Siamo grati al Dr. Thierry Pourcher per averci permesso di utilizzare lo spettrometro a fluorescenza e per il suo aiuto. Il laboratorio è finanziato dalla Ligue Nationale Contre le Cancer (équipe labellisée), l&#39;Associazione pour la Recherche contre le Cancer, l&#39;Institut National du Cancer (INCA), l&#39;Agence Nationale pour la Recherche, METOXIA (programma dell&#39;UE 7 ° PQ), il Centro A. Lacassagne, il Centre National de la Recherche Scientifique, l&#39;Institut National de la Santé et de la Recherche Médicale e l&#39;Università di Nizza ( <a href="http://www.unice.fr/isdbc/" target="_blank">http://www.unice.fr/isdbc/</a> ). Ringraziamo il Dott. M Christiane Brahimi-Horn per la lettura critica e la correzione editoriale.

Titolazione biochimica di glicogeno 1. Lisi cellulare <ol><li> Alveoli ad una concentrazione di 0,5-2 x 10 6 per ogni 100 mm di diametro piatto. </li><li> Trattamento: incubare cellule 24, 48, o 72 ore a bassa concentrazione di ossigeno (ipossia) in una stazione di lavoro anaerobico Bug-Box (Ruskinn Tecnologia Biotrace International Plc, Bridgend, Regno Unito) fissato al 1% o 0,1% O 2, 94% o 94,9% N 2, e 5% di CO 2. Parallelamente, incubare le cellule in normossia (21% O 2, 5% CO 2). Cambiare mezzo (25 mM mezzo contenente glucosio) ogni 24 ore per minimizzare variazioni della concentrazione di glucosio durante il periodo dell&#39;esperimento. </li><li> Dopo il trattamento, le cellule lavaggio 2x con PBS per rimuovere tracce di glucosio dal mezzo di coltura. Raschiare le cellule in PBS freddo. </li><li> Centrifugare a 1.000 rpm per 5 min, scartare il surnatante e lavare il pellet una volta con PBS. Il pellet può essere congelato a -20 ° C prima di procedere ulteriormente.</li><li> Risospendere il pellet in 100 ml di acqua distillata. Aggiungere 100 ml di idrolisi Buffer glicogeno forniti con il glicogeno Assay Kit (Abcam). Bollire il lisato a 95 ° C per 5 minuti per inattivare gli enzimi. </li><li> Vortex e centrifugare il lisato a 13.000 rpm per 10 minuti per rimuovere i prodotti insolubili. Conservare il surnatante. </li><li> Per normalizzare i livelli di glicogeno al contenuto proteico tra i campioni, quantificazione delle proteine ​​può essere fatto con 25-35 ml del supernatante utilizzando il test Acid bicinconinico (BCA-Interchim). </li></ol> Alcuni di lisato può essere utilizzato per misurare la concentrazione di glucosio libero all&#39;interno delle cellule. 2. L&#39;idrolisi del glicogeno <ol><li> Mescolare 50 ml di surnatante (passo 1) con 1 ml di miscela di idrolisi enzimatica. Questa miscela contiene glucoamilasi. Incubare per 30 minuti a temperatura ambiente. </li><li> Preparare la curva di taratura diluendo il glicogeno standard (2 mg / ml) supplied con il kit per una soluzione di 10 mg / ml nel tampone di idrolisi. Quindi, preparare sei provette separate con 0, 4, 8, 12, 16, e 20 ml di 10 mg / ml di serie e regolare ogni tubo ad un volume finale di 50 ml con tampone di idrolisi per ottenere una concentrazione di glicogeno di 0, 0,04 , 0,08, 0,12, 0,16 e 0,2 mg / ml. Aggiungere 1 ml di idrolisi enzimatica Mix nelle norme e incubare per 30 minuti a temperatura ambiente. </li></ol> La concentrazione di glucosio sfondo della linea cellulare, data dal valore della concentrazione di glucosio libero, deve essere sottratta dal valore della concentrazione di glucosio totale (glucosio dal glicogeno idrolisi + senza glucosio) per determinare il livello di glicogeno nella cellula. 3. Sviluppo e lettura di fluorescenza <ol><li> Per ogni condizione, preparare un tubo (tubo 1) per misurare la concentrazione di glucosio libero e due tubi (tubi 2 e 3) per misurare la concentrazione di glucosio totale (ogni wit tuboha diverso volume di glicogeno idrolizzato). </li><li> Aggiungere 15 ml di surnatante estratti alla fine del passaggio 1 nella provetta 1. Aggiungere 35 ml di tampone di idrolisi per completare ad un volume finale di 50 microlitri. Fluorescenza Ottenuto darà la concentrazione di glucosio libero. </li><li> Aggiungi X ml di glicogeno idrolizzato (ottenuta nella fase 2) in tubo 2. Regolare il volume finale di 50 microlitri. Volume del campione (X microlitri) deve essere regolata in funzione della densità cellulare e / o tipo cellulare al fine di ottenere valori di fluorescenza che non vadano oltre concentrazioni della curva di calibrazione. </li><li> Aggiungere 2 X ml di glicogeno idrolizzato nel tubo 3. Regolare il volume finale di 50 microlitri. </li><li> Preparare una soluzione di sviluppo per i campioni e gli standard miscelando per ogni tubo 48,7 ml di tampone Sviluppo, 1 ml di Sviluppo Enzyme Mix e 0,3 ml di OxiRed Probe (fornita nel glicogeno Assay Kit). Ad esempio, per 2 condizioni, preparare una miscela per 12 tubos (6 per gli standard, 2 per il glucosio libero e 4 per la fluorescenza totale di glucosio). </li><li> Aggiungere 50 ml di questa miscela a ciascuna provetta e incubare per 30 minuti a temperatura ambiente al buio. </li><li> Misurare la fluorescenza ad una lunghezza d&#39;onda di eccitazione di 535 nm, lunghezza d&#39;onda di emissione di 587 nm come raccomandato, e una fessura di 3 nm di eccitazione e di emissione. </li></ol>

