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

Adattato da progetto preliminare 1. 1. Coniugazione di annessina V SPIO <ol><li> Ossidare le particelle SPIO (Ocean Nanotech Inc., 5 mg / ml) per 1 ora a 20 ° C al buio in una soluzione con 0,15 M di sodio periodato (Naio 4) (04:01 peso: rapporto in peso) 2. </li><li> Incubare la SPIO ossidato per 12 ore con una proteina purificata ANX (rapporto 1:1, peso: peso) 0,15 M borato di sodio (Na 2 B 4 O 7 10H 2 O) a 20 ° C. </li><li> Ridurre la miscela per 1 ulteriore ora con boroidruro di sodio (NaBH 4) sotto la cappa aspirante e poi bloccato con 0,15 M Tris-HCl. </li><li> Separare il ANX libero da ANX-SPIO mediante centrifugazione a 14000 g (75 micron pori Microcon filtro Millipore, Billerica, MA). </li><li> Raffinare ulteriormente il composto rimuovendo le impurità non associati utilizzando un dispositivo magnetico di separazione (Ocean Nanotech, Inc.). </li><li> Quantificare il filtro retentato (ANX-SPIO) livelli di proteinamediante saggio Bradford proteine ​​e conservare a -80 ° C a 2 mg / ml in 0,1 M Tris-HCl con 1% di albumina e di inibitori della proteasi (proteasi Halt, Thermo Scientific, 1-50 mg / mg ANX). </li></ol> 2. Light Scattering dinamico <ol><li> Separatamente diluire il SPIO e ANX-SPIO in PBS ad una densità ottica di 0,1 e analizzare mediante diffusione di luce dinamica in una macchina Zetasizer Nano DLS (Malvern Inc., Worcestershire, UK) 3. </li><li> Ottenere picchi monomodale (idealmente) per il composto, con Z dimensione particellare in media e indice di polidispersità (PDI). Confrontare questa dimensione al DLS-misurato dimensioni delle nanoparticelle ossido di ferro da solo. ANX-SPIO DLS risultati hanno mostrato un coniugato idrodinamico diametro delle particelle di 78 nm con un PDI di 0,13 1, riflettendo una omogenea ANX-SPIO specie 3. </li></ol> 3. Coltura cellulare, induzione di apoptosi in cultura, e la RM di cellule in coltura <ol><li> La cultura degli adulti mesenchimali del midollo osseo di topo scelle TEM (mMSCs) in DMEM supplementato con 10% FBS e 1% di penicillina-streptomicina (Invitrogen, Inc.), preferibilmente al passaggio 20-30 Aprile. </li><li> Indurre l&#39;apoptosi nelle cellule di coltura mediante trattamento per periodi variabili a 37 ° C con DOX (Sigma, 1 mM in 0,9% soluzione salina, una concentrazione di 1 pM in media) 12. </li><li> Quantificare la quantità di apoptosi nella cultura utilizzando MRI con ANX-SPIO (descritto in 3.4) o usando la microscopia dopo TUNEL macchia (APO-BrdUTM TUNEL Assay Kit, Molecular Probes / Invitrogen, Eugene, OR) o dopo la macchia con ANX-FITC (Roche, Svizzera). </li><li> Etichettare le cellule con ANX-SPIO (10 mg di proteina / ml mezzo eccezione di quanto indicato), oppure con un peso equivalente di libera SPIO (5 mg), per 15 min a 37 ° C. Lavare 4 volte con PBS, e sovrapposizione con 1 ml di 1% agarosio per impedire la diffusione delle nanoparticelle SPIO. Eseguire MRI dei piatti (descritto sotto). In alternativa, lavare le cellule e Tripsinizzare dopo DOX e ANX-SPIO etichettatura, quindi consentire alle cellule diformare pellet in provette Eppendorf. </li></ol> 4. In Vitro MRI <ol><li> Usare un 1.5 o 3 Tesla GE scanner Signa EXCITE con batteria standard del ricevitore al ginocchio per l&#39;analisi in vitro MRI. </li><li> Porre le capsule di coltura o provette in un fantasma agar e immagini usando un gradiente-echo sequenza viziato gradiente: tempo di ripetizione (TR) 550 ms, tempo di eco (TE) 10 ms, flip angle 15 °, matrice 256 x 256, campo di vista di 3 cm x 3 cm, spessore 1,3 mm, il numero delle medie (NEX) 2, distanza 0 mm 5. Immagine assialmente attraverso lo strato di cellule sulla piastra di T2 * perdita di segnale, utilizzando le regioni d&#39;interesse (ROI) di una zona fissa (per esempio, 0,8 mm 2). Regolare per lo sfondo utilizzando il controllo del segnale ROI dal fantasma agar. Misurare il rumore di fondo dal segnale dell&#39;aria circostante il fantasma. </li><li> Calcolare il contrasto MRI-to-Noise Ratio (CNR: [SI campione ROI-SI di controllo ROI] / [SD del SI del rumore di fondo], dove SI è l&#39;intensità del segnale eSD è la deviazione standard) è stato calcolato per ciascun piatto di coltura. </li></ol> 5. Risultati rappresentativi Misura del T2 * perdita di segnale in vitro richiede un fantoccio omogeneo agar e la mancanza di bolle d&#39;aria, che creerà nulla artefatto segnale. Questo artefatto è difficile da distinguere da vera e propria perdita di segnale T2 * di SPIO, ed è fondamentale per i risultati interpretabili. Si veda la Figura 1 per un esempio di un manufatto aria così come ossido di ferro effettivo T2 * perdita di segnale. Per nell&#39;imaging piatto di coltura in vitro, applicare un volume adeguato di gel di agar per ciascun piatto di coltura, in modo che sufficienti sezioni assiali MRI, che sono circa 1 mm di spessore, possono essere adeguatamente immagine l&#39;intera piastra. Figura 2 illustra il segnale previsto MRI da piastre di coltura individuali in agar. Vi è tipicamente una certa variazione del segnale ai bordi della piastra, così analisi delle regioni di intcresta dovrebbero evitare queste aree irregolari. <img alt="Figura 1" src="/files/ftp_upload/3775/3775fig1.jpg" /> Figura 1. ANX-SPIO rileva l&#39;apoptosi in colture di cardiomiociti neonatali di ratto. Figura 1A mostra una foto di pellet cardiomiociti neonatali trattati con soluzione di controllo (tubo a sinistra), ANX-SPIO (tubo al centro) e DOX + ANX-SPIO (tubo a destra) . Notare il colore brunastro della DOX + ANX-SPIO cellule trattate, riflettendo un aumento della ritenzione ANX-SPIO composto in cellule apoptotiche. Figura 1B in vitro immagini MRI mostrano l&#39;intensa perdita del segnale T2 * dei + DOX ANX-SPIO cellule. Inoltre, una bolla d&#39;aria è visibile tra il 4 ° e 5 ° tubi Eppendorf (piccolo vuoto segnale nero), che è simile al segnale di vuoto ANX-SPIO, e che può influire analisi del CNR. <img alt="Figura 2" src="/files/ftp_upload/3775/3775fig2.jpg" /> Figura 2. ANX-SPIOlegame è specifico per le cellule apoptotiche in coltura. Figura 2A mostra cardiomiociti di ratto apoptotici neonatali sui piatti cultura, sottoposti a uno ANX-FLUOS colorazione o ANX-SPIO colorazione, con concentrazioni basse e alte (sinistra e destra, rispettivamente) di ANX-SPIO. Si noti la diversa T2 * perdita di segnale (segnale nero) di cellule apoptotiche che sono stati trattati con fluorescente ANX (ANX-FLUOS) e bassi livelli rispetto ai livelli elevati di ANX-SPIO. L&#39;aumento della perdita di segnale T2 * a destra è associata ad una marcata riduzione ANX-FLUOS segnale, che è stato gareggiato fuori dai maggiori ASX-SPIO concentrazioni. Figura 2B illustra il legame competitivo di ANX-SPIO rispetto ANX-FLUOS segnale e la dissociazione costante, K i. L&#39;asse X indica la concentrazione di proteine ​​Annexin in scala logaritmica. Movie 1. <a href="http://www.jove.com/files/ftp_upload/3775/3775movie1.mov" target="_blank">Clicca qui per vedere supplementare</a>film.

