Noi riconosciamo il supporto di finanziamento da CPRIT (RP100119) e la Welch Foundation (I-1665).

Intero protocollo impiega circa 4 giorni. 1. Preparazione di cellule per siRNA e Reporter Transfezione Introdurremo qui un protocollo per interrogare librerie di siRNA utilizzando un piccolo insieme di siRNA (384 diversi siRNA piscine array in formato a 96 pozzetti con ogni pool composto da siRNA 4x Targeting per un singolo gene) da una libreria siRNA genoma a scopo illustrativo. Per questo particolare studio, sfrutteremo HCT116 cellule che esibiscono devianti segnalazione Wnt e Kras, e l&#39;attività di p53. La linea cellulare appropriata in studi volti a interrogare altri processi cellulari di interesse dovrà essere identificato e valutato in modo simile. <ol><li> Lavare 5 x 10 cm 2 piatti di 80-90% confluenti HCT116 cellule con PBS poi raccolto mediante tripsinizzazione cellule con 1 ml di soluzione di tripsinizzazione / piastra seguito da neutralizzazione con 10 ml di Dulbecco Modified Eagle Medium (DMEM) contenente 2% feTal siero bovino (FBS) e 1% di penicillina / streptomicina / piastra. </li><li> Trasferire le cellule in sospensione in una provetta da 50 ml e centrifugare a ~ 130 xg per 5 minuti, rimuovere il surnatante e risospendere il pellet cellulare in 10 ml di DMEM / 2% FBS / 1% di penicillina / streptomicina. </li><li> Contare il numero di cellule con un contatore di cellule, e poi fare una soluzione di cellule con 10 5 cellule / ml, 150 ml mantenere sospensione cellulare per il passaggio successivo. Piastra 10 4 cellule (100 microlitri) in ciascun pozzetto di una piastra di coltura cellulare 96 utilizzando un dosatore automatico multipunto (d&#39;ora in poi multipunto). La sospensione cellulare dovrebbe essere sufficiente in questo caso per placcatura 12 x 96 pozzetti. </li><li> Incubare le piastre con cellule a 37 ° C in un incubatore con il 5% di CO 2 durante la preparazione della miscela di trasfezione. </li></ol> 2. Preparazione della Construct Reporter Archivio Solution <ol><li> Isolare giornalista di alta qualità costruire DNA utilizzando kit standard di midiprep (NucleoBond Xtra MIDI prodotti daClontech) e diluire il DNA per una concentrazione finale di 1 mg / ml per ciascun giornalista. </li><li> Preparare 100 ml di soluzione madre giornalista costruire combinando 30 ml di p53-FL, 30 ml di 8X TCF-RL, 30 ml di ELK1-Gal4, 6 microlitri UAS-CL e 4 ml di H 2 O per ottenere una miscela di DNA con un 1:1:1:0.2 rapporto finale dei giornalisti. Concentrazione di DNA finale di questa soluzione madre di DNA misto è di 0,3 mg / ml. </li></ol> 3. Preparazione del siRNA / DNA Transfezione Mix In questa parte del protocollo, prepareremo siRNA / DNA mix di trasfezione che saranno sufficienti per trasfezione 12 x 96 pozzetti. Per questa libreria di test di 4 x 96 pozzetti di siRNA, saremo trasfettare ogni pool siRNA in triplice copia. Il reagente di trasfezione che useremo si chiama Effectene (Qiagen). Nelle nostre mani questo è l&#39;unico reagente che funziona per l&#39;erogazione contemporanea di siRNA e DNA in cellule coltivate. <ol><li> Diluire 80 ml di giornalista costrutto magazzino solution in 8 ml di tampone CE (Effectene Kit) per ottenere una soluzione di DNA con una concentrazione finale di 3 mg / ml. In generale, preparare abbastanza soluzione di miscela di DNA per uso immediato. </li><li> Piastra 20 ml di questa soluzione di DNA a ciascun pozzetto di una ben 96 / piastra. Il numero delle piastre a 96 pozzetti dipenderà da quanti saranno testati piscine siRNA. Ad esempio, in questo caso avremo bisogno di 4 x 96 pozzetti per valutare 384 diverse piscine siRNA. </li><li> I siRNA da testare devono essere ad una concentrazione magazzino di 5 micron. Se non, possono essere replica placcato e diluito a questa concentrazione utilizzando il tampone di diluizione raccomandata associato a ciascuna libreria. </li><li> Trasferire 2 ml di ogni 5 micron siRNA magazzino al corrispondente pozzetto della