<ol>
	<li>Pelletier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+Synthesis+is+Induced+in+Hypoxia+by+the+Hypoxia-Inducible+Factor+and+Promotes+Cancer+Cell+Survival.">Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival.</a> Front Oncol. 2, (2012).</li><li>Alonso, M. D., Lomako, J., Lomako, W. M., Whelan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+the+biogenesis+of+glycogen.">A new look at the biogenesis of glycogen.</a> FASEB J. 9, 1126-1137 (1995).</li><li>Bollen, M., Keppens, S., Stalmans, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+features+of+glycogen+metabolism+in+the+liver.">Specific features of glycogen metabolism in the liver.</a> Biochem. J. 336 (Pt 1), 19-31 (1998).</li><li>Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J., Stapleton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP-activated+protein+kinase+does+not+associate+with+glycogen+alpha-particles+from+rat+liver.">AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver.</a> Biochem. Biophys. Res. Commun. 362, 811-815 (2007).</li><li>Sullivan, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+alpha+and+beta+particles+in+glycogen+using+molecular+size+distributions.">Nature of alpha and beta particles in glycogen using molecular size distributions.</a> Biomacromolecules. 11, 1094-1100 (2010).</li><li>Chee, N. P., Geddes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+liver+glycogen.">The structure of liver glycogen.</a> FEBS Lett. 73, 164-166 (1977).</li><li>Roach, P. J., Depaoli-Roach, A. A., Hurley, T. D., Tagliabracci, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+and+its+metabolism:+some+new+developments+and+old+themes.">Glycogen and its metabolism: some new developments and old themes.</a> Biochem. J. 441, 763-787 (2012).</li><li>Moses, S. W., Bashan, N., Gutman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+metabolism+in+the+normal+red+blood+cell.">Glycogen metabolism in the normal red blood cell.</a> Blood. 40, 836-843 (1972).</li><li>Agbanyo, M., Taylor, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+3-deoxy-3-fluoro-D-glucose+into+glycogen+and+trehalose+in+fat+body+and+flight+muscle+in+Locusta+migratoria.">Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle in Locusta migratoria.</a> Biosci. Rep. 6, 309-316 (1986).</li><li>Louzao, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Fluorescent+glycogen"+formation+with+sensibility+for+in+vivo+and+in+vitro+detection.">"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection.</a> Glycoconj. J. 25, 503-510 (2008).</li><li>Sheehan, D. C. H., B, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and practice of histotechnology. , (1980).</li><li>Baba, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+monoclonal+antibody+that+recognizes+glycogen+and+its+application+for+immunohistochemistry.">Production of monoclonal antibody that recognizes glycogen and its application for immunohistochemistry.</a> Kokubyo Gakkai Zasshi. 60, 264-287 (1993).</li></ol>

Gli autori dichiarano assenza di conflitto di interessi.

