Gli autori desiderano ringraziare John Woolford per la base iniziale di questo metodo, TGK è sostenuto da NIH GM057483 e AI076245 e PRC è supportato da GM77073.

1. Preparazione di gradienti di saccarosio 7-47% <ol><li> Preparare gradienti di saccarosio un giorno prima per permettere l&#39;uso gradienti di diventare continua. Rendere sterile il 7% e soluzioni di saccarosio al 47% contenente 50 mM NH 4 Cl, 50 mM Tris-acetato pH 7,0 e 12mm MgCl 2 e conservare a 4 ° C 1. </li><li> Per 6 gradienti di mescolare il 7% e il 47% soluzioni madri di saccarosio per fare 48 ml di ciascuna delle seguenti concentrazioni di saccarosio. Aggiungi DTT (1M magazzino) per ogni soluzione di saccarosio a una concentrazione finale di 1 mm. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Concentrazione finale </td><td> 7% di saccarosio </td><td> 47% di saccarosio </td></tr><tr><td> 7% </td><td> 48 ml </td><td> 0 </td></tr><tr><td> 17% </td><td> 36 ml </td><td> 12 ml </td></tr><tr><td> 27% </td><td> 24 ml </td><td> 24 ml </td></tr><tr><td> 37% </td><td> 12 ml </td><td> 36 ml </td></tr><tr><td> 47% </td><td> 0 </td><td> 48 ml </td></tr></tbody></table></li><li> Al fine di versare i gradienti, allegare una pipetta Pasteur di una siringa da 20 ml di fissaggio e di tenuta con Parafilm o utilizzare un lungo ago. Aggiungere 7 ml di saccarosio 7% al fondo di una x 1 &quot;tubo 3,5 polyallomer ultracentrifuga. Successiva aggiungere 7 ml di soluzione di saccarosio 17% sotto la soluzione al 7% mettendo la punta della pipetta Pasteur vicino fondo della provetta e pipettaggio non più veloce di 0,3 mL / sec. Ripetete con 7 ml ciascuna del 27%, 37% e soluzioni di saccarosio al 47%. Si dovrebbe osservare chiare linee tra gli strati, ad indicare che la miscelazione minimo si è verificato. gradienti Conservare a 4 ° C per una notte insieme con i rotori, le bottiglie e tubi che verranno utilizzati per raccogliere campioni. </li></ol> 2. Preparazione dell&#39;estratto di lievito <ol><li> Grow 125 mL di coltura di lievito ad un OD 600 di 0,8-1,0. Aggiungi cicloeximide alla cultura ad una concentrazione finale di 100 mg / ml e continuare agitazione a 30 ° C per 15 min. </li><li> Nel frattempo, preparare buffer di lisi contenente 80 mg / ml cicloeximide, 200 mg / mL di eparina, 0,2% DEPC, e 10 mM Tris-HCl pH 7,5, 0,1 M NaCl, 30mm MgCl 2. Tenere tutto in ghiaccio da questo punto in poi. </li><li> Versare la cultura in una bottiglia da 250 ml centrifuga e riempire con ghiaccio. Centrifugare a 8000 xg (7.250 giri in un SLA-1500 rotore) per 5 minuti a 4 ° C. Lavare il pellet una volta con 10 ml di tampone di lisi ghiaccio freddo e trasferimento in tubi 15 ml in polipropilene. Centrifugare a 5900 g (circa 6.000 giri in un SLA-600TC rotore) per 3 minuti a 4 ° C. Risospendere il pellet in 0,5 ml di tampone di lisi, trasferimento a tubo di 1,5 ml e aggiungere 0,5 ml di acqua refrigerata, al forno lavata con acido perle di vetro. Vortex le cellule per quattro intervalli di 30 secondi, il raffreddamento della provetta in ghiaccio per 30 secondi tra ogni intervallo. Aggiungere un altro 0,5 mL di tampone di lisi nel tubo 1,5 ml. Centrifugare alla massima velocità in una microcentrifuga (14.000 giri al minuto in una microcentrifuga Eppendorf 5417R) per 10 minuti a 4 ° C. Trasferire il supernatante in un nuovo tubo di 1,5 ml microcentrifuga. Rimuovere 10 microlitri di un tubo separato per determinare la 260 / 280 OD OD rapporto (vedi punto 3.1). Estratti di conservare a -80 ° C. </li></ol> 3. Preparazione di estratti da cellule di mammifero <ol><li> Per le cellule aderenti, cresca in 100 piatto mm a confluenza ~ 70%. Avrete bisogno di un piatto di 100 mm per 11 ml di gradiente (rendimento tipico è ~ 20 OD 260 unità). Scalare la quantità linearmente per dimensioni gradiente più o meno grandi. Per i non aderenti cellule, circa 1 x 10 7 cellule sono necessari per 11 ml di gradiente. </li><li> Prima di lisi, aggiungere cicloeximide a 100 mg / ml. Incubare a 37 ° C per 15 min. Trasferire le piastre di ghiaccio e lavare le cellule due volte in PBS freddo ghiaccio. Tutte le fasi futuro deve essere effettuata a 0-4 ° C. </li><li> Rimuovere tutte le tracce di PBS per aspirazione (per non piastre di drenaggio su un letto inclinato di ghiaccio) e aggiungere 1 mL di tampone di lisi, raschiare e trasferimento in pre-raffreddata tubo 1,5 ml. Componenti del tampone di lisi dovranno essere ottimizzati per il tipo di cellula e condizioni di crescita. Un buffer di partenza sarà di 20 mM HEPES-KOH, pH 7,4, 15 mM MgCl 2, 200 mM KCl, 1% Triton X-100 (v / v), 100 mg / ml cicloeximide, 2 mM DTT, e 1 mg / mL di eparina. Le variabili chiave sono le concentrazioni di magnesio e cloruro di potassio, nonché l&#39;inclusione o l&#39;esclusione di detergenti non ionici per migliorare l&#39;estrazione di polisomi legati citoscheletro o membrana. Lisare cellule, passando attraverso 27 gauge siringa o due volte da colpi 5-10 di omogeneizzazione dounce utilizzando tipo B pestello. La siringa o omogeneizzatore devono essere pre-raffreddata prima dell&#39;uso. </li><li> Spin a 14000 g per 5 min in microcentrifuga refrigerata. Trasferimento lisato fresco al gradiente