Gli autori desiderano ringraziare Drs. Rick Ryan, Umesh Patel, Jeff Till, Matteo Hsu, Jehangir Mistry e Michele Hatler per il supporto durante lo sviluppo di questi progetti e protocolli.

Preparazione delle cellule: <ol><li> Prima della semina delle cellule per i neuroni cultura saggio, / astrociti in terreni di crescita fino a ~ confluente 70-80% (a meno che la semina di cellule direttamente dal disgelo o isolamento). </li><li> Staccare le cellule da fiaschi cultura / piastre tramite metodo appropriato per il tipo di cellule di interesse. Se necessario, cappotto pozzetti piastra con poli-D-lisina o di proteine ​​della matrice extracellulare per migliorare l&#39;adesione delle cellule. Co-colture di astrociti e neuroni possono essere seminate contemporaneamente o in sequenza. Per le teste di serie sequenziali, placcatura di astrociti prima come livello &quot;basale&quot; è consigliato, seguito da placcatura neuronale e la differenziazione, se necessario. Regolare la densità cellulare come appropriato per il tipo di cellule, il tempo della cultura successiva, i parametri di interesse, ecc A seconda dell&#39;età della cultura, fonte di cellule e la densità di semina, colture primarie possono variare notevolmente in velocità di proliferazione, espressione di GFAP o crescita dei neuriti - è importante caratterizzare e ottimizzare il sistema cellulare ea garantire la rilevanza biologica per il modello sperimentale, così come per fornire efficaci per l&#39;imaging, la segmentazione e l&#39;analisi utilizzando HCS (vedi Figura 1). Dopo aver aggiunto le cellule a piastra (cellule possono essere seminate in 90 microlitri mezzi di comunicazione, di semplificare il trattamento tossina come descritto nel passaggio 3 di seguito), permettono di sedere piatto su una superficie piana a temperatura ambiente per 15-30 minuti, consentendo una distribuzione uniforme delle celle. Dopo questo periodo, le cellule incubare in terreni di coltura (37 ° C / 5% CO 2) per almeno 24 ore, quindi passare a condizioni di coltura differenziazione (per esempio, bassi livelli sierici / NGF per cellule PC12), a seconda dei casi. Continuare la cultura fino cellule raggiungere il livello desiderato di confluenza o di differenziazione, la sostituzione del supporto ad intervalli appropriati per il tipo di cellule. </li><li> Trattamenti con le cellule (composti di controllo, composti di prova, ecc) può essere introdotto in qualsiasi momento durante questo periodo la cultura, come appropriato per il tempo-ciclo di trattamento di interesse. Acrilamide, perossido di idrogeno e K-252bis sono forniti come composti controllo neurotossici. Reagenti siano sufficienti per le curve di duplicare la dose di 12 punti risposta (di cui uno dH 2 O o DMSO-control trova all&#39;interno del dose-risposta) per tutte e cinque le piastre a 96 pozzetti. I composti sono forniti concentrazione 250X (per K-252bis, assumendo una terapia massima di 1 microM) o 10X (di acrilamide e perossido di idrogeno, assumendo trattamenti massima di 100 mm e 10 mm, rispettivamente). Preparazione trattamento raccomandato coinvolge mezza log (1: √ 10) diluizione seriale del composto 250X in DMSO, seguita da diluizione in tampone di diluizione Compound a 10 volte (10 volte composti controllo originari dH 2 O può essere diluito in serie direttamente in sterili dH 2 O o di diluizione Compound). 