayısı (q) Belirlenmesi <ol><li> H 2 O karmaşık (~ 1 mM) içeren III-Eu bir çözüm ve D 2 O. aynı konsantrasyonu başka bir çözüm hazırlayın Analizden önce, D 2 O çözümü kalan H 2 O. kaldırmak için üç kez buharlaştırılır ve D 2 O içinde çözünmüş olmalıdır </li><li> Temiz bir küvet su çözeltisi ekleyin ve bir spectrofluorometer küvet yerleştirmek. </li><li> Eksitasyon ve emisyon taramalar her (~ 395 nm ve 595 nm idi) maxima belirlemek için gerçekleştirin. </li><li> Eksitasyon ve emisyon dalga boylarında adım 5.3, eksitasyon ve emisyon yarık genişlikleri (5 nm), flaş sayısı (100), ilk gecikme (0.01 ms), maksimum gecikme (13 ms) belirlenen aşağıdaki parametreleri kullanarak bir fosforesans zaman çürüme deney yapın ve gecikme artış (0,1 ms). Bu koşullar, en kompleksleri uygun, ancak azami gecikme ve artım değerleri son derece uzun veya çok kısa bir çürüme kez türler için artırılabilir veya azaltılabilir. </li><li> Adım adım 5.1 &#39;de hazırlanan D 2 O çözeltisi ile 5.4 tekrarlayın. </li><li> 5.4 ve 5.5 &#39;de elde edilen lüminesans-çürüme verilere göre, şiddeti zamana doğal günlük arsa. Bu satırların eğimi çürüme oranları (τ -1) (Şekil 2). Bu video, Microsoft Excel 2007, ham veri doğal günlük araziler oluşturmak için kullanılır oldu. Horrocks tarafından geliştirilen ve arkadaşları (eq 1) 3 denklemi çürüme oranları kullanın. Ligand metal koordine OH veya NH grupları varsa, o denklemi kullanmak 3 önce değiştirilmiş olmalıdır. </li></ol> eq 1: <img src="/files/ftp_upload/2844/2844eq1.jpg" alt="Denklem 1" /> 6. Relaksivite ölçümler <ol><li> T 1 (longitudinal relaksasyon zamanı) veya T 2 (transvers relaksasyon zamanı): relaksasyon zamanı analizörü istediğiniz uygulamayı modunu seçin. </li><li> Gd farklı konsantrasyonlarda III içeren sulu bir çözücü kompleks içeren örneklerin bir dizi hazırlayın. Bu video, su, 0.625, 1.25, 2.50, 5.00, 10.0, solvent ve çözümleri olarak kullanılan ve 0 mM hazırlanmıştır. Diğer sulu solventler veya tamponlar kullanılabilir, ancak boş olarak solvent kullanılması önemlidir olabilir. Son örnek hacmi, kullanılan cihaz için özeldir. </li><li> Aracı bir örnek koyun ve alet (37 ° C Bu video) sıcaklık dengelenmesi için 5 dakika bekletin. </li><li> T 1 ve T 2 (T 1 ve T 2 temsilcisi eğrileri Şekil 3&#39;te gösterilmiştir) için düzgün bir üstel eğriyi elde etmek için yazılım parametrelerini ayarlayarak gevşeme zamanı (s birimleri) belirleyin. </li><li> 6.3 ve 6.4 örnekleri boş da dahil olmak üzere tüm adımları tekrarlayın. </li><li> S -1 birimleri