di saccarosio (per cellule di mammifero è costituita in tampone di lisi privo Triton X-100 ed eparina ma per il resto come descritto nei punti 1.2 e 1.3) o congelare flash in azoto liquido per un uso successivo. </li></ol&gt; 4. Centrifugazione di gradienti <ol><li> Disgelo lisati polyribosome sul ghiaccio. Determinare la concentrazione di acido nucleico del lisato aggiungendo la riservato un campione di 10 microlitri di 1 ml di acqua e leggere il 260 / 280 OD OD rapporto con uno spettrofotometro. Rendimento tipico di una cultura lievito cresciuto come descritto è di 50 unità OD. </li><li> Utilizzando una pipetta con la punta appoggiata alla parete del tubo vicino alla superficie del gradiente, delicatamente layer da 25 OD 260 unità del lisato in cima al gradiente. Per un gradiente di 35 ml 500 microlitri di lisato è tipicamente caricato. Tampone di lisi può essere usato per assicurare a tutti le sfumature vengono caricati con un uguale volume. Minori quantità di materiale resa migliore risoluzione. Prendere con cautela abbassare le pendenze in secchi di un rotore oscillante. </li><li> Centrifugare i gradienti a 100.000 xg (23.000 rpm in un rotore 630 Surespin) per 4 ore a 4 ° C. Questo protocollo può essere adattato per le sfumature volume più piccolo. </li></ol> 5. Raccolta dei dati e delle frazioni <ol><li> Circa 30 minuti prima del completamento dello spin 4 ore collegare l&#39;ago il piercer tubo Modello 184. Collegare la penna al modello in linea Isco UA-5 Assorbanza / Monitor fluorescenza. Accendere l&#39;assorbanza monitor per consentire una linea di base stabile da raggiungere prima all&#39;analisi di campioni. Acquisizione elettronica del profilo polisomi può facilmente essere ottenuta collegando un DI-148U unità di acquisizione dati (DATAQ Instruments) al registratore grafico. I profili possono poi essere raccolti in tempo reale e salvati per la successiva annotazione utilizzando il software che accompagna WinDaq. </li><li> Riempire la pompa a siringa con 50 mL di Fluorinert utilizzando il flusso inverso, presa rapida. Assicurarsi che non vi siano bolle d&#39;aria presenti. Collegare il tubo dalla siringa al tubo di piercer e brevemente accendere il flusso di Fluorinert a 3 ml / min in avanti per eliminare il tubo di eventuali bolle d&#39;aria. </li><li> Posizionare un bicchiere di rifiuti o di una serie di tubi, se raccolta frazioni sotto il tubo al termine della cella di flusso per la raccolta del campione scappare. </li><li> Rimuovere con attenzione i tubi polyallomer centrifuga contenente il gradiente di saccarosio dal rotore della centrifuga e disporli sul ghiaccio (pre-forma dei pozzi nel ghiaccio con un tubo vuoto per evitare gradienti di disturbo). </li><li> Per analizzare estratti cellulari, posizionare il tubo di pendenza sul piercer tubo. Collegare la parte superiore del tubo alla presa e sicuro. Sollevare il palco fondo alla provetta in modo che l&#39;ago penetra il fondo della provetta. Una volta che l&#39;ago è sporgevano attraverso il fondo della provetta, inizia il flusso di Fluorinert in avanti a 6mL/min. </li><li> Una volta che il Fluorinert ha iniziato scorre nel tubo, monitorare il movimento dell&#39;ago sul monitor assorbanza online. Una volta che l&#39;ago inizia a muoversi, accendere il movimento grafico in una impostazione di velocità di 60. Il monitor registra il diametro esterno 254 a partire dal materiale libero seguita da rilevamento subunità 40S ribosomiale e continuando fino ai complessi polyribosome. Se si stanno raccogliendo frazioni di RNA o analisi delle proteine, tipicamente 0,2 frazioni min sono prese (1 ml). Per l&#39;analisi dell&#39;RNA, le frazioni devono essere raccolti direttamente in tubi contenenti Trizol (Invitrogen), fenolo o SDS / proteinasi K, a seconda del metodo preferito di estrazione. </li><li> Quando alla fine del profilo polyribosome è stato raggiunto, spegnete il flusso di Fluorinert alla provetta e fermare il movimento grafico della assorbanza / monitor fluorescenza. </li><li> Ritrarre il Fluorinert dalla provetta nella siringa inizio il flusso in direzione inversa a un ritmo rapido facendo attenzione a non trarre saccarosio dal tubo nella siringa. Svitare il tubo da centrifuga il piercer tubo, abbassare l&#39;ago, e rimuovere il tubo. </li><li> Ripetere i passaggi 5.5 tramite 5.8 per ogni gradiente di saccarosio. </li><li> Quando tutti i gradienti di saccarosio sono stati analizzati, ritrarre il Fluorinert dal tubo nella siringa. Rimuovere ogni parte del frazionatore (piercer tubo e cella a flusso), tra cui il tubo dei rifiuti situato nella parte superiore e risciacquare bene con acqua. </li></ol> 6. Rappresentante Risultati <img src="/files/ftp_upload/1948/1948fig1.jpg" alt="Figura 1" /> Figura 1. Traccia rappresentante di estratto ribosoma preparata da un ceppo di lievito BY4741 (Mat A Δ his3 1 leu2 met15 Δ Δ 0 0 ura3 Δ 0) in presenza di cicloesimide. L&#39;estratto ribosomiale è stato frazionato utilizzando un gradiente di saccarosio 7 47% e analizzati utilizzando un apparato di siringa pompa collegata ad un rilevatore di raggi UV. Vette contenenti poliribosomi e 80S, 60S e 40S ribosomi sono indicati.

