Name: synthesis of an in vivo mri-detectable apoptosis probe
Uploaded: 2012-07-31T12:00:00
Description: Watch this Scientific Journal Video about Synthesis of an In vivo MRI-detectable Apoptosis Probe at JoVE.com

National Institutes of Health, NHLBI (RD, PY, PS). American Heart Association (JL). Stanford University, VPUE Grant (JL).

Tilpasset fra tidligere studie 1. 1. Bøyning av Annexin V til SPIO <ol><li> Oksidere SPIO partikler (Ocean Nanotech Inc., 5 mg / ml) for 1 time ved 20 ° C i mørket i løsningen med 0,15 M natrium periodate (NaIO 4) (04:01 vekt: vekt ratio) 2. </li><li> Inkuber oksidert SPIO for 12 t med renset ANX protein (1:1 ratio, vekt: vekt) i 0,15 M natrium borate (Na 2 B 4 O 7 10H 2 O) ved 20 ° C. </li><li> Reduser bland...

<ol>
	<li>Dash, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+MRI+probe+to+detect+treatment+of+cardiac+apoptosis+in+vivo.">A molecular MRI probe to detect treatment of cardiac apoptosis in vivo.</a> Magn. Reson. Med. , (2011).</li><li>Khym, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reaction+of+methylamine+with+periodate-oxidized+adenosine+5'-phosphate.">The reaction of methylamine with periodate-oxidized adenosine 5'-phosphate.</a> Biochemistry. 2, 344-350 (1963).</li><li>Liu, Y. Y., Yu, Y., Zhang, G. B., Tang, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+controlled+release+of+novel+nanoparticles+based+on+MMA/beta-CD+copolymers.">Preparation, characterization, and controlled release of novel nanoparticles based on MMA/beta-CD copolymers.</a> Macromol. Biosci. 7, 1250-1257 (2007).</li><li>Seo, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FeCo/graphitic-shell+nanocrystals+as+advanced+magnetic-resonance-imaging+and+near-infrared+agents.">FeCo/graphitic-shell nanocrystals as advanced magnetic-resonance-imaging and near-infrared agents.</a> Nat. Mater. 5, 971-976 (2006).</li><li>Hsiao, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticle+labeling+of+mesenchymal+stem+cells+without+transfection+agent:+cellular+behavior+and+capability+of+detection+with+clinical+1.5+T+magnetic+resonance+at+the+single+cell+level.">Magnetic nanoparticle labeling of mesenchymal stem cells without transfection agent: cellular behavior and capability of detection with clinical 1.5 T magnetic resonance at the single cell level.</a> Magn. Reson. Med. 58, 717-724 (2007).</li><li>Zhao, M., Beauregard, D. A., Loizou, L., Davletov, B., Brindle, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+detection+of+apoptosis+using+magnetic+resonance+imaging+and+a+targeted+contrast+agent.">Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent.</a> Nat. Med. 7, 1241-1244 (2001).</li><li>van Tilborg, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internalization+of+annexin+A5-functionalized+iron+oxide+particles+by+apoptotic+Jurkat+cells.">Internalization of annexin A5-functionalized iron oxide particles by apoptotic Jurkat cells.</a> Contrast Media Mol. Imaging. 4, 24-32 (2009).</li><li>Sosnovik, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+cardiomyocyte+apoptosis+with+a+novel+magneto-optical+nanoparticle.">Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle.</a> Magn. Reson. Med. 54, 718-724 (2005).</li><li>Sosnovik, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+MRI+detects+low+levels+of+cardiomyocyte+apoptosis+in+a+transgenic+model+of+chronic+heart+failure.">Molecular MRI detects low levels of cardiomyocyte apoptosis in a transgenic model of chronic heart failure.</a> Circ. Cardiovasc. Imaging. 2, 468-475 (2009).</li><li>Dumont, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+death+induced+by+myocardial+ischemia+and+reperfusion:+measurement+with+recombinant+human+annexin-V+in+a+mouse+model.">Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model.</a> Circulation. 102, 1564-1568 (2000).</li><li>Blankenberg, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+and+imaging+of+phosphatidylserine+expression+during+programmed+cell+death.">In vivo detection and imaging of phosphatidylserine expression during programmed cell death.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 6349-6354 (1998).</li><li>Schellenberger, E. A., Sosnovik, D., Weissleder, R., Josephson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magneto/optical+annexin+V,+a+multimodal+protein.">Magneto/optical annexin V, a multimodal protein.</a> Bioconjug. Chem. 15, 1062-1067 (2004).</li></ol>