10 L di ogni trattamento può poi essere aggiunto al 90 microlitri di terreni di coltura già presenti in ciascun pozzetto, per una concentrazione finale 1X (0,4% o al 10% DMSO dH 2 O). Dati di esempio viene fornito per 24 o 96 ore di trattamento composto a 37 ° C prima della fissazione. </li></ol> Fissazione e colorazione delle cellule immunofluorescenza: Nota: il tempo di colorazione è ~ 2,5 ore dopo la fissazione. Non permettere pozzetti si asciughino tra i passaggi colorazione. Aspirazione e dispensazione dei reagenti deve essere condotta a basse portate a diminuire la perdita di cellule dovuta al taglio del fluido. Tutti raccomandati &#39;per bene&#39; i volumi si riferiscono a un singolo pozzetto di una micropiastra a 96 pozzetti. Tutti raccomandati &#39;per 96 pozzetti&#39; volumi includono il 25% la perdita di liquido in eccesso di volume di movimentazione. Tutte le fasi di colorazione vengono eseguite a temperatura ambiente (RT). Tutti i tamponi e le soluzioni di anticorpi sono stabili per almeno 24 ore a temperatura ambiente. <ol start="4"><li> Alla fine del periodo di coltura, pre-riscaldare Soluzione Fissazione HCS (2X) a temperatura ambiente (RT) o di 37 º C se lo si desidera (12 piastra mL/96-well). In una cappa, aggiungere 100 l / pozzetto direttamente ai terreni di coltura e consentono di fissare per 30 minuti a RT. Rimuovere fissativo / tossina contenenti i media e smaltire in conformità alle norme per i rifiuti pericolosi (vedi scheda di sicurezza). Se procedere immediatamente alle macchie, lavare ogni pozzetto due volte con 200 ml di tampone HCS immunofluorescenza. In alternativa, se le targhe devono essere colorati in un secondo momento, lavare due volte con 200 ml di soluzione di lavaggio, quindi lasciare secondo risciacquo volume in pozzi e piastre di conservare ben chiusi a 4 ° C fino a colorazione. </li><li> Se i campioni sono stati conservati fisso a 4 ° C prima della colorazione, lavare due volte con 200 microlitri Buffer HCS immunofluorescenza prima di procedere con il protocollo di colorazione. </li><li> Preparare soluzione di lavoro di coniglio anti-tubulina βIII / Mouse anti-GFAP Anticorpi HCS primaria (6 piastra mL/96-well) come segue: Aggiungere 60 ml di ogni anticorpo primario scongelati a 5,88 ml di tampone HCS immunofluorescenza. Mescolare bene. Rimuovere precedenti Buffer immunofluorescenza risciacquare. Aggiungere 50 ml di soluzione di anticorpo primario in ciascun pozzetto e incubare per 1 ora a temperatura ambiente. </li><li> Rimuovere Anticorpo primariosoluzione. Lavare tre volte con 200 ul Buffer HCS immunofluorescenza. </li><li> Preparare la soluzione di lavoro di anticorpi HCS secondaria / Hoechst HCS nucleare Stain (6 piastra mL/96-well) come segue: aggiungere 30 ml di ogni anticorpo secondario scongelati e 30 ml di scongelato Hoechst HCS nucleare Stain a 5,91 ml di tampone HCS immunofluorescenza. Mescolare bene, proteggendo la soluzione dalla luce. Rimuovere precedenti HCS Buffer immunofluorescenza risciacquare. Aggiungere 50 ml di anticorpo HCS secondaria / Hoechst HCS soluzione nucleare Stain e incubare per 1 ora a temperatura ambiente, al riparo dalla luce. </li><li> Rimuovere HCS secondaria Anticorpo / Hoechst HCS soluzione nucleare Stain. Sciacquare due volte con 200 ul Buffer HCS immunofluorescenza. </li><li> Rimuovere precedenti HCS Buffer immunofluorescenza risciacquare. Sciacquare due volte con 200 ml di tampone HCS lavaggio, lasciando secondo risciacquo volume in