<ol>
	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <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+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Le informazioni ottenute dal profilo polyribosome può fornire informazioni preziose sullo stato traslazionale della cellula. Inoltre, lo stato del gruppo di subunità ribosomiali si può essere determinato 2. Per esempio la presenza di halfmers o 80S e grandi vette con una spalla leggermente a destra sul profilo indica una subunità 40S legato in attesa di entrare subunità 60S. Esecuzione l&#39;esperimento in assenza di qualsiasi cicloeximide aggiunto o inibitore altri allungamento permette l&#39;analisi del tasso di scappare, che indica se non viene alterata o allungamento 3. Le stesse frazioni sono una ricca fonte di reagenti per la determinazione successiva l&#39;associazione di un mRNA specifico o di proteine ​​con sottopopolazioni ribosomiale tamponando Nord Occidentale o delle frazioni. L&#39;intero gruppo di mRNA associati ribosomi attivo può essere determinato tramite un associato microarray 4 o profonda analisi di sequenziamento 5. Le associazioni proteina può anche essere stabilizzato con l&#39;aggiunta appropriata di un reticolazione reagente 6. Analisi Polyribosome dalle cellule dei mammiferi è da segnalare come un protocollo distinto in termini di apparente difficoltà nello stabilire le condizioni necessarie per ottenere polisomi stabile. I sistemi tampone riportati in letteratura sono ampiamente variable 7-10, quindi non vi può essere un requisito per il tipo cellulare ottimizzazione specifica del tampone di lisi, o è altrettanto plausibile che il sistema tampone non è importante quanto l&#39;attenzione desiderosi di lavorare con lisati fresca conservati a basse temperature. A nostra conoscenza una valutazione sistematica dei parametri che influenzano la formazione di polisomi mammiferi non è stata riportata.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>polyallomer ultracentrifuge tube</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>326823</td>
<td>1 x 3.5 in. (25x89mm)</td>
</tr>
<tr>
<td>Fluorinert</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9755</td>
<td>&nbsp;</td>
<tr>
<td>Cycloheximde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-7698</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3393</td>
<td>&nbsp;</td>
<tr>
<td>BY4741</td>
<td>&nbsp;</td>
<td>Open Biosystems</td>
<td>YSC1048</td>
<td>Other strains work equally well</td>
</tr>		</tbody>
		</table>
		</div>