ölçülen T 1 ve T 2 değerleri ters hesaplayın. </li><li> T 1 -1 veya T karşı 2 -1 Gd III konsantrasyonu değerleri (mM birimleri) çizilir. Gd III içeren kompleksler higroskopik doğası nedeniyle, Gd III konsantrasyonu atomik absorpsiyon spektrofotometresi veya indüktif eşleşmiş plazma kütle spektrometresi kullanarak onaylayın. Düz bir çizgi ile arsa sığdır. Bir temsilci komplo Şekil 4&#39;te gösterilmiştir. </li><li> Monte hattının eğimi relaksivite (T 1 ve T 2, sırasıyla r 1 r 2) ve mM -1 s -1 birimleri vardır. </li></ol> 7. Temsilcisi Sonuçlar Tablolar ve Şekiller bölümünde bu protokolü adımları Temsilcisi veriler dahil edilmiştir. Protokolde belirtilen su koordinasyon sayısı ve relaksivite karakterizasyonu yanı sıra, nihai ürünlerin standart kimyasal teknikleri kullanılarak karakterize etmek için önemlidir. Eu III içeren kompleksler Şekil 5&#39;te gösterilmiştir bileşik kimlik kütle spektrometresi, ve temsili kitle spektrumu Gd III tanı izotop desen kullanılarak elde edilebilir. Ayrıca, non-Gd III. </s&gt; lantanide içeren kompleksler, NMR spektroskopisi ürünün tanımlanması için kullanılabilir. Karmaşık saflık, HPLC veya elementel analiz karakterize etmek için veya her ikisi de kullanılabilir. <img src="/files/ftp_upload/2844/2844fig1.jpg" alt="Şekil 1" /> Şekil 1 metalation ve saflaştırılması için genel şeması: Programı metalation ve nedenleri farklı arıtma yolları seçmek için genel bir prosedür resmeden . <img src="/files/ftp_upload/2844/2844fig2.jpg" alt="Şekil 2" /> Şekil 2 Lüminesans yoğunluğu arsa: bölüm 5 yoğunluğu karşısında zaman doğal günlük Temsilcisi arsa. Hatları, su ve D 2 O çözümleri için edinilen benzer eğrileri yamaçları, Eu su koordinasyon sayısı III içeren kompleksler karakterize eq 1 ile kullanılır. <img src="/files/ftp_upload/2844/2844fig3.jpg" alt="Şekil 3" /> Şekil 3 Gevşeme çürüme zaman eğrileri: (sol) T 1 ve (sağda) T 2 satın alma Temsilcisi veri. Bu eğri şekilleri sapmalar güvenilir veri üretmek. <img src="/files/ftp_upload/2844/2844fig4.jpg" alt="Şekil 4" /> Şekil 4 relaksivite belirlenmesi: Gd III konsantrasyonu karşı 1 / T 1 bir temsilci arsa. Monte hattının eğimi relaksivite ve mM -1 s -1 birimleri vardır. <img src="/files/ftp_upload/2844/2844fig5.jpg" alt="Şekil 5" /> Şekil 5 Kütle spektrumları: tanı izotop desen gösteren Temsilcisi kütle spektrumları (sol) Gd III (sağda) içeren kompleksler ve Eu III içeren kompleksler. Siyah Gauss doruklarına teorik izotop dağılımı temsil eder ve kırmızı çizgiler gerçek verilerdir.