pozzi. </li><li> Piatto di tenuta e di immagine subito, o conservare a 4 ° C al riparo dalla luce fino al momento per l&#39;imaging. </li></ol> Acquisizione di immagini e analisi: <ol start="12"><li> Imaging e analisi di piatti colorati possono essere eseguite su una varietà di piattaforme disponibili HCS, tra cui il IN Analyzer Cell (GE Healthcare), ArrayScan (ThermoFisher scientifico) o Opera (Perkin Elmer). Alcune linee guida per l&#39;imaging e di analisi sono riportati nella Tabella 1: </li></ol><table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4"> HCS222 Image Acquisition Linee guida </td></tr><tr><td> Rilevazione dei reagenti </td><td> Obiettivo obiettivo </td><td> Intervallo di eccitazione Filter [picco / larghezza di banda (nm)] </td><td> Intervallo di emissione Filtro [picco / larghezza di banda (nm)] </td></tr><tr><td> Hoechst HCS nucleare Stain </td><td> 20X </td><td> 360/40 </td><td> 460/40 (o 535/50 se necessario) </td></tr><tr><td> HCS anticorpo secondario, coniugati con Donkey anti-IgG </td><td> 20X </td><td> 480/40 </td><td> 535/50 </td></tr><tr><td> HCS anticorpo secondario, Cy3-Donkey anti-mouse IgG </td><td> 20X </td><td> 535/50 </td><td> 600/50 </td></tr></tbody></table> <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4" width="545"> HCS222 Image Analysis Linee guida </td></tr><tr><td> Cella di </td><td> Rivelazione </td><td> Segmentazione / misurazione </td><td> Fondamento logico </td></tr><tr><td> Numero di cellulare, Caratteristiche nucleare </td><td> Hoechst HCS nucleare Stain </td><td> Regione nucleare (460 nm di emissione del canale). Contare il numero di nuclei. Contenuto di DNA (intensità nucleare) o analisi settore nucleare sono inoltre possibili. </td><td> Usa numero cellulare, caratteristiche nucleari per determinare la perdita di cellule, fenotipi tossicità, ecc Può nuclei &quot;filtro&quot; per quelli associati βIII-tubulina o espressione GFAP di ottenere separare conta delle cellule neuronali e astrocitaria / caratterizzazioni (fortemente consigliato). </td></tr><tr><td> βIII-tubulina di espressione, neuriti escrescenza </td><td> HCS anticorpo secondario, coniugato FITC </td><td> Regione citoplasmatica (535 nm di emissione del canale). Segnale FITC possono essere utilizzati per distinguere corpi cellulari neuronali da neuriti (ad esempio, attraverso le zone delle cellule minimo / medio corpo, la lunghezza dei neuriti minimo / massimo e larghezza). Determinare parametri quali la lunghezza dei neuriti totale, conta neuriti / cellulari, ecc </td><td> Misure crescita dei neuriti può essere modulata durante la differenziazione neuronale o come risultato di danno chimico, stati di malattia, ecc Can &quot;filtro&quot; corpi cellulari per quelli associati βIII-tubulina espressione di ottenere distinte caratterizzazione neuronale (fortemente consigliato). </td></tr><tr><td> GFAP Espressione, Area astrociti </td><td> HCS anticorpo secondario, coniugato con Cy3- </td><td> Regione citoplasmatica (600 nm di emissione del canale). Cy3 segnale può essere utilizzato per definire astrocitaria segmentazione citoplasmatica. Determinare parametri come intensità media del segnale citoplasmatici, area delle celle, ecc </td><td> Espressione di GFAP e la zona delle cellule astrociti possono essere modulata durante lo sviluppo degli astrociti o come risultato di danno chimico, stati di malattia, ecc Can &quot;filtro&quot; corpi cellulari per quelli associati