Titolazione biochimica di glicogeno in vitro permette una quantificazione precisa delle cellule contenuto di glicogeno. Rispetto ad altre tecniche (PAS, immunofluorescenza con un anticorpo glicogeno, ecc.), Questa titolazione è molto specifico, sensibile e riproducibile. Inoltre, il metodo è conveniente in quanto richiede nessuna radioattività ma uno spettrometro a fluorescenza. Tuttavia, questa tecnica è puramente quantitativa e non fornisce informazioni sulla distribuzione glicogeno nella cella. ...

Il glicogeno è un polimero con molti rami di residui di glucosio, che è presente nel citoplasma di molti tipi di cellule. È una delle principali forme di immagazzinamento dell&#39;energia nelle cellule e svolge un ruolo importante nel metabolismo del glucosio. La maggior parte delle cellule di mammiferi sono capaci di produrre e immagazzinare glicogeno, che può essere rapidamente degradato in glucosio per promuovere la glicolisi e produzione di ATP durante lo stress metabolico. Gli epatociti producono enormi quantità di glicogeno per regolare il livello di zucchero nel sangue fornendo così un approvvigionamento continuo di glucosio nel corpo. Al contrario, la concentrazione di glicogeno in altre cellule (muscoli, globuli rossi, ecc) è relativamente bassa. Tuttavia, localmente, queste quantità sono sufficienti a fornire energia a breve termine, quando le cellule sono improvvisamente esposti a un ambiente privo di sostanze nutritive. Sintesi di glicogeno e la ripartizione del glicogeno segue gli stessi passi in tutti i tessuti (Figura 1). In primo luogo, il glucosioentra cellule trasportatori del glucosio (glutei), e viene rapidamente convertito dal glucosio-6-fosfato (G6P) in glucosio-1-fosfato (G1P) da fosfoglucomutasi. G1P viene quindi modificato in UDP-glucosio e il carbonio C1 di UDP-glucosio è attaccato ad un residuo di tirosina glycogenin, la proteina di ancoraggio di glicogeno. Questa molecola, considerato un primer glicogeno, viene esteso mediante attacco del UDP-glucosio al glucosio terminale da un α (1 → 4) bond tramite il glicogeno sintasi. Infine, quando si forma una catena lineare di oltre 11 residui di glucosio, l&#39;enzima di ramificazione trasferisce un oligosaccaride terminale costituito da un minimo di 6 residui di glucosio all&#39;altro glucosio della catena vicina attraverso un α (1 → 6) linkage. La ripetizione di questo processo dà una struttura frattale massiccia contenente rami che formano un&#39;elica con 6,5 glucosio ogni turno. Il glicogeno può essere inversamente idrolizzato al glucosio con l&#39;azione concertatadebranching di enzimi che idrolizzano il α (1 → 6) vincolato e glicogeno fosforilasi che idrolizza il α (1 → 4) glicosidico limite tra l&#39;ultimo residuo di glucosio di un ramo e la molecola di glicogeno. Questa reazione chiamato glicogenolisi viene attivato aumentando i livelli di AMP (riflettente consumo di ATP), e inibito dal glucosio e ATP 2,3. Mediante microscopia elettronica, le molecole di glicogeno sono stati descritti in molti tipi cellulari particelle β libere (o monoparticelle glicogeno) di 15-30 nm di diametro. In specifici tipi cellulari quali epatociti, particelle β possono essere assemblate in un complesso per formare rosette, noto anche come particelle α che variano in diametro da 80 nm a un massimo di 200 nm 4 </sup&gt;. Il modo in cui queste particelle β sono legati per formare grandi aggregati di particelle α non è ancora completamente chiarito. Alcune prove tende a dimostrare che le particelle β possono essere legati da legami covalenti 5, legame a idrogeno, o anche attraverso le interazioni proteina-proteina 6. La quantità di glicogeno immagazzinato nelle cellule dipende da molti parametri: (I) la quantità di glycogenin nella cella che inizia la sintesi del glicogeno, (ii) l&#39;attività di glicogeno sintasi e