piastra di PCR contenente il brodo DNA diluito con un Biomek Automated Liquid Handler. Seconda della scala dello studio, una pipetta multicanale può essere sufficiente. </li><li> Ora, aggiungeremo 1 ml di soluzione Enhancer (kit Effectene) in ciascun pozzetto del DNA / siRNA plaTE. Anche questo può essere realizzato sia con un gestore di liquido automatizzato o multicanale pipetta. </li><li> Lasciare che la reazione Enhancer avvenga a temperatura ambiente per 5 minuti poi aggiungere 3 ml di reagente di trasfezione Effectene a ciascun pozzetto e incubare a temperatura ambiente per altri 10 min. </li><li> Per interrompere trasfezione complesso formazione, aggiungere 10 ml di DMEM / 2% FBS / 1% di penicillina / streptomicina a ciascun pozzetto della piastra PCR. </li><li> Trasferire 10 microlitri della miscela di trasfezione per ciascun pozzetto della piastra a 96 pozzetti contenenti le cellule (recuperati dal termostato cella). Come ogni trasfezione verrà effettuata in triplicato, la stessa miscela sarà applicato a due ulteriori piastre 96 pozzetti contenenti cellule. </li><li> Incubare le cellule con miscele di trasfezione aggiunti a 37 ° C in ambiente umidificato al 5% di CO 2 per 36 hr. </li></ol> 4. Determinare attività luciferasi da campioni di cellule Dopo 36 ore, le attività di luciferasi sono pronti per la misurazionein cellule trasfettate. Il punto finale dovrebbe essere determinato su una base per-esperimento. Nel nostro studio, un tempo di incubazione di 36 ore aveva già dato un segnale sufficientemente robusto per significato determinazione esito. Poiché questo studio incorpora reporter multipli (uno secreta nel terreno di coltura, e altri due espressa nel citoplasma cellulare), otterremo misurazioni sia da mezzo di coltura così come un lisato cellulare. <ol><li> Rivelazione dell&#39;attività CL nel mezzo di coltura: <ol><li> 20 ml di terreno di coltura è replica-placcato in un bianco opaco 96 pozzetti (sia utilizzando il Liquid Handler Biomek o multicanale pipetta). </li><li> Aggiungere 20 ml di tampone CL (sistemi di puntamento) in ciascun pozzetto. </li><li> Aggiungere 10 ml di Cypridina substrato luciferina (Sistemi Targeting) a ciascun pozzetto e rilevare attività CL usando un luminometro (il lettore di piastre Pherastar prodotto da BMG per esempio). </li></ol></li><li> Rilevazione di FL e attività RL: <ol><li> Lyse il cells rimuovendo prima il terreno di coltura. Questo può essere realizzato rapidamente girando piastra capovolta e lanciare medie in un lavandino. Rimanendo mezzo può essere rimosso battendo leggermente la piastra di testa in giù su carta assorbente. Aggiungere 30 ml di 1X Passive Lysis Buffer (Promega) in ciascun pozzetto di celle usando il multipunto e posto su una sedia a dondolo piattaforma (Bellco è una ditta che produce questi) impostare una velocità media per 5 min a RT. </li><li> Aggiungere 20 microlitri della Luciferase Assay Reagent II (parte del kit Promega dual luciferasi) utilizzando l&#39;attività FL Multidrop e subito misurare usando il luminometro. Successivamente, aggiungere Stop &amp; Glo Reattivo (Promega) che spegnerà l&#39;attività FL e consentire la misurazione delle attività di RL. Nota: se non si sta utilizzando miscele di substrato che permettono durata del segnale prolungato (come Dual-Glo Kit luciferasi da Promega), l&#39;uso di kit flash richiede una rapida misurazione su substrato oltre (entro 5 minuti). </li></ol></li></ol>

<ol>
