Los Institutos Nacionales de Salud, NHLBI (RD, Paraguay, PS). Asociación Americana del Corazón (JL). La Universidad de Stanford, VPUE Grant (JL).

Adaptado de un estudio previo. 1. La conjugación de Anexina V a SPIO <ol><li> Oxidar partículas Spio (Mar Nanotech Inc., 5 mg / ml) durante 1 hora a 20 ° C en la oscuridad en solución con 0,15 M de peryodato de sodio (NaIO 4) (04:01 peso: peso) 2. </li><li> Incubar el SPIO oxidado durante 12 horas con la proteína purificada ANX (relación 1:1, el peso: peso) en 0,15 M de borato de sodio (Na 2 B 4 O 7 10H 2 O) a 20 ° C. </li><li> Reducir la mezcla durante 1 hora más con borohidruro sódico (NaBH 4) bajo la campana de humos y después se apaga con 0,15 M de Tris-HCl. </li><li> Separar la ANX libre de ANX-SPIO por centrifugación a 14.000 xg (75 micras de poro Microcon filtro, Millipore, Billerica, MA). </li><li> Además refinar el compuesto mediante la eliminación de impurezas no unidas mediante un dispositivo separador magnético (Mar Nanotech, Inc.). </li><li> Cuantificar el filtro retenido (ANX-SPIO) los niveles de proteínapor ensayo de proteínas Bradford y almacenar a -80 ° C a 2 mg / ml en 0,1 M Tris-HCl con 1% de suero de albúmina inhibidores de la proteasa y (inhibidor de la proteasa Alto, Thermo Scientific, 1-50 mg / ANX mg). </li></ol> 2. Dispersión de luz dinámica <ol><li> Separadamente diluir la SPIO y ANX SPIO-en PBS a una densidad óptica de 0,1 y analizar por dispersión dinámica de luz en una máquina Zetasizer nano DLS (Malvern Inc., Worcestershire, Reino Unido) 3. </li><li> Obtener picos monomodales (idealmente) para el compuesto, con un tamaño de partícula promedio Z y el índice de polidispersidad (PDI). Comparar este tamaño al tamaño DLS-medida de la nanopartícula de óxido de hierro solo. ANX-DLS Spio resultados mostraron un diámetro de partícula hidrodinámico conjugado de 78 nm con un PDI de 0,13 1, lo que refleja un homogéneos Spio ANX especies 3. </li></ol> 3. Cultivo celular, la inducción de la apoptosis en cultivos, y la resonancia magnética de las células cultivadas <ol><li> Cultura ratón adulto de médula ósea mesenquimales scélulas TEM (MMSCs) en DMEM suplementado con FBS al 10% y 1% de penicilina-estreptomicina (Invitrogen, Inc.), preferiblemente en pasaje 4 20-30. </li><li> Inducir la apoptosis en las células de cultivo mediante el tratamiento de diferentes tiempos a 37 ° C con DOX (Sigma, 1 mM en solución salina al 0,9%, 1 mM de concentración en los medios de comunicación) 12. </li><li> Cuantificar la cantidad de apoptosis en cultivos utilizando resonancia magnética con ANX-SPIO (descrito en el punto 3.4 más adelante) o utilizando un microscopio después de la tinción de TUNEL (APO-BrdUTM Kit de ensayo de TUNEL, Molecular Probes / Invitrogen, Eugene, OR) o después de la tinción con ANX-FITC (Roche, Suiza). </li><li> Etiquete las células con ANX-SPIO (10 mg de proteína / ml de medio excepto como se indica), o con un peso equivalente de libre SPIO (5 mg), durante 15 min a 37 ° C. Lavar 4 veces con PBS, y de superposición con 1 ml de agarosa al 1% para evitar la difusión de las nanopartículas Spio. Realizar resonancia magnética de los platos (descrito más adelante). Alternativamente, se lavan las células y después trypsinize DOX y etiquetado ANX-SPIO, a continuación, permitir que las célulasformar gránulos en tubos Eppendorf. </li></ol> 4. In vitro RM <ol><li> Use un 1,5 o 3 Tesla GE Signa Excite escáner con la batería estándar de rodilla para el receptor in vitro análisis de resonancia magnética. </li><li> Coloque las placas de cultivo o tubos en un fantasma de agar, y la imagen utilizando una secuencia de eco de gradiente gradiente de mal estado: tiempo de repetición (TR) 550 ms, tiempo de eco (TE) de 10 ms, ángulo de inclinación de 15 °, matriz de 256 x 256, sobre el terreno de vista de 3 cm x 3 cm, grosor de corte de 1,3 mm, el número de promedios (NEX) 2, el espaciado de 0 5 mm. Imagen axialmente a través de la capa de células sobre la placa para la pérdida de señal * T2, utilizando regiones de interés (ROIs) de un área fija (por ejemplo, 0,8 mm 2). Ajustar para obtener el fondo usando la señal de control de retorno de la inversión el fantasma de agar. Medir el ruido de fondo de la señal del aire que rodea al fantasma. </li><li> Calcular la resonancia magnética con contraste-ruido (CNR: [SI-SI retorno de la inversión de la muestra de control de retorno de la inversión] / [SD de SI de ruido de fondo], en la SI es la intensidad de la señal, ySD es la desviación estándar) fue calculada para cada placa de cultivo. </li></ol> 5. Los resultados representativos Medición de la pérdida de la señal T2 * in vitro requiere un fantasma de agar homogénea y la falta de burbujas de aire, lo que creará artefactos de señal sin efecto. Este artefacto es difícil de distinguir de la verdadera pérdida de la señal T2 * de SPIO, y es fundamental para los resultados interpretables. Por favor, vea la Figura 1 para un ejemplo de un artefacto de aire, así como óxido de hierro auténtica T2 * la pérdida de señal. Para la formación de imágenes in vitro placa de cultivo, aplicar un volumen apropiado de gel de agar para cada placa de cultivo, de modo que suficientes cortes axiales de resonancia magnética, que son aproximadamente 1 mm de espesor, se pueden hacer de manera adecuada la imagen de la placa entera. Figura 2 ilustra la señal de resonancia magnética esperado a partir de placas de cultivo individuales en agar. Normalmente hay alguna variación de la señal en los bordes de la placa, de modo análisis de las regiones de interest deben evitar estas zonas irregulares. <img alt="Figura 1" src="/files/ftp_upload/3775/3775fig1.jpg" /> Figura 1. ANX-SPIO detecta la apoptosis en cultivos de cardiomiocitos de ratas neonatales. Figura 1A muestra una foto de granulados cardiomiocitos neonatales tratados con solución de control (tubo izquierdo), ANX-SPIO (tubo de media), y DOX + ANX-SPIO (tubo de la derecha) . Tenga en cuenta el color marrón de la DOX + ANX-Spio las células tratadas, lo que refleja una mayor retención del compuesto ANX-SPIO en las células apoptóticas. Figura 1B in vitro de imágenes de resonancia magnética muestran la intensa pérdida de la señal T2 * de los DOX + ANX-Spio células. Además, una burbuja de aire se ve entre la 4 ª y 5 ª tubos Eppendorf (vacío pequeña señal de negro), que es similar a la señal de vacío ANX-SPIO, y que puede afectar el análisis de CNR. <img alt="Figura 2" src="/files/ftp_upload/3775/3775fig2.jpg" /> Figura 2. ANX-SPIOunión es específica para las células apoptóticas en la cultura. Figura 2A muestra de apoptosis cardiomiocitos de rata neonatal en placas de cultivo, sometidos a cualquiera de ANX-Fluos manchas o ANX-SPIO tinción, con concentraciones bajas y altas (izquierda y derecha, respectivamente) de Ans-SPIO. Tenga en cuenta los diferentes T2 * la pérdida de señal (señal de negro) de las células apoptóticas que fueron tratados con fluorescentes ANX (ANX-Fluos) y niveles bajos en comparación con los altos niveles de ANX-SPIO. El aumento de la pérdida de T2 * señal a la derecha está asociada con una marcada reducción ANX-Fluos señal, que se ha competido por fuera superior Spio ASX-concentraciones. Figura 2B ilustra la unión competitiva de ANX-SPIO versus señal ANX-Fluos y la disociación del constante, K i. El eje X indica la concentración de proteína Anexina en escala logarítmica. Película 1. <a href="http://www.jove.com/files/ftp_upload/3775/3775movie1.mov" target="_blank">Haga clic aquí para ver el suplementario</a>película.