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	<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. 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Il metodo descritto per il collegamento coniugazione SPIO per annessina V sfrutta i gruppi amminici della catena laterale di annessina e le frazioni carbossilici della nanoparticelle SPIO. Attraverso specifici ossidoriduzione passaggi, legame covalente di questi composti può essere raggiunto, e il risultante nanoparticella funzionalizzato può essere isolato. Questo metodo può essere generalizzato ad altre proteine ​​e nanoparticelle di interesse. Le fasi più critiche sono le condizio...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Catalogo # </td></tr><tr><td> Superparamagnetico ossido di ferro </td><td> Ocean Nanotech, Inc. </td><td> ICK-40-005 </td></tr><tr><td> Doxorubicina </td><td> Sigma </td><td> D1515-10MG </td></tr><tr><td> Supermag Separator </td><td> Ocean Nanotech, Inc. </td><td> N / A </td></tr><tr><td> Zetasizer Nano DLS macchina </td><td> Malvern, Inc </td><td> N / A </td></tr></tbody></table>

synthesis of an in vivo mri-detectable apoptosis probe

Apoptosi cellulare è una caratteristica importante di molte malattie, e questa morte cellulare programmata in genere si verifica prima di manifestazioni cliniche della malattia sono evidenti. Un mezzo per rilevare l&#39;apoptosi nelle sue prime fasi e reversibili che garantisca loro un pre-clinica &#39;finestra&#39; nel corso della quale le misure preventive o terapeutiche potrebbero essere prese per proteggere il cuore dai danni permanenti. Vi presentiamo qui un metodo semplice e robusto di coniugare umana annessina V (ANX), che si lega alle cellule in avidamente le prime, le fasi reversibili di apoptosi, superparamagnetiche di ossido di ferro (SPIO) nanoparticelle, che servono come una risonanza magnetica-rilevabile agente di contrasto. Il metodo inizia con un coniugazione ossidazione delle nanoparticelle SPIO, che ossida gruppi carbossilici sul guscio di polisaccaride SPIO. ANX proteina purificata è poi aggiunto nel contesto di una soluzione di tetraborato di sodio per facilitare l&#39;interazione covalente di ANX con SPIO in un buffer riduzione. Una fase di riduzione finale con sodio borohydride viene eseguita per completare la riduzione, e quindi si spegne la reazione. ANX non coniugato viene rimosso dalla miscela per filtrazione microcentrifuga. La dimensione e la purezza del ANX-SPIO prodotto viene verificata mediante dynamic light scattering (DLS). Questo metodo non richiede aggiunta a, o modifica, il polisaccaride SPIO guscio, al contrario di reticolati metodi particelle di ossido di ferro coniugazione o biotina-etichettati nanoparticelle. Come risultato, questo metodo rappresenta un approccio semplice, robusta che può essere estesa a coniugazione di altre proteine ​​di 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

La diagnosi precoce di apoptosi può identificare a rischio le popolazioni di cellule in una varietà di malattie. Qui mostriamo un metodo per collegare un precoce rilevamento della proteina-apoptosi (annessina V) per una risonanza magnetica-rilevabile nanoparticelle di ossido di ferro (SPIO). Questo metodo può essere esteso ad altre proteine ​​di interesse per generare MRI-rilevabili sonde di imaging molecolare.

Sintesi di uno<em&gt; In vivo</em&gt; RM-rilevabile Apoptosis Probe

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 Views.  Stanford University Medical Center. La diagnosi precoce di apoptosi può identificare a rischio le popolazioni di cellule in una varietà di malattie. Qui mostriamo un metodo per collegare un precoce rilevamento della proteina-apoptosi (annessina V) per una risonanza magnetica-rilevabile nanoparticelle di ossido di ferro (SPIO). Questo metodo può essere esteso ad altre proteine ​​di interesse per generare MRI-rilevabili sonde di imaging molecolare.

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.

Mutagenesi sito fatto in casa Regia di Plasmidi 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 ...

Ricerca

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.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

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.

Monitoraggio Cambiamenti dei livelli di calcio mitocondriale durante l&#39;apoptosi mediante un sensore di calcio geneticamente codificato

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.

Attivazione di apoptosi da Microiniezione citoplasmatici 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.

Il pomodoro / GFP-FLP / FRT Metodo per Live Imaging del Mosaico adulti<em&gt; Drosophila</em&gt; cellule fotorecettrici

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.

Analisi di apoptosi in embrioni di zebrafish da Whole-mount immunofluorescenza per rilevare caspasi 3 attivata

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 La reticolazione Approccio per isolare complessi proteici da Drosophila Embrioni

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.

La misurazione dei livelli Heme sintesi in cellule di mammifero

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; Cultura sistema per studiare lo sviluppo della tiroide

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.

Strategie per l&#39;inseguimento Anastasis, una cellula di sopravvivenza fenomeno che Inverte 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 ...