<ol>
	<li>Barge, A., Cravotto, G., Gianolio, E., Fedeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+determine+free+Gd+and+free+ligand+in+solution+of+Gd+chelates.+A+technical+note.">How to determine free Gd and free ligand in solution of Gd chelates. A technical note.</a> Contrast Med. Mol. Imaging. 1, 184-188 (2006).</li><li>Nagaraja, T. N., Croxen, R. L., Panda, S., Knight, R. A., Keenan, K. A., Brown, S. L., Fenstermacher, J. D., Ewing, 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=Application+of+arsenazo+III+in+the+preparation+and+characterization+of+an+albumin-linked,+gadolinium-based+macromolecular+magnetic+resonance+contrast+agent.">Application of arsenazo III in the preparation and characterization of an albumin-linked, gadolinium-based macromolecular magnetic resonance contrast agent.</a> J. Neurosci. Methods. 157, 238-245 (2006).</li><li>Supkowski, R. M., Horrocks, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+determination+of+the+number+of+water+molecules,+q,+coordinated+to+europium(III)+ions+in+solution+from+luminescence+decay+lifetimes.">On the determination of the number of water molecules, q, coordinated to europium(III) ions in solution from luminescence decay lifetimes.</a> Inorg. Chim. Acta. 340, 44-48 (2002).</li><li>Menjoge, A. R., Kannan, R. M., Tomalia, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer-based+drug+and+imaging+conjugates:+design+considerations+for+nanomedical+applications.">Dendrimer-based drug and imaging conjugates: design considerations for nanomedical applications.</a> Drug Discovery Today. 15, 171-185 (2010).</li><li>Que, E. L., Chang, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsive+magnetic+resonance+imaging+contrast+agents+as+chemical+sensors+for+metals+in+biology+and+medicine.">Responsive magnetic resonance imaging contrast agents as chemical sensors for metals in biology and medicine.</a> Chem. Soc. Rev. 39, 51-60 (2010).</li><li>Uppal, R., Caravan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+probes+for+cardiovascular+MR+imaging.">Targeted probes for cardiovascular MR imaging.</a> Future Med. Chem. 2, 451-470 (2010).</li><li>Major, J. L., Meade, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioresponsive,+cell-penetrating,+and+multimeric+MR+contrast+agents.">Bioresponsive, cell-penetrating, and multimeric MR contrast agents.</a> Acc. Chem. Res. 42, 893-903 (2009).</li><li>Datta, A., Raymond, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gd-hydroxypyridinone+(HOPO)-based+high-relaxivity+magnetic+resonance+imaging+(MRI)+contrast+agents.">Gd-hydroxypyridinone (HOPO)-based high-relaxivity magnetic resonance imaging (MRI) contrast agents.</a> Acc. Chem. Res. 42, 938-947 (2009).</li><li>Le&#243;n-Rodr&#237;guez, L. M. D., Lubag, A. J. M., Malloy, C. R., Martinez, G. V., Gillies, R. J., Sherry, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsive+MRI+agents+for+sensing+metabolism+in+vivo.">Responsive MRI agents for sensing metabolism in vivo.</a> Acc. Chem. Res. 42, 948-957 (2009).</li><li>Castelli, D. D., Gianolio, E., Crich, S. G., Terreno, E., Aime, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+containing+nanosized+systems+for+MR-molecular+imaging+applications.">Metal containing nanosized systems for MR-molecular imaging applications.</a> Coord. Chem. Rev. 252, 2424-2443 (2008).</li><li>Caravan, P., Ellison, J. J., McMurry, T. J., Lauffer, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gadolinium(III)+chelates+as+MRI+contrast+agents:+structure,+dynamics,+and+applications.">Gadolinium(III) chelates as MRI contrast agents: structure, dynamics, and applications.</a> Chem. Rev. 99, 2293-2352 (1999).</li><li>Lauffer, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paramagnetic+metal+complexes+as+water+proton+relaxation+agents+for+NMR+imaging:+theory+and+design.">Paramagnetic metal complexes as water proton relaxation agents for NMR imaging: theory and design.</a> Chem. Rev. 87, 901-927 (1987).</li><li>Yoo, B., Pagel, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+responsive+MRI+contrast+agents+for+molecular+imaging.">An overview of responsive MRI contrast agents for molecular imaging.</a> Front. Biosci. 13, 1733-1752 (2008).</li><li>Pandya, S., Yu, J., Parker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+emissive+europium+and+terbium+complexes+for+molecular+imaging+and+sensing.">Engineering emissive europium and terbium complexes for molecular imaging and sensing.</a> Dalton Trans. 23, 2757-2766 (2006).</li><li>Nwe, K., Xu, H., Regino, C. A. S., Bernardo, M., Ileva, L., Riffle, L., Wong, K. J., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+in+the+preparation+of+dendrimer-based+bifunctional+diethylenetriaminepentaacetic+acid+MR+contrast+agent+derivatives.">A new approach in the preparation of dendrimer-based bifunctional diethylenetriaminepentaacetic acid MR contrast agent derivatives.</a> Bioconjugate Chem. 20, 1412-1418 (2009).</li><li>Nwe, K., Bernardo, M., Regino, C. A. S., Williams, M., Brechbiel, M. W. <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+MRI+properties+between+derivatized+DTPA+and+DOTA+gadolinium-dendrimer+conjugates.">Comparison of MRI properties between derivatized DTPA and DOTA gadolinium-dendrimer conjugates.</a> Bioorg. Med. Chem. 18, 5925-5931 (2010).</li><li>Caravan, P., Das, B., Deng, Q., Dumas, S., Jacques, V., Koerner, S. K., Kolodziej, A., Looby, R. J., Sun, W. -. C., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lysine+walk+to+high+relaxivity+collagen-targeted+MRI+contrast+agents.">A lysine walk to high relaxivity collagen-targeted MRI contrast agents.</a> Chem. Commun. , 430-432 (2009).</li><li>Le&#243;n-Rodr&#237;guez, L. M. D., Kovacs, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+and+chelation+chemistry+of+DOTA-peptide+conjugates.">The synthesis and chelation chemistry of DOTA-peptide conjugates.</a> Bioconjugate Chem. 19, 391-402 (2008).</li><li>Boswell, C. A., Eck, P. K., Regino, C. A. S., Bernardo, M., Wong, K. J., Milenic, D. E., Choyke, P. L., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization,+and+biological+evaluation+of+integrin+&#945;V&#946;3-targeted+PAMAM+dendrimers.">Synthesis, characterization, and biological evaluation of integrin &#945;V&#946;3-targeted PAMAM dendrimers.</a> Mol. Pharm. 5, 527-539 (2008).</li></ol>

