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

Adaptado a partir de estudo prévio 1. 1. Conjugação de anexina V para SPIO <ol><li> Oxidar partículas SPIO (oceano Nanotech Inc., 5 mg / mL) durante 1 hora a 20 ° C no escuro em solução com 0,15 M de sódio periodato (NaIO4) (4:1 em peso: proporção em peso) 2. </li><li> Incubar a SPIO oxidado durante 12 horas com purificado ANX proteína (proporção 1:1, peso: peso) em borato de sódio 0,15 M (Na 2 B 4 O 7 10H 2 O) a 20 ° C. </li><li> Reduzir ainda mais a mistura durante 1 hora com boro-hidreto de sódio (NaBH4), sob a hotte e, em seguida, diluiu-se com 0,15 M de Tris-HCl. </li><li> Separa-se a ANX livre de ANX-SPIO por centrifugação a 14.000 xg (75 mM de poro Microcon filtro, Millipore, Billerica, MA). </li><li> Refinar ainda mais o composto através da remoção de impurezas não acoplados usando um dispositivo separador magnético (Oceano Nanotech, Inc.). </li><li> Quantificar o retentado filtro (ANX-SPIO) níveis de proteínapor Bradford ensaio de proteína e armazenar a -80 ° C a 2 mg / mL em 0,1 M Tris-HCl com 1% de soro de albumina e os inibidores de protease (Inibidor de Protease Halt, Thermo Scientific, 1-50 mg / mg ANX). </li></ol> 2. Espalhamento dinâmico de luz <ol><li> Separadamente diluir a SPIO e ANX-SPIO em PBS para uma densidade óptica de 0,1 e analisar por dispersão dinâmica de luz em uma máquina de Zetasizer DLS Nano (Malvern Inc., Worcestershire, Reino Unido) 3. </li><li> Obter picos monomodal (idealmente) para o composto, com um tamanho de partícula Z-média e do índice de polidispersão (PDI). Comparar este tamanho ao tamanho DLS-medidos das nanopartículas de óxido de ferro sozinho. Resultados ANX-SPIO DLS mostrou um diâmetro hidrodinâmico conjugado de partículas de 78 nm com um PDI de 0,13 um, reflectindo um homogéneos ANX-SPIO espécies 3. </li></ol> 3. Cultura Celular, indução de apoptose em Cultura, e ressonância magnética das células em cultura <ol><li> Cultura adulto rato de medula óssea mesenquimal scélulas TEM (mMSCs) em DMEM suplementado com 10% de FBS e 1% de penicilina-estreptomicina (Invitrogen, Inc.), de preferência a passagem 20-30 abril. </li><li> Induzir a apoptose nas células de cultura por tratamento para diferentes tempos a 37 ° C com DOX (Sigma, 1 mM em solução salina a 0,9%, 1 uM em meio de concentração) 12. </li><li> Quantificar a quantidade de apoptose em cultura utilizando ressonância magnética com ANX-SPIO (descrito em 3.4 abaixo) ou usando microscopia após TUNEL mancha (APO-BrdUTM Assay Kit TUNEL, Molecular Probes / Invitrogen, Eugene, OR) ou após a mancha com ANX-FITC (Roche, Suíça). </li><li> Rotular as células com ANX-SPIO (10 mg médio de proteína / mL, excepto como notado), ou com um peso equivalente de livre SPIO (5 mg), durante 15 min a 37 ° C. Lavar 4 vezes com PBS, e de sobreposição com 1 mL de agarose a 1% para evitar a difusão das nanopartículas SPIO. Realizar MRI dos pratos (descrito abaixo). Alternativamente, lavar as células e depois trypsinize DOX e ANX-SPIO rotulagem, então permitir que as célulasformar pelotas em tubos Eppendorf. </li></ol> 4. In Vitro RM <ol><li> Use um de 1,5 ou 3 Tesla GE scanner de EXCITE Signa com bobina padrão joelho receptor in vitro para a análise de ressonância magnética. </li><li> Coloque as placas de cultura ou tubos em um phantom de ágar, e imagens utilizando um gradiente de eco-seqüência gradiente mimada: tempo de repetição (TR) 550 ms, tempo de eco (TE) 10 