fosforilasi regolato dalla proteina fosforilazione / defosforilazione; (III) la concentrazione di glucosio nelle cellule, che dipende da diversi parametri, quali la fornitura di glucosio dal sistema vascolare e l&#39;assorbimento del glucosio da parte delle cellule. Le riserve di glicogeno sono strettamente regolati da regolazione allosterica di ormoni biosintetici attraverso metaboliti intermedi, da ormoni che regolano il metabolismo energetico, e attraverso vie di segnalazione di rilevamento nutrienti 7. È importante esseregrado di quantificare glicogeno in campioni biologici per meglio comprendere l&#39;importanza del metabolismo del glicogeno in tutto il corpo e livello cellulare. Descriviamo qui un preciso, riproducibile e conveniente biochimico prova in vitro per glicogeno. Questa tecnica si basa sulla fluorescenza quantificazione glucosio prima e dopo specifico idrolisi di glicogeno. Altri metodi esistono per stimare il livello di glicogeno nelle cellule, ma la maggior parte di essi non sono quantitative. Una delle prime tecniche descritte per la quantificazione di glicogeno nelle cellule era basato sulla misurazione di costituzione [14 C]-glucosio in glicogeno 8,9. L&#39;uso di radioattività rende questo processo più difficile da gestire, ma ha il vantaggio di fornire la velocità di incorporazione del glucosio in glicogeno e nel distinguere tra la distribuzione di residui di glucosio sui rami esterni e nel nucleo della molecola (richiede anche un ulteriori β-amylolysis ed una fase cromatografica). Un&#39;altra tecnica è stata sviluppata più recentemente e si basa sull&#39;incorporazione di 2-NBDG (2 - {N-[7-nitrobenz-2-oxa-1 ,3-diazol-4-il] ammino}-2-deossiglucosio), un derivato fluorescente 2-deossiglucosio, in glicogeno 10. L&#39;intensità di fluorescenza misurata riflette la quantità di glicogeno prodotta e può essere misurata con un lettore di fluorescenza. Distribuzione di fluorescenza nella cella può essere valutata mediante microscopia confocale. Tra le altre tecniche nonquantitative, Periodic Acid Schiff colorazione (PAS) è forse il più comune. Può essere usato per la rilevazione di glicogeno in cellule fissate, sezioni di tessuto in paraffina o congelati. Questo istologici colori tecnica non specificamente polisaccaridi, glicolipidi, glicoproteine, cellulosa e mucine neutre in viola. La specificità di questo test può essere aumentata mediante trattamento di cellule fissate o sezioni di tessuto con diastasi, che digerisce specificamente glicogeno. Successivamente,il livello di glicogeno può essere qualitativamente stimata confrontando campioni non idrolizzati (tutti macromolecole carboidrati modificati) per campioni idrolizzati (carboidrati modificato macromolecole tranne glicogeno). Colorazione PAS e analisi microscopica, a differenza saggi biochimici di glicogeno, fornisce informazioni relative alla distribuzione di glicogeno nella cellula, che può essere diffusa o concentrata in una determinata parte della cellula. Tuttavia, anche se le differenze stime PAS colorazione in accumulo glicogeno tra condizioni diverse, non è quantitativa 11. Un anticorpo monoclonale di topo originariamente realizzato utilizzando condilare mandibolare cartilagine come antigene ha dimostrato di reagire con glicogeno in cellule e con glicogeno purificato in vitro 12. Dato che questo anticorpo riconosce specificamente catene di zuccheri glicogeno riscontrato, è un utile strumento per la rilevazione di glicogeno mediante immunoistochimica in maniera più specifica della colorazione PAS. </p&gt; La microscopia elettronica è un&#39;altra tecnica che permette la visualizzazione di grani di glicogeno nelle cellule e la valutazione del grado di glicogeno accumulo. Infatti, le particelle β glicogeno sono facilmente riconoscibili con un microscopio elettronico come elettroni densi granuli 1.