Lantanide bazlı kontrast ajanlar 4-14 yayınların sayısının artması göz önüne alındığında, bakım, hazırlama, arındırıcı ve tekrarlanabilir ve karşılaştırılabilir sonuçlar sağlamak için ürün karakterize alınması çok önemlidir . Bu kompleksler, genellikle arındırmak ve paramanyetik doğa ve bioconjugation için kullanılan olabilir herhangi bir fonksiyonel grupların duyarlılığı nedeniyle organik moleküllerin göreceli karakterize zorlu kabul edilir. Biz lantanide komplek...

<table border="1"><tbody><tr><th> Reaktifler ve Ekipmanları </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> EuCl 3 • 6H 2 O </td><td> Sigma-Aldrich </td><td> 203.254-5G </td></tr><tr><td> p-SCN-Bn-DTPA </td><td> Macrocyclics </td><td> B-305 </td></tr><tr><td> amonyum hidroksit </td><td> EMD </td><td> AX1303-3 </td></tr><tr><td> Spectra / Por Biotech Selüloz Ester (CE) Diyaliz Membran - 500 D MWCO </td><td> Fisher Scientific </td><td> 68-671-24 </td></tr><tr><td> Millipore IC Millex-LG Filtre Üniteleri </td><td> Fisher Scientific </td><td> SLLG C13 NL </td></tr><tr><td> xylenol turuncu tetrasodyum tuz </td><td> Alfa Aesar </td><td> 41379 </td></tr><tr><td> asetik asit </td><td> Fluka </td><td> 49199 </td></tr><tr><td> D 2 O </td><td> Cambridge İzotop Laboratories, Inc. </td><td> DLM-4-25 </td></tr><tr><td> su arıtma </td><td> ELGA </td><td> Purelab Ultra </td></tr><tr><td> yüksek performanslı sıvı kromatografisi ve kütle spektrometresi </td><td> Shimadzu </td><td> LCMS-2010EV </td></tr><tr><td> relaksasyon zamanı çözümleyicisi </td><td> Bruker </td><td> mq60 minispec </td></tr><tr><td> UV-vis spektrofotometre </td><td> Fisher Scientific </td><td> 20-624-00092 </td></tr><tr><td> dondurarak kurutma makinası </td><td> Fisher Scientific </td><td> 10-030-133 </td></tr><tr><td> pH metre </td><td> Hanna Instruments </td><td> HI 221 </td></tr><tr><td> spectrofluorometer </td><td> Horiba Jobin Yvon </td><td> Fluoromax-4 </td></tr><tr><td> Molekül Ağırlığı Hesaplama sürümü 6.46 Matthew Monroe, indirilen 17 Ekim 2009 </td><td> <a href="http://ncrr.pnl.gov/software/">http://ncrr.pnl.gov/software/</a> </td><td> Molekül Ağırlığı Hesaplama </td></tr></tbody></table>

preparation, purification, and characterization of lanthanide complexes for use as contrast agents for magnetic resonance imaging