Den beskrives konjugering metode for å koble SPIO til Annexin V utnytter sidekjettingen amin grupper av Annexin og karboksylgrupper moieties av SPIO nanopartikler. Gjennom konkrete oksidasjon-reduksjon trinn, kan kovalent kobling av disse forbindelsene kan oppnås, og den resulterende functionalized nanopartikler kan isoleres. Denne metoden kan generaliseres til andre proteiner og nanopartikler av interesse. De mest kritiske trinnene er de oksidasjon og reduksjon forhold, utført i de aktue...

<table border="1"><tbody><tr><td> Navn på Reagens </td><td> Firma </td><td> Katalog # </td></tr><tr><td> Superparamagnetiske jernoksid </td><td> Ocean Nanotech, Inc. </td><td> ICK-40-005 </td></tr><tr><td> Doksorubicin </td><td> Sigma </td><td> D1515-10mg </td></tr><tr><td> SuperMag Separator </td><td> Ocean Nanotech, Inc. </td><td> N / A </td></tr><tr><td> Zetasizer Nano DLS maskin </td><td> Malvern, Inc </td><td> N / A </td></tr></tbody></table>

synthesis of an in vivo mri-detectable apoptosis probe

Cellular apoptose er et fremtredende trekk ved mange sykdommer, og dette programmert celledød oppstår vanligvis før kliniske manifestasjoner av sykdommen er tydelig. En måte å oppdage apoptose i de tidligste stadiene, reversible ville ha råd til en pre-klinisk &quot;vindu&quot; hvor forebyggende eller terapeutisk tiltak kan treffes for å beskytte hjertet fra permanent skade. Vi presenterer her en enkel og robust metode for å konjugat menneskelig Annexin V (ANX), som ivrig binder seg til cellene i de tidligste, reversible stadier av apoptose, til superparamagnetiske jernoksid (SPIO) nanopartikler, som fungerer som en MR-detekterbar kontrastmiddel. Den konjugering metoden begynner med en oksidasjon av SPIO nanopartikler, som oksiderer karboksylgrupper på polysakkaridet skallet av SPIO. Renset ANX protein blir deretter lagt i innstillingen av en natrium borate løsning for å lette kovalente samspill av ANX med SPIO i en redusere buffer. En endelig reduksjon takt med natrium borohydride er utført for å fullføre reduksjon, og deretter reaksjonen er slukket. Ukonjugert ANX fjernes fra blandingen av mikrosentrifuge filtrering. Størrelsen og renhet ANX-SPIO produktet er bekreftet av dynamisk lysspredning (DLS). Denne metoden krever ikke tillegg til, eller modifisering av, polysakkaridet SPIO skallet, i motsetning til krysskoblede jernoksid partikkel konjugasjonsforsøkene metoder eller biotin-merket nanopartikler. Som et resultat, representerer denne metoden en enkel, robust tilnærming som kan utvides til konjugering av andre proteiner av interesse.

Watch this Scientific Journal Video about Synthesis of an In vivo MRI-detectable Apoptosis Probe at JoVE.com

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Tidlig deteksjon av apoptose kan identifisere risikopersoner celle populasjoner i en rekke sykdommer. Her demonstrerer vi en metode for å koble en tidlig apoptose-deteksjon protein (Annexin V) til en MR-påvisbar jernoksid nanopartikler (SPIO). Denne metoden kan utvides til andre proteiner av interesse å generere MRI-sporbare Molecular Imaging sonder.

Syntese av en<em&gt; In vivo</em&gt; MR-påvisbare Apoptose Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Research

JoVE Journal

Biology

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

Tomat / GFP-FLP / FRT Metode for Live Imaging av Mosaic Voksen<em&gt; Drosophila</em&gt; fotoreseptorcellene

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

En In vivo Crosslinking Approach å isolere protein komplekser Fra Drosophila Embryo

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

An Ex vivo Culture System to Study Thyroid Development

An Ex vivo Culture System to Study Thyroid Development

En<em&gt; Ex vivo</em&gt; Kultur system for å studere Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Strategier for sporing Anastasis, A Cell Survival fenomen som reverserer apoptose

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...