con l&#39;espressione di GFAP di ottenere distinte caratterizzazione astrocitaria (fortemente consigliato). </td></tr></tbody></table> . Tabella 1 Immagine Linee guida acquisizione e l&#39;analisi - HCS222 βIII-Tubulin/GFAP (Co-Cultura) Assay Rappresentante dei risultati: <img src="/files/ftp_upload/1173/figure31173.png" alt="Figura 3" /> Figura 1. βIII-tubulina e GFAP immunofluoreScence misti di colture di cellule neuronali di ratto. Co-colture di astrociti primari di ratto sia con i neuroni dell&#39;ippocampo di ratto primaria dell&#39;ippocampo di ratto o cellule PC12 sono state generate dalla pre-placcatura astrociti per diversi giorni di cultura, seguito da semina neuronale. Neuroni primari sono state coltivate per altri 11 giorni, mentre PC12s sono stati coltivati ​​per altri 2 giorni in condizioni di differenziazione (bassi livelli sierici / NGF). Manipolazione delle cellule, la fissazione e immunostaining sono stati eseguiti secondo protocolli HCS222 test. Le cellule sono state proietta sulla GE IN cella Analyzer 1000 (3.3) a 20X (neuroni primario) o 10X (PC12) ingrandimento obiettivo e analizzati utilizzando il GE IN Workstation cellulare Analyzer 1000 (3.4) crescita dei neuriti e algoritmi di analisi multi target Pannello superiore.: immagini fuse di Hoechst HCS Stain nucleare (blu), βIII-tubulina (verde) e GFAP (rosso) fluorescenza. Analisi separata dei βIII-tubulina canale di fluorescenza permette di segmentazione crescita dei neuriti (al centro del pannello: corpi cellulari delineate nel blu (neuroni primari) o giallo (PC12), neuriti in verde (neuroni primario) o azzurro (PC12)). Analisi del canale GFAP permette di segmentazione astrociti (pannello di fondo: i nuclei in blu, citoplasma in verde). Segmentazione GFAP per il primario dei neuroni dell&#39;ippocampo di ratto / astrociti co-coltura dimostra la capacità di distinguere tra nuclei / celle corpi che sono GFAP (+) (verde contorni) e quelli che sono GFAP (-) (rosso), che consente la conta delle cellule separate per neuroni e astrociti in un ambiente cultura mista. <img src="/files/ftp_upload/1173/figure41173.png" alt="Figura 4" /> <img src="/files/ftp_upload/1173/figure4.51173.png" alt="Figura 4 part2" /> Figura 2. Risposte dose di primari di ratto neurone dell&#39;ippocampo / astrociti co-colture agli stress tossico. Astrociti primari di ratto dell&#39;ippocampo (P6) sono stati placcati in piastre a 96 pozzetti in terreni di coltura e coltura per 6 giorni. Neuroni dell&#39;ippocampo di ratto primario (diretto da disgelo) sono poi stati seminati in cima alla pre-plated astrociti e coltivate in terreni di coltura per 11 giorni. Le cellule sono state trattate con diluizioni seriali di perossido di idrogeno o acrilamide (concentrazione max = 100mm e 10mm, rispettivamente) per le ultime 24 ore di cultura. Manipolazione delle cellule, la fissazione e immunostaining sono stati eseguiti secondo protocolli HCS222 test. Le cellule sono state proietta sulla GE IN cella Analyzer 1000 (3.3) a 20X (10 campi / pozzetto) ed analizzati (nucleare / citoplasma / segmentazione dei neuriti) utilizzando il cellulare di GE IN Workstation Analyzer 1000 (3.4) crescita dei neuriti e algoritmi di analisi multi target. I dati presentati sono media ± SEM, n = 3. Più parametri (crescita dei neuriti, intensità GFAP, zona astrociti e conta delle cellule neuronali o astrociti) sono stati indagati per dose-dipendente tendenze.