<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>

No hay conflictos de interés declarado.

El método de conjugación descrito para ligarse SPIO de Anexina V explota los grupos amino de cadena lateral de Anexina y los restos carboxilo de la nanopartícula SPIO. A través de específicas de oxidación-reducción pasos, unión covalente de estos compuestos se puede lograr, y la nanopartícula funcionalizado resultante puede ser aislado. Este método puede ser generalizado a otras proteínas y nanopartículas de interés. Los pasos más importantes son las condiciones de oxidación y...

<table border="1"><tbody><tr><td> Nombre de Reactivo </td><td> Empresa </td><td> Catálogo # </td></tr><tr><td> Óxido de hierro superparamagnético </td><td> Ocean Nanotech, Inc. </td><td> ICK-40-005 </td></tr><tr><td> La doxorrubicina </td><td> Sigma </td><td> D1515-10MG </td></tr><tr><td> Supermag Separador </td><td> Ocean Nanotech, Inc. </td><td> N / A </td></tr><tr><td> Zetasizer Nano DLS máquina </td><td> Malvern, Inc </td><td> N / A </td></tr></tbody></table>

synthesis of an in vivo mri-detectable apoptosis probe

La apoptosis celular es una característica prominente de muchas enfermedades, y esta muerte programada de las células normalmente se produce antes de las manifestaciones clínicas de la enfermedad son evidentes. Un medio para la detección de la apoptosis en sus primeras etapas, los reversibles que pagar un pre-clínica &quot;ventana&quot; durante el cual las medidas preventivas o terapéuticas que podrían adoptarse para proteger el corazón de un daño permanente. Se presenta aquí un método sencillo y robusto para conjugar humana anexina V (ANX), que se une ávidamente a las células en las primeras etapas, los reversibles de la apoptosis, que superparamagnéticas de óxido de hierro (SPIO) nanopartículas, que sirven como un agente de contraste para RM-detectable. El método de conjugación comienza con una oxidación de las nanopartículas Spio, que oxida grupos carboxilo en la cáscara de polisacárido SPIO. La proteína purificada ANX se añade a continuación en la configuración de una solución de borato de sodio para facilitar la interacción covalente de ANX con SPIO en un tampón de reducción. Una medida de reducción final con borohydrid de sodioe se lleva a cabo para completar la reducción y, a continuación la reacción se apaga. ANX no conjugado se elimina de la mezcla por filtración microcentrífuga. El tamaño y la pureza del producto ANX-SPIO es verificada por dispersión dinámica de luz (DAL). Este método no requiere además de, o la modificación de, el polisacárido SPIO cáscara, en oposición a reticulados métodos de óxido de hierro conjugación de partículas o nanopartículas marcado con biotina. Como resultado, este método representa un método sencillo, robusto que puede extenderse a la conjugación de otras proteínas de interés.

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

La detección temprana de la apoptosis puede identificar en riesgo las poblaciones de células en una variedad de enfermedades. Aquí se demuestra un método para unir una de las primeras apoptosis de detección de la proteína (anexina V) a una nanopartícula de hierro óxido de la RM-detectable (SPIO). Este método puede extenderse a otras proteínas de interés para generar resonancia magnética detectables sondas de imagen molecular.

Síntesis de un<em&gt; En vivo</em&gt; RM-detectable apoptosis de la sonda

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 vistas.  Stanford University Medical Center. La detección temprana de la apoptosis puede identificar en riesgo las poblaciones de células en una variedad de enfermedades. Aquí se demuestra un método para unir una de las primeras apoptosis de detección de la proteína (anexina V) a una nanopartícula de hierro óxido de la RM-detectable (SPIO). Este método puede extenderse a otras proteínas de interés para generar resonancia magnética detectables sondas de imagen molecular.

Video: 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.

Mutagénesis dirigida casera de plásmidos enteros

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

Investigación

Biología

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.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

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.

Seguimiento de los cambios dinámicos en niveles de calcio mitocondrial de la apoptosis mediante un sensor de calcio genéticamente codificados

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.

La activación de la apoptosis mediante microinyección citoplasmática del citocromo 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.

El tomate / GFP-FLP / FRT Método para imágenes en vivo del mosaico adultos<em&gt; Drosophila</em&gt; Las células fotorreceptoras

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.

Análisis de la apoptosis en embriones de pez cebra en Todo el montaje de inmunofluorescencia para detectar la caspasa 3 activada

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.

Un In vivo Enfoque de reticulación para aislar Complejos de Proteína Desde Drosophila Los embriones

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.

Medición de niveles hemo síntesis en células de mamífero

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.

Un<em&gt; Ex vivo</em&gt; Sistema de Cultura para estudiar el desarrollo de la tiroides

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.

Evaluación del selectivo ARNm traducción en células de mamífero por polisoma perfiles

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