Lantanide iyonları şelat tabanlı Polyaminopolycarboxylate ligandlar yaygın kullanılan ve ortaya çıkan komplekslerinin manyetik rezonans görüntüleme (MRG) kontrast ajanlar olarak yararlıdır. Birçok ticari olarak kullanılabilir ligandlar, fonksiyonel, hızlı, yüksek saflıkta izin grupları ve amin-reaktif aktif esterleri ve izotiyosiyanat grupları veya tiol-reaktif maleimides üzerinden makromoleküllerin ve biyomoleküllerin yüksek getirili konjugasyon içerdiğinden özellikle yararlıdır. Bu ligandların metalation bioconjugation kimya, metalation prosedürleri ince farklar alanında ortak bilgi olarak kabul edilirken, metal başlangıç ​​malzemeleri seçerken dikkate alınmalıdır. Ayrıca, saflaştırılması ve karakterizasyonu var ve en etkili yöntemdir seçimi için birden fazla seçenek kısmen başlangıç ​​materyallerinin seçimine bağlıdır. Bu ince farklar yayınlanan protokoller genellikle ihmal edilir. Burada amacımız, metalation, arıtma, ve MRI (Şekil 1) kontrast ajanlar olarak kullanılabilir lantanide komplekslerin karakterizasyonu için ortak yöntemler göstermektir. Bu yayının, biyomedikal bilim adamları, başlangıç ​​maddeleri ve arıtma yöntemleri seçimi kolaylaştırılması lantanide kompleks reaksiyonlar sık ​​kullanılan tepkimelerin kendi repertuarına dahil olacağını bekliyoruz.

Watch this Scientific Journal Video about Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging at JoVE.com

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Biz metalation, arıtma, ve lantanide komplekslerin karakterizasyonu göstermektedir. Burada açıklanan komplekslerinin manyetik rezonans görüntüleme kullanarak bu moleküllerin izlemeyi etkinleştirmek için makromoleküllerin konjuge olabilir.

Manyetik Rezonans Görüntüleme için hazırlanması, Saflaştırılması ve lantanide Kompleksleri Kontrast Ajanlar Kullanım Karakterizasyonu

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

preparation-purification-characterization-lanthanide-complexes-for

Research

JoVE Journal

Biology

14.9K Görüntüleme.  Wayne State University. Biz metalation, arıtma, ve lantanide komplekslerin karakterizasyonu göstermektedir. Burada açıklanan komplekslerinin manyetik rezonans görüntüleme kullanarak bu moleküllerin izlemeyi etkinleştirmek için makromoleküllerin konjuge olabilir.

Video: Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Demir Oksit hESC ve hMSCs Etiketleme MR Görüntüleme in vivo Takip Non-Invasive Nanopartiküller

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Biyoloji

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optik Görüntüleme ile non-invaziv Algılama için Floresan Boyalar Kök Hücreler Etiketleme

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

İnsan Embriyonik Kök Hücreleri Manyetik Rezonans Görüntüleme için in vitro Etiketleme

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Faz Kontrast ve Diferansiyel Girişim Kontrast (DIC) Mikroskopi

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

İzolasyon ve Arıtma Drosophila</emManyetik Boncuk Sıralama> Periferik Nöronlar

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Model Genetik Organizma olarak kullanmak için Medaka Embriyolar, Mikroenjeksiyon

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

Protein Saflaştırma Kullanım için Otomatik Hidrofobik Etkileşim Kromatografisi Sütun Seçimi

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

Manyetik Rezonans Görüntüleme kullanarak Karışım miktarının

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Görüntüleme ve İmmünhistokimyanın için Retina Wholemount Hazırlık için optimize edilmiş Protokolü

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Nesil ve İnsan INO80 Kromatin Yaptırma Kompleksleri ve subcomplexes saflaştırılması

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...