<ol>
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M., Gass&#243;, S., V&#237;lchez, D., Rodr&#237;guez-Farr&#233;, E., Sanfeliu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+effects+of+trimethyltin+and+triethyltin+on+human+fetal+neuron+and+astrocyte+cultures:+a+comparative+study+with+rat+neuronal+cultures+and+human+cell+lines.">Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: a comparative study with rat neuronal cultures and human cell lines.</a> Toxicol Lett. 152, 35-46 (2004).</li></ol>

Gli autori sono tutti i dipendenti di Millipore Corporation.

Fino ad oggi, in esperimenti in vitro progettato per studiare la valutazione del rischio di neurotossicità utilizzando colture miste di neuroni e astrociti sono stati limitati. E &#39;ben accettato che le cellule gliali sono parte integrante nel fornire supporto spaziale e metabolico ai neuroni, e svolgono un ruolo cruciale nel modulare diverse funzioni neuronali, compresa la migrazione neuronale, la differenziazione e l&#39;attività sinaptica. Astrociti gliali anche rilascio di citochine e altri fattori solubili, che sono in grado di indurre sia tolleranza effetti avversi su tessuti circostanti neuronale, oltre a promuovere neuronale di molte tossine. Dimostrare la profondità dei neuroni-gliali interazioni, in co-coltura studi, gli astrociti hanno dimostrato di proteggere i neuroni contro la tossicità di stress ossidativo, mentre l&#39;alterazione delle funzioni astrociti, come la manutenzione di difesa antiossidante ed i livelli di energia cellulare, è stato dimostrato che criticamente influenzano la sopravvivenza neuronale. Recenti studi hanno suggerito che l&#39;uso di astrociti in una neurotossicità in vitro di test del sistema può rivelarsi più rilevanti per la salute umana struttura del sistema nervoso centrale e la funzione di cellule neuronali da solo. Nonostante la crescente consapevolezza del significato fisiologico di neuroni, astrociti interazioni, è già stato difficile effettuare analisi quantitative di queste interazioni in cellule intatte. L&#39;avvento di screening ad alta Contenuto consente lo sviluppo di potenti nuove analisi per affrontare questo. In questo studio, abbiamo dimostrato che le analisi quantitative di più fenotipi neuronali e astrociti all&#39;interno di un singolo test sono rese possibili da HCA. Nei neuroni primari abbiamo effettuato misurazioni quantitative dei marcatori di neurotossicità quali il numero dei neuroni, il conteggio dei neuriti e la lunghezza dei neuriti. Negli astrociti, gliosi reattiva è un biomarker noto di neurotossicità, caratterizzata da alterazioni nell&#39;espressione GFAP, il numero di astrociti e la morfologia degli astrociti. Abbiamo dimostrato che ognuno di questi endpoint possono facilmente essere valutata tramite HCA. Questi studi supportano il concetto che HCA di neurali specifici biomarcatori potrebbe essere utilizzato come parte di una batteria di test in vitro per lo screening rapidamente un gran numero di sostanze chimiche per gli endpoint neurotossici. Millipore HCS222 Alta kit Content Analysis per co-coltura di neuroni e astrociti si compone di alta qualità, reagenti e protocolli per il rilevamento simultaneo profilazione βIII-tubulina e proteina acidica fibrillare gliale (GFAP) in una vasta gamma di modelli cellulari di neurotossicità. La altamente convalidato reagenti forniti con questo kit consentono all&#39;utente di standardizzare i test, ridurre al minimo dosaggio a dosaggio variabilità, riproducibile e per generare immagini con un elevato rapporto segnale-background rapporto.

<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>Rabbit Anti-&#x03B2;III-Tubulin HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201672</td>
<td>1 vial containing 300 &micro;L 
Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-rabbit IgG, FITC conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201649</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Mouse Anti-GFAP HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201671</td>
<td>1 vial containing 300 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-mouse IgG, Cy3 conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200437</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hoechst HCS Nuclear Stain, 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200438</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Fixation Solution with Phenol Red, 2X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200434</td>
<td>1 bottle containing 100 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Immunofluorescence Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200435</td>
<td>1 bottle containing 1000 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Wash Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. 2007643</td>
<td>1 bottle containing 500 mL Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>Acrylamide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201679</td>
<td>1 vial containing 500 &micro;L of 1M acrylamide in dH2O Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hydrogen Peroxide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201730</td>
<td>1 vial containing 500 &micro;L of 100 mM hydrogen peroxide in dH2O Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>K-252a (Control Compound), 250X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201741</td>
<td>1 vial containing 100 &micro;L of 250 &micro;M K-252a in DMSO Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>DMSO for Compound Serial Dilution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200441</td>
<td>1 bottle containing 10 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Compound Dilution Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200442</td>
<td>1 bottle containing 25 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Plate Sealers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200443</td>
<td>10 each Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>tissue culture-treated black/clear bottom microplates </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HCS imaging/analysis system</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>

a neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity

Alta Content Analysis (HCA) saggi combinano le cellule e reagenti di rilevamento automatico e con immagini potenti algoritmi di analisi delle immagini, consente la misurazione di diversi fenotipi cellulari all&#39;interno di un singolo test. In questo studio, abbiamo utilizzato HCA per sviluppare un nuovo metodo di analisi di neurotossicità. Neurotossicità di valutazione rappresenta una parte importante della valutazione sulla sicurezza dei farmaci, oltre ad essere un focus significativo degli sforzi di protezione ambientale. Inoltre, la neurotossicità è anche ben accettato<em&gt; In vitro</emMarker&gt; dello sviluppo di malattie neurodegenerative come l&#39;Alzheimer e di Parkinson. Recentemente, l&#39;applicazione di HCA allo screening neuronale è stata riportata. Mediante l&#39;etichettatura cellule neuronali con βIII-tubulina, saggi di HCA in grado di fornire high-throughput, non soggettivo, misurazioni quantitative dei parametri quali il numero dei neuroni, il conteggio dei neuriti e la lunghezza dei neuriti, che può indicare effetti neurotossici. Tuttavia, il ruolo degli astrociti rimane inesplorato in questi modelli. Astrociti ha un ruolo fondamentale nel mantenimento del sistema nervoso centrale (SNC) omeostasi, e sono associati con entrambi neuroprotezione e neurodegradation quando sono attivati ​​in risposta a sostanze tossiche o stati di malattia. GFAP è una proteina filamenti intermedi espressi prevalentemente astrociti del sistema nervoso centrale. Attivazione degli astrociti (gliosi) porta alla upregulation di GFAP, comunemente accompagnato da proliferazione degli astrociti e ipertrofia. Questo processo di gliosi reattiva è stata proposta come marker precoce di danni al sistema nervoso. Il metodo tradizionale per la quantificazione GFAP è da immunologico. Questo approccio è limitato dalla incapacità di fornire informazioni sulla localizzazione cellulare, la morfologia e il numero di cellulare. Abbiamo determinato che HCA potrebbero essere utilizzati per superare queste limitazioni e per misurare simultaneamente molteplici funzionalità associate a gliosi - cambiamenti nell&#39;espressione GFAP, ipertrofia astrociti, e la proliferazione degli astrociti - all&#39;interno di un singolo test. In co-coltura studi, gli astrociti hanno dimostrato di proteggere i neuroni contro diversi tipi di insulto tossico e di influenzare in modo critico la sopravvivenza neuronale. Recenti studi hanno suggerito che l&#39;uso di astrociti in<em&gt; In vitro</emNeurotossicità&gt; sistema di test possono rivelarsi più rilevanti per la struttura umana SNC e la funzione di cellule neuronali da solo. Di conseguenza, abbiamo sviluppato un saggio HCA per co-coltura di neuroni e astrociti, composto di protocolli e validati, target-specifici reagenti di rilevamento per il profiling βIII-tubulina e proteina acida gliale fibrillare (GFAP). Questo test consente l&#39;analisi simultanea di neurotossicità, crescita dei neuriti, gliosi, morfologia neuronale e astrociti e lo sviluppo neuronale e astrociti in una vasta gamma di modelli cellulari, che rappresentano un romanzo, non soggettivo, high-throughput test per la valutazione neurotossicità. Il saggio ha un grande potenziale per il rilevamento di neurotossicità maggiore e una migliore produttività della ricerca nel campo delle neuroscienze e la scoperta della droga.

Watch this Scientific Journal Video about A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity at JoVE.com

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

Questo articolo descrive un nuovo protocollo e reagenti impostare progettato per la misura sensibile degli effetti neurotossici di composti e trattamenti sulla co-culture di neuroni e astrociti utilizzando l&#39;analisi ad alto contenuto. Risultati dimostrano che l&#39;analisi ad alto contenuto rappresenta una tecnologia entusiasmante romanzo per la valutazione neurotossicità.

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 ...

a-neuronal-astrocyte-co-culture-assay-for-high-content-analysis

Research

JoVE Journal

Biology

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

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Colture cellulari neuronali da Aplysia per imaging ad alta risoluzione di coni di crescita

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Ricerca

Biologia

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Un multi-vano SNC Neuron-glia Co-cultura Microfluidic piattaforma

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidica Co-coltura di cellule epiteliali e batteri per le indagini solubile del segnale mediato da Interazioni

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Analisi di Schwann-astrociti Interazioni utilizzo In Vitro Saggi

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantificazione di dsDNA con il F-7000 Hitachi Fluorescenza spettrofotometro e PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Absolute Quantum la misurazione del rendimento dei campioni di polvere

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Un alto contenuto di flusso di lavoro di imaging per studiare GRB2 Centri di segnalazione di espressione clonazione

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Un saggio di legame High Throughput MHC II per l&#39;analisi quantitativa dei peptidi epitopi

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Analisi qPCRTag - A High Throughput, Real Time PCR Assay per Sc2.0 genotipizzazione

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

Alta Analisi Screening contenuto di valutare gli effetti tossicologici di componenti nocivi e potenzialmente dannoso (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...