	<li>Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G. S., Kricka, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescent+and+bioluminescent+reporter+gene+assays.">Chemiluminescent and bioluminescent reporter gene assays.</a> Analytical Biochemistry. 219, 169-181 (1994).</li><li>Miraglia, L. J., King, F. J., Damoiseaux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+light:+luminescent+reporter+gene+assays.">Seeing the light: luminescent reporter gene assays.</a> Combinatorial Chemistry &amp; High Throughput Screening. 14, 648-657 (2011).</li><li>McGuire, S. E., Roman, G., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+systems+in+Drosophila:+a+synthesis+of+time+and+space.">Gene expression systems in Drosophila: a synthesis of time and space.</a> Trends in Genetics: TIG. 20, 384-391 (2004).</li><li>Smirnova, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Neh2-luciferase+reporter+and+its+application+for+high+throughput+screening+and+real-time+monitoring+of+Nrf2+activators.">Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators.</a> Chem. Biol. 18, 752-765 (2011).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule-mediated+disruption+of+Wnt-dependent+signaling+in+tissue+regeneration+and+cancer.">Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.</a> Nat. Chem. Biol. 5, 100-107 (2009).</li><li>Tang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+RNAi+screen+for+Wnt/beta-catenin+pathway+components+identifies+unexpected+roles+for+TCF+transcription+factors+in+cancer.">A genome-wide RNAi screen for Wnt/beta-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9697-9702 (2008).</li><li>Lum, L. D., Kulak, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Luciferase+Reporter+Assay+Systems.">Multiplexed Luciferase Reporter Assay Systems.</a> United States patent. , (2012).</li><li>Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptionally+active+DNA-binding+site+for+human+p53+protein+complexes.">A transcriptionally active DNA-binding site for human p53 protein complexes.</a> Mol. Cell Biol. 12, 2866-2871 (1992).</li><li>DasGupta, R., Kaykas, A., Moon, R. T., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+the+Wnt-wingless+signaling+pathway.">Functional genomic analysis of the Wnt-wingless signaling pathway.</a> Science. 308, 826-833 (2005).</li><li>Korinek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+epithelial+stem-cell+compartments+in+the+small+intestine+of+mice+lacking+Tcf-4.">Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.</a> Nat. Genet. 19, 379-383 (1998).</li><li>Korinek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+members+of+the+Tcf+family+implicated+in+Wnt/beta-catenin+signaling+during+embryogenesis+in+the+mouse.">Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse.</a> Mol. Cell Biol. 18, 1248-1256 (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+characterization+of+human+colon+and+rectal+cancer.">Comprehensive molecular characterization of human colon and rectal cancer.</a> Nature. 487, 330-337 (2012).</li><li>Koboldt, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+portraits+of+human+breast+tumours.">Comprehensive molecular portraits of human breast tumours.</a> Nature. , (2012).</li><li>Wood, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+landscapes+of+human+breast+and+colorectal+cancers.">The genomic landscapes of human breast and colorectal cancers.</a> Science. 318, 1108-1113 (2007).</li><li>Berndt, J. D., Biechele, T. L., Moon, R. T., Major, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+genome-wide+RNA+interference+screens.">Integrative analysis of genome-wide RNA interference screens.</a> Science Signaling. 2, pt4 (2009).</li><li>Falschlehner, C., Steinbrink, S., Erdmann, G., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+to+dissect+cellular+pathways:+a+how-to+guide.">High-throughput RNAi screening to dissect cellular pathways: a how-to guide.</a> Biotechnology Journal. 5, 368-376 (2010).</li><li>Boutros, M., Bras, L. P., Huber, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell-based+RNAi+screens.">Analysis of cell-based RNAi screens.</a> Genome Biology. 7, R66 (2006).</li><li>Gilbert, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multiplex+cell+viability+assay+for+high-throughput+RNAi+screening.">A novel multiplex cell viability assay for high-throughput RNAi screening.</a> PLoS ONE. 6, e28338 (2011).</li></ol>

OK e LL sono autori di un brevetto relativo alla tecnologia multiplex luciferasi.