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			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>31966.047</td>
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		 <tr>
			<td>Glycogen Assay Kit</td>
			<td>Abcam</td>
			<td>ab65620 </td>
			<td></td>
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			<td>PBS</td>
			<td></td>
			<td></td>
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 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
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			<td>Company</td>
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			<td>Fluorescence Spectrometer</td>
			<td>PerkinElmer</td>
			<td>LS 50B</td>
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biochemical titration of glycogen in vitro

Il glicogeno è la principale polimero energetico del glucosio negli animali vertebrati e svolge un ruolo fondamentale nel metabolismo del corpo intero e nel metabolismo cellulare. Esistono già molti metodi per rilevare glicogeno, ma solo pochi sono di tipo quantitativo. Descriviamo qui un metodo che utilizza il kit di analisi Abcam Glicogeno, che si basa sulla specifica degradazione del glicogeno in glucosio da glucoamilasi. Il glucosio viene ossidato specificamente ad un prodotto che reagisce con la sonda OxiRed per produrre fluorescenza. La titolazione è preciso, sensibile e può essere realizzato su estratti cellulari o sezioni di tessuto. Tuttavia, a differenza di altre tecniche, non dà informazioni sulla distribuzione del glicogeno nella cellula. Come esempio di questa tecnica, descriviamo qui la titolazione di glicogeno in due linee cellulari, cinese CCL39 fibroblasti di polmone di criceto e LS174 carcinoma del colon umano, incubati in normossia (21% O 2) rispetto ipossia (1% O 2). Abbiamo ipotizzato che l&#39;ipossia è un segnalenale che prepara le cellule per sintetizzare e conservare glicogeno per sopravvivere 1.

Un basso livello di ossigeno (ipossia) in segnali tumori a cellule tumorali la necessità di immagazzinare energia per gestire conseguente esaurimento degli elementi nutritivi, in modo da sopravvivere. Come glicogeno è il principale polimero energetico del glucosio nelle cellule di mammifero, abbiamo studiato la regolazione della glicogenosi in ipossia. Il calcolo e la standardizzazione della concentrazione di glicogeno in lisati cellulari devono essere eseguite sui dati grezzi di fluorescenza come mostrato nelle <stro...

Watch this Scientific Journal Video about Biochemical Titration of Glycogen In vitro at JoVE.com

Biochemical Titration of Glycogen In vitro

Descriviamo qui un metodo biochimico accurato, riproducibile e conveniente per la titolazione del glicogeno in vitro. Questa tecnica utilizza il kit di analisi Abcam glicogeno e si basa sulla successiva idrolisi del glicogeno in glucosio e titolazione mediante fluorescenza.

Titolazione biochimica di glicogeno<em&gt; In vitro</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

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JoVE Journal

Biology

Biochemical Titration of Glycogen In vitro

28.3K Views.  University of Nice - Sophia Antipolis.  Descriviamo qui un metodo biochimico accurato, riproducibile e conveniente per la titolazione del glicogeno in vitro. Questa tecnica utilizza il kit di analisi Abcam glicogeno e si basa sulla successiva idrolisi del glicogeno in glucosio e titolazione mediante fluorescenza. 

Video: Biochemical Titration of Glycogen In vitro

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

Titolazione del coronavirus umani utilizzando un test immunoperossidasi

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

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Biologia

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish Cervello iniezione ventricolo

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

Raccolta variabile concentrazione dataset Calorimetria isotermica di titolazione per determinare meccanismi vincolanti

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Ricostituzione biochimica dei recettori di steroidi • Complessi proteina Hsp90 e la riattivazione del Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Il prelievo di sangue per l&#39;analisi biochimica in Zebrafish Adult

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Biochimica e High Throughput valutazione microscopica di massa grassa in<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Saggi biochimici per le attività di analisi ATP-dipendente rimodellamento della cromatina Enzimi

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

High-throughput Titolazione della luciferasi che esprimono ricombinanti virus

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Titolazione biochimica di glicogeno

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