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

Übernommen aus vorherigen Studien ein. 1. Konjugation von Annexin V SPIO <ol><li> Oxidieren SPIO Teilchen (Ocean Nanotech Inc., 5 mg / ml) für 1 Stunde bei 20 ° C im Dunkeln in Lösung mit 0,15 M Natriumchlorid (NaIO 4) (4:1 Gewicht: Gewicht-Verhältnis) 2. </li><li> Inkubieren des oxidierten SPIO für 12 Stunden mit gereinigtem ANX Protein (im Verhältnis 1:1, Gewicht: Gewicht) in 0,15 M Natriumborat (Na 2 B 4 O 7 10H 2 O) bei 20 ° C </li><li> Reduzieren der Mischung weiter für 1 Stunde mit Natriumborhydrid (NaBH 4) unter dem Abzug und dann Löschen mit 0,15 M Tris-HCl. </li><li> Trennen Sie den kostenlosen ANX von ANX-SPIO durch Zentrifugation bei 14.000 xg (75 um Pore Microcon Filter, Millipore, Billerica, MA). </li><li> Weiter verfeinern, die Verbindung durch Entfernen ungebundener Verunreinigungen mit Hilfe eines Magnet-Separator-Gerät (Ocean Nanotech, Inc.). </li><li> Quantifizieren Sie den Filter Retentat (ANX-SPIO) Protein-Spiegelvon Bradford-Protein-Assay und lagern bei -80 ° C bei 2 mg / ml in 0,1 M Tris-HCl mit 1% Serumalbumin und Protease-Inhibitoren (Halt Protease-Inhibitor, Thermo Scientific, 1-50 mg / mg ANX). </li></ol> 2. Dynamische Lichtstreuung <ol><li> Getrennt verdünnen SPIO und AX-SPIO in PBS zu einer optischen Dichte von 0,1 und Analyse durch dynamische Lichtstreuung in einem Zetasizer Nano DLS Maschine (Malvern Inc., Worcester, UK) 3. </li><li> Erhalten monomodale Peaks (im Idealfall) für die Verbindung mit einer Z-mittlere Teilchengröße und der Polydispersitätsindex (PDI). Vergleichen Sie diese Größe auf die DLS-gemessene Größe der Eisenoxid-Nanopartikel allein. AX-SPIO DLS Ergebnisse zeigten ein Konjugat Teilchen hydrodynamischen Durchmesser von 78 nm mit einem PDI von 0,13 1, reflektierenden ein homogenes AX-SPIO Spezies 3. </li></ol> 3. Cell Culture, Induktion von Apoptose in Kultur und MRT von kultivierten Zellen <ol><li> Kultur erwachsenen Maus Knochenmark mesenchymale sTEM-Zellen (MMSCs) in DMEM, ergänzt mit 10% FBS und 1% Penicillin-Streptomycin (Invitrogen, Inc.), vorzugsweise in der Passage 20-30 April. </li><li> Apoptose in den kultivierten Zellen durch Behandeln für unterschiedliche Zeiten bei 37 ° C mit DOX (Sigma, 1 mM in 0,9% Kochsalzlösung, 1 nM-Konzentration in den Medien) 12. </li><li> Quantifizierung der Menge an Apoptose in Kultur entweder mit MRT mit ANX-SPIO (beschrieben in 3.4 unten) oder über Mikroskopie nach TUNEL-Färbung (APO-BrdUTM TUNEL Assay Kit, Molecular Probes / Invitrogen, Eugene, OR) oder nach der Färbung mit ANX-FITC (Roche, Schweiz). </li><li> Kennzeichnen der Zellen mit AX-SPIO (10 mg Protein / ml Medium nicht anders angegeben), oder ein gleichwertiges Gewicht der freien SPIO (5 mg), 15 min bei 37 ° C 4 x waschen mit PBS und Überlagerung mit 1 ml 1% Agarose um die Diffusion der SPIO Nanopartikel zu verhindern. Führen MRT der Schalen (unten beschrieben). Alternativ, waschen Sie die Zellen und trypsinieren nach DOX und ANX-Kennzeichnung SPIO, dann lassen sich die Zellen zubilden Pellets in Eppendorf-Röhrchen. </li></ol> 4. In-vitro-MRT <ol><li> Verwenden Sie einen 1,5 oder 3 Tesla GE Signa EXCITE Scanner mit Standard-Knie-Empfangsspule für In-vitro-MRT-Analyse. </li><li> Legen Sie die Kulturschalen oder Rohre in einem Agar-Phantom und Bild unter Verwendung eines Gradienten-Echo-verwöhnte Gradientensequenz: Repetitionszeit (TR) 550 ms, Echozeit (TE) 10 