Ci sono diversi fornitori dei sistemi reporter luciferasi-based (come ad esempio Promega, sistemi di puntamento, New England Biolabs, e Thermo Fisher) che forniscono utili risorse on-line per coloro intrattenere uno schermo ad alta produttività per la prima volta. Inoltre, una serie di ottime recensioni riguardo ottimizzare l&#39;uso di schermi high-throughput per la scoperta del gene e come base di RNAi risultati di screening possono essere integrati con altri dataset-omiche basate dovrebbe essere utile 15,16.</s...

Una varietà di reporter luciferasi-based per il monitoraggio di una gamma diversificata di processi biologici cellulari sono disponibili in commercio. La maggioranza di questi DNA costruisce codificare proteine ​​luciferasi che sono indotte ad esprimere da stimoli specifici delle cellule o perturbazioni. La maggior parte dei giornalisti robusti transcriptionally basati inserire l&#39;espressione di un enzima luciferasi sotto il controllo di elementi enhancer sintetici o regioni promotore del gene ben stabiliti per la loro affidabilità nel riportare una fisiologicamente rilevanti 1,2 evento trascrizionale. Ci sono anche sistemi trans-reporter luciferasi che utilizzano GAL4-UAS per guidare l&#39;espressione della proteina luciferasi. GAL4 è un lievito attivatore trascrizionale e la SUP è un enhancer di GAL4 che si lega specificamente ad attivare la trascrizione di sequenze geniche poste a valle di esso 3. Questi tipi di sistemi di luciferasi sono tipicamente supportate da due plasmidi DNA - una codifica la proteina GAL4 fusa al regulatory porzione di una proteina di interesse, e il secondo codifica una proteina luciferasi posto sotto il controllo di una o più sequenze UAS. Così, il segnale luciferasi cellulare riflette il livello di attività della proteina di interesse. Un altro uso comune di reporter luciferasi è basati per monitorare la stabilità proteica quale una proteina di interesse è fuso ad un enzima luciferasi 4. Indipendentemente dal tipo di giornalista, un caveat emptor viene qui notare come non si può assumere la specificità di qualsiasi reporter di essere stato ben interrogato. Così, due diligence è richiesta la costituzione di nuovi costrutti reporter di qualsiasi strategia di ricerca. La selezione dell&#39;enzima luciferasi read-out può essere importante quanto l&#39;affidabilità dei elementi di DNA che controllano l&#39;espressione. Considerando lucciola luciferasi (FL) è l&#39;enzima più comunemente usato in costrutti disponibili in commercio, l&#39;avvento di nuovi sistemi di luciferasi che non richiedono ATP (come nel casodi reazioni FL-based), o che incorporano enzimi più stabili che emettono segnali forti promettono di migliorare l&#39;efficienza e l&#39;affidabilità di questa piattaforma di ricerca. Una nota importante per quanto riguarda la selezione dei reporter luciferasi in studi chimici è la suscettibilità di FL di inibizione chimica che potrebbe dare luogo a falsi rapporti. Nella nostra esperienza, altri enzimi come Renilla o Gaussia luciferasi (RL e GL, rispettivamente) che utilizzano