eukaryotic polyribosome profile analysis

Sintesi proteica è un processo complesso cellulare che è regolata a diversi livelli. Per esempio, la traduzione globale può essere inibito in fase di inizio o la fase di allungamento da una varietà di stress cellulare come fame di aminoacidi o fattore di crescita ritiro. In alternativa, la traduzione di mRNA individuo può essere regolato dalla localizzazione mRNA o la presenza di microRNA affini. Studi di sintesi proteica frequentemente utilizzano analisi polyribosome a far luce sui meccanismi di regolazione traduzione o difetti di sintesi proteica. In questo saggio, mRNA / ribosoma complessi sono isolate da cellule eucariotiche. Un gradiente di densità di saccarosio separa mRNA legata ai ribosomi più conosciuto come poliribosomi da mRNA associato a un ribosoma singola o monosome. Frazionamento dei gradienti consente l&#39;isolamento e la quantificazione delle diverse popolazioni ribosomiale e il loro mRNA associati o proteine. Le differenze nel rapporto tra poliribosomi monosomes a determinate condizioni può essere indicativo di difetti in entrambe le iniziazione traduzione o allungamento / terminazione. L&#39;esame dei mRNA presenti nelle frazioni polyribosome può rivelare se la coorte di mRNA individuale in corso di traduzione cambia con le condizioni sperimentali. Inoltre, il montaggio ribosoma può essere monitorato attraverso l&#39;analisi dei picchi piccoli e grandi subunità ribosomiale che sono anche separati dal gradiente. In questo video, vi presentiamo un metodo per la preparazione di estratti grezzi di ribosomiale da cellule di lievito, la separazione dell&#39;estratto dal gradiente di saccarosio e l&#39;interpretazione dei risultati. Questa procedura è facilmente adattabile alle cellule di mammifero.