ms, Flip-Winkel 15 °, Matrix 256 x 256, Feld- of-View 3 cm x 3 cm, Schichtdicke 1,3 mm, Anzahl der Mittelungen (NEX) 2, 0 mm Abstand 5. Bild axial durch die Zellschicht auf der Platte für T2 * Signalverlust, mit Regionen-von-Interesse (ROI) von einem festen Bereich (zum Beispiel 0,8 mm 2). Stellen Sie für den Hintergrund mit Kontrolle ROI Signal von der Agar-Phantom. Messen des Hintergrundrauschens von dem Signal der umgebenden Luft Phantom. </li><li> Berechnen Sie die MRT-Kontrast-zu-Rausch-Verhältnis (CNR: [SI Probe ROI-SI-Steuerung ROI] / [SD von SI von Hintergrundgeräuschen], wobei SI ist Signalintensität undSD ist die Standardabweichung) wurde für jede Kulturschale berechnet. </li></ol> 5. Repräsentative Ergebnisse Die Messung der T2 * Signalverlust in vitro erfordert eine homogene Agar Phantom und das Fehlen von Luftblasen, die Signal nichtig Artefakt schaffen wird. Dieses Artefakt ist schwierig, aus echtem T2 *-Signal Verlust von SPIO zu unterscheiden, und ist entscheidend für interpretierbare Ergebnisse. Bitte siehe Abbildung 1 für ein Beispiel eines Luft-Artefakt als auch echte Eisenoxid T2 * Signalverlust. Für In-vitro-Kulturschale Bildgebung, legen Sie eine entsprechende Volumen der Agar-Gel zu jedem Kulturschale, so dass eine ausreichende axiale MRT-Scheiben, die etwa 1 mm dick sind, angemessen zu Bild die ganze Schale gemacht werden können. Abbildung 2 zeigt die erwartete MRT-Signal aus einzelnen Platten Kultur in Agar. Es ist typischerweise ein Signalverlauf an den Kanten der Platte, so Analyse von Regionen von intERest sollten es vermeiden, diese Unebenheiten. <img alt="1" src="/files/ftp_upload/3775/3775fig1.jpg" /> Abbildung 1. ANX-SPIO erkennt Apoptose in kultivierten neonatalen Kardiomyozyten der Ratte. 1A zeigt ein Foto von pelletiert neonatalen Kardiomyozyten mit Control-Lösung (links Rohr), ANX-SPIO (Mitte Rohr), und DOX + ANX-SPIO (rechts Rohr) behandelt . Notieren Sie sich die bräunliche Farbe des DOX + ANX-SPIO behandelten Zellen, was auf erhöhte Retention des ANX-SPIO Verbindung in apoptotischen Zellen. 1B In-vitro-MRT-Bilder zeigen die intensiven T2 *-Signal Verlust der DOX + ANX-SPIO-Zellen. Auch wird eine Luftblase zwischen dem 4. und 5. Eppendorf-Röhrchen (kleine schwarze Signal void), die ähnlich wie die Leere des Signals ANX-SPIO, und die Analyse kann CNR beeinflussen ist zu sehen. <img alt="2" src="/files/ftp_upload/3775/3775fig2.jpg" /> Abbildung 2. ANX-SPIOBindung ist spezifisch für apoptotische Zellen in Kultur. 2A zeigt apoptotische Ratte neonatalen Kardiomyozyten auf Kulturschalen, unterzogen, um entweder ANX-FLUOS Färbung oder ANX-SPIO-Färbung, mit niedrigen und hohen Konzentrationen (links bzw. rechts) von ANX-SPIO. Beachten Sie die unterschiedlichen T2 * Signalverlust (schwarz-Signal) von apoptotischen Zellen, die mit fluoreszierenden ANX (ANX-FLUOS) und das niedrige Niveau im Vergleich zu hohen ANX-SPIO behandelt wurden. Die erhöhte T2 * Signalverlust auf der rechten Seite ist mit einem deutlich reduzierten ANX-FLUOS Signal, das wurde bestritten hat, durch höhere ASX-SPIO Konzentrationen assoziiert. 2B zeigt die kompetitive Bindung von ANX-SPIO gegen ANX-FLUOS Signal und der Dissoziation Konstante, K i. Die X-Achse bezeichnet die Annexin Proteinkonzentration im logarithmischen Maßstab. Movie 1. <a href="http://www.jove.com/files/ftp_upload/3775/3775movie1.mov" target="_blank">Klicken Sie hier, um zusätzliche ansehen</a>Film.