ms, flip ângulo de 15 °, matriz de 256 x 256, em campo de-vista de 3 cm x 3 cm, espessura de fatia 1,3 mm, número de médias (NEX) 2, mm de espaçamento 0 5. Imagem axialmente através da camada de células na placa para a perda de sinal T2 *, utilizando-se regiões de interesse (ROIs) de uma área fixa (por exemplo, 0,8 mm 2). Ajuste para o fundo usando o controle de sinal de ROI do phantom de ágar. Medir o ruído de fundo a partir do sinal do ar que circunda a tracejado. </li><li> Calcular a relação de contraste-ruído MRI (CNR: [amostra SI-SI controle ROI ROI] / [SD de SI de ruído de fundo], onde SI é a intensidade do sinal eSD é o desvio padrão) foi calculada para cada prato de cultura. </li></ol> 5. Os resultados representativos Medição da perda de sinal T2 * in vitro requer um fantasma agar homogénea e à falta de bolhas de ar, o que irá criar artefacto vazio sinal. Este artefato é difícil de distinguir de uma verdadeira perda de sinal T2 * de SPIO, e é fundamental para resultados interpretáveis. Favor ver Figura 1 para um exemplo de um artefacto de ar, bem como o óxido de ferro genuína T2 perda de sinal *. Para in vitro de imagem prato de cultura, aplicar um volume apropriado de gel de agar a cada prato de cultura, de modo que suficientes axiais fatias de ressonância magnética, que são cerca de 1 mm de espessura, podem ser feitas de forma adequada a imagem do prato inteiro. A Figura 2 ilustra o sinal esperado RM a partir de placas de cultura individuais em agar. Há tipicamente alguma variação de sinal nas bordas da placa, de modo a análise das regiões de interest devem evitar essas áreas irregulares. <img alt="A Figura 1" src="/files/ftp_upload/3775/3775fig1.jpg" /> Figura 1. ANX-SPIO detecta apoptose em cultura de cardiomiócitos de ratos neonatos. Figura 1A mostra uma foto de peletizadas cardiomiócitos neonatais tratados com solução de controle (tubo à esquerda), ANX-SPIO (tubo central), e + DOX ANX-SPIO (tubo à direita) . Observe a cor acastanhada da DOX + ANX-SPIO células tratadas, refletindo maior retenção do composto ANX-SPIO em células apoptóticas. Figura 1B Em imagens de ressonância magnética in vitro mostram a intensa perda de sinal T2 * do + DOX ANX-SPIO células. Além disso, uma bolha de ar é visto entre os dias 4 e 5 th tubos Eppendorf (void pequeno sinal preto), que é semelhante ao vazio sinal de ANX-SPIO, e que podem afectar a análise de CNR. <img alt="A Figura 2" src="/files/ftp_upload/3775/3775fig2.jpg" /> Figura 2. ANX-SPIOa ligação é específica para células apoptóticas em cultura. Figura 2A demonstra cardiomiócitos de ratos neonatais apoptóticas em pratos de cultura, submetidos a qualquer coloração ANX-FLUOS ou ANX-SPIO coloração, com concentrações baixas e altas (esquerda e direita, respectivamente) de ANX-SPIO. Note-se a diferentes T2 * perda de sinal (sinal preto) de células apoptóticas que foram tratados com ANX fluorescente (ANX-FLUOS) e níveis baixos, versus os níveis elevados de ANX-SPIO. O aumento T2 * perda de sinal no lado direito está associado com uma marcadamente reduzida sinal ANX-FLUOS, que tem sido competiu fora por mais elevados ASX SPIO-concentrações. Figura 2B ilustra a ligação competitiva de ANX-SPIO contra ANX-FLUOS sinal ea dissociação constante, K i. O eixo dos X indica a concentração de proteína anexina em escala logarítmica. Filme 1. <a href="http://www.jove.com/files/ftp_upload/3775/3775movie1.mov" target="_blank">Clique aqui para ver suplementar</a>filme.