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

Questo articolo descrive un protocollo per l&#39;estrazione di tradurre ribosomi delle cellule eucariotiche. Una volta estratto, i ribosomi sono separati in monosomes e poliribosomi per frazionamento gradiente di saccarosio per consentire diverse popolazioni ribosomiale da analizzare. Come tale, questo metodo è il gold standard per l&#39;esame della regolamentazione della traduzione.

Eucarioti Analisi Profilo Polyribosome

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Research

JoVE Journal

Biology

52.4K Views.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. Questo articolo descrive un protocollo per l'estrazione di tradurre ribosomi delle cellule eucariotiche. Una volta estratto, i ribosomi sono separati in monosomes e poliribosomi per frazionamento gradiente di saccarosio per consentire diverse popolazioni ribosomiale da analizzare. Come tale, questo metodo è il gold standard per l'esame della regolamentazione della traduzione.

Video: Eukaryotic Polyribosome Profile Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Ricerca

Biologia

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

Profilo Esperimento bioenergetica utilizzando cellule dei mioblasti C2C12

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

Luce Correlativo e Microscopia Elettronica (CLEM) come strumento per visualizzare molecole microiniezione e dei loro sub-cellulari eucariotiche Obiettivi


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Istituzione di microbiche colture di arricchimento eucariotiche da un chimicamente Stratificati Antarctic Lake e valutazione del potenziale fissazione del carbonio

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Analisi Profilo metabolico di embrioni di zebrafish

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Real Time Analisi di profilo metabolico in<em&gt; Ex Vivo</em&gt; Culture del mouse intestinale Cripta organoide

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Purificazione genoma a livello di extrachromosomal circolare DNA dalle cellule eucariotiche

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Protocollo di laboratorio per analisi genetiche del contenuto dell'intestino del rDNA acquatica macroinvertebrati Using Group-specific Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Preparazione di organismi procarioti ed eucarioti mediante essiccazione chimica per analisi morfologica in microscopia elettronica (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, postmortem computed tomography (PMCT) acquisition, postprocessing, and evaluation were successfully established. In this pioneer cetacean virtopsy stranding response program, PMCT was performed on 193 stranded cetaceans, providing postmortem findings to aid necropsy and shed light on the biological health and profile of the animals. This study aimed to assess 8 image rendering techniques in PMCT, including multiplanar reconstruction, curved planar reformation, maximum intensity projection, minimum intensity projection, direct volume rendering, segmentation, transfer function, and perspective volume rendering. Illustrated with practical examples, these techniques were able to identify most of the PM findings in stranded cetaceans and served as a tool to investigate their biological health and profile. This study could guide radiologists, clinicians and veterinarians through the often difficult and complicated realm of PMCT image rendering and reviewing.

The Hong Kong cetacean stranding response program has incorporated postmortem computed tomography, which provides valuable information on the biological health and profile of the deceased animals. This study describes 8 image rendering techniques that are essential for the identification and visualization of postmortem findings in stranded cetaceans, which will help clinicians, veterinarians and stranding response personnel worldwide to fully utilize the radiological modality.

Tecniche di rendering delle immagini nella tomografia computerizzata post-mortem: valutazione della salute biologica e del profilo nei cetacei bloccati

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, ...