<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>

Keine Interessenskonflikte erklärt.

Die beschriebene Methode der Konjugation zum Verbinden SPIO Annexin V nutzt die Seitenkette Amingruppen von Annexin und die Carboxylgruppen der SPIO Nanopartikel. Durch gezielte Oxidations-Reduktions-Schritte können kovalente Verknüpfung dieser Verbindungen erreicht werden, und die resultierende funktionalisierte Nanopartikel können isoliert werden. Dieses Verfahren kann verallgemeinert werden, um andere Proteine ​​und Nanopartikel in der Umgebung. Die wichtigsten Schritte sind die Ox...

<table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td><td> Katalog # </td></tr><tr><td> Superparamagnetische Eisenoxid </td><td> Ozean Nanotech, Inc. </td><td> ICK-40-005 </td></tr><tr><td> Doxorubicin </td><td> Sigma </td><td> D1515-10MG </td></tr><tr><td> Supermag Separator </td><td> Ozean Nanotech, Inc. </td><td> N / A </td></tr><tr><td> Zetasizer Nano DLS Maschine </td><td> Malvern, Inc </td><td> N / A </td></tr></tbody></table>

synthesis of an in vivo mri-detectable apoptosis probe

Zellulärer Apoptose ist ein hervorstechendes Merkmal von vielen Krankheiten, und das den programmierten Zelltod tritt in der Regel vor der klinischen Manifestation der Erkrankung offensichtlich sind. Ein Mittel, um Apoptose in ihren frühesten, reversiblen Stadium zu erkennen wäre leisten einen vorklinischen &quot;Fenster&quot;, in der präventiven oder therapeutischen Maßnahmen ergriffen, um das Herz vor bleibenden Schäden schützen könnte. Hier stellen wir ein einfaches und robustes Verfahren für die menschliche Annexin V (ANX), die eifrig an Zellen bindet, in den ersten, reversiblen Stadium der Apoptose, superparamagnetische Eisenoxid (SPIO)-Nanopartikel, die als MRI-Kontrastmittel nachweisbaren dienen konjugieren. Die Methode der Konjugation beginnt mit einer Oxidation der SPIO-Nanopartikel, die Carboxylgruppen oxidiert auf dem Polysaccharid Schale der SPIO. Gereinigtes ANX Protein wird dann in der Einstellung eines Natrium-Borat-Lösung, um eine kovalente Wechselwirkung mit ANX SPIO erleichtern in einer reduzierenden Puffer zugesetzt. Ein abschliessender Reduktionsschritt mit Natrium borohydridE wird durchgeführt, um die Reduktion abgeschlossen ist, und dann wird die Reaktion gestoppt. Unkonjugiertes ANX wird aus der Mischung von Mikrozentrifuge Filtration entfernt. Die Größe und Reinheit der ANX-SPIO Produkt zeichnet sich durch dynamische Lichtstreuung (DLS) verifiziert. Diese Methode erfordert keine zusätzlich oder Modifikation, das Polysaccharid SPIO-Shell, um vernetztes Eisenoxid-Partikel-Konjugation Methoden oder Biotin-markierten Nanopartikeln dagegen. Im Ergebnis stellt diese Methode einen einfachen, robusten Ansatz, der Konjugation von anderen Proteinen von Interesse verlängert werden kann.

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

Früherkennung von Apoptose gefährdeten Zellpopulationen in einer Vielzahl von Krankheiten zu identifizieren. Hier zeigen wir ein Verfahren, um eine der frühen Apoptose-Detektion Protein (Annexin V) mit einem MRI-nachweisbare Eisenoxid-Nanopartikel (SPIO) zu verbinden. Diese Methode kann auch auf andere Proteine ​​von Interesse zu MRT-nachweisbar molekulare Bildgebung Sonden erzeugen verlängert werden.

Synthese eines<em&gt; In vivo</em&gt; MRT-Probe nachweisbar Apoptose

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

synthesis-of-an-in-vivo-mri-detectable-apoptosis-probe

Research

JoVE Journal

Biology

Synthesis of an In vivo MRI-detectable Apoptosis Probe

9.1K Aufrufe.  Stanford University Medical Center. Früherkennung von Apoptose gefährdeten Zellpopulationen in einer Vielzahl von Krankheiten zu identifizieren. Hier zeigen wir ein Verfahren, um eine der frühen Apoptose-Detektion Protein (Annexin V) mit einem MRI-nachweisbare Eisenoxid-Nanopartikel (SPIO) zu verbinden. Diese Methode kann auch auf andere Proteine ​​von Interesse zu MRT-nachweisbar molekulare Bildgebung Sonden erzeugen verlängert werden.

Serie: 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 target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Hausgemachte Mutagenese von Whole Plasmide

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

Forschung

Biologie

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 of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

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

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In-vivo-Imaging und therapeutische Behandlungen in einem orthotopen Mausmodell des Eierstockkrebs

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, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Überwachung dynamischen Veränderungen in der mitochondrialen Kalzium während der Apoptose mit einem genetisch kodierten Kalzium-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

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Die Aktivierung der Apoptose durch cytoplasmatische Mikroinjektion von Cytochrom C</em

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 Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

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

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Die Tomate / GFP-FLP / FRT Verfahren zur Live-Imaging von Mosaic Erwachsene<em&gt; Drosophila</em&gt; Sehzellen

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 in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Die Analyse der Apoptose in Zebrafischembryonen von Whole-mount Immunfluoreszenz an aktivierte Caspase 3 Detect

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

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

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

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Ein In vivo Vernetzung Ansatz zur Proteinkomplexe Von Isolieren Drosophila Embryonen

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

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Die Messung der Hämsynthese Levels in Säugerzellen

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

An Ex vivo Culture System to Study 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 produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Ein<em&gt; Ex-vivo-</em&gt; Kultur-System, um Schilddrüsenentwicklungsstudie

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 ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Bewertung der Selective mRNA Translation in Säugerzellen durch Polysomen Profilieren

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

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