<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>

Não há conflitos de interesse declarados.

O método de conjugação descrito para a ligação de SPIO a anexina V explora os grupos de cadeia lateral de amina de anexina e as porções carboxilo da nanopartícula SPIO. Através específicos de oxidação-redução passos, ligação covalente destes compostos pode ser alcançado, e as nanopartículas resultante funcionalizado pode ser isolado. Este método pode ser generalizado a outras proteínas e nanopartículas de interesse. Os passos mais críticos são as condições de oxidaç...

<table border="1"><tbody><tr><td> Nome do Reagente </td><td> Companhia </td><td> Catálogo # </td></tr><tr><td> Óxido de ferro superparamagnético </td><td> Oceano Nanotech, Inc. </td><td> ICK-40-005 </td></tr><tr><td> A doxorrubicina </td><td> Sigma </td><td> D1515-10MG </td></tr><tr><td> SuperMag Separator </td><td> Oceano Nanotech, Inc. </td><td> N / A </td></tr><tr><td> Zetasizer Nano máquina DLS </td><td> Malvern, Inc </td><td> N / A </td></tr></tbody></table>

synthesis of an in vivo mri-detectable apoptosis probe

Apoptose celular é uma característica proeminente de muitas doenças, e esta morte celular programada normalmente ocorre antes de manifestações clínicas da doença são evidentes. Um meio para detectar apoptose em suas primeiras etapas reversíveis e iria pagar um pré-clínica &quot;janela&quot; durante o qual as medidas preventivas ou terapêuticas podem ser tomadas para proteger o coração contra danos permanentes. Apresentamos aqui um método simples e robusto para conjugar humano anexina V (ANX), que avidamente se liga às células durante as primeiras, etapas reversíveis de apoptose, a superparamagnéticas óxido de ferro (SPIO) nanopartículas, que servem como um agente de contraste para IRM-detectável. O método de conjugação começa com uma oxidação das nanopartículas SPIO que oxida grupos carboxilo na casca de polissacárido de SPIO. Proteína purificada ANX é então adicionado na configuração de uma solução de borato de sódio para facilitar a interacção covalente de ANX com SPIO num tampão de redução. Um passo de redução final com borohydrid de sódioe é realizada para completar a redução, e, em seguida, a reacção é arrefecida. ANX não conjugada é removido da mistura por filtração microcentrifugadora. O tamanho e pureza do produto ANX-SPIO é verificada por dispersão dinâmica de luz (DLS). Este método não necessita de adição a, ou a modificação de, o polissacárido SPIO concha, em oposição a ligação cruzada de partículas de óxido de ferro métodos de conjugação ou biotina-rotulados nanopartículas. Como resultado, este método representa uma abordagem simples, robusto que pode ser alargado a conjugação de outras proteínas de 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

A detecção precoce de apoptose pode identificar populações de risco de células em uma variedade de doenças. Aqui demonstramos um método para ligar uma proteína de apoptose de detecção precoce (anexina V) a uma nanopartícula de óxido de MRI-detectável de ferro (SPIO). Este método pode ser estendido para outras proteínas de interesse para gerar sondas de ressonância magnética-detectáveis ​​de imagens moleculares.

Síntese de um<em&gt; Em vivo</em&gt; Sonda Apoptosis MRI-detectável

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 visualizações.  Stanford University Medical Center. A detecção precoce de apoptose pode identificar populações de risco de células em uma variedade de doenças. Aqui demonstramos um método para ligar uma proteína de apoptose de detecção precoce (anexina V) a uma nanopartícula de óxido de MRI-detectável de ferro (SPIO). Este método pode ser estendido para outras proteínas de interesse para gerar sondas de ressonância magnética-detectáveis ​​de imagens moleculares.

Vídeo: 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.

Mutagênese do site caseiros Direção de Plasmids Whole

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

Biologia

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 de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

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.

Mudanças Dinâmicas de monitoramento dos níveis de cálcio mitocondrial durante a apoptose através de um sensor de cálcio geneticamente codificado

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.

Ativação da apoptose por microinjeção citoplasmática de 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.

O tomate / GFP-FLP / FRT Method for Imaging ao vivo de Mosaico Adulto<em&gt; Drosophila</em&gt; As 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.

Análise de apoptose em embriões Zebrafish por Whole-mount imunofluorescência para detectar Caspase 3 ativado

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.

Uma In vivo A reticulação Abordagem para isolar Complexos de proteínas a partir de Drosophila Os embriões

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.

Medição dos Níveis de heme em células de mamíferos Síntese

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.

Uma<em&gt; Ex vivo</em&gt; Sistema Cultura para estudar o desenvolvimento da tireóide

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

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Estratégias para Rastreamento Anastasis, uma célula de sobrevivência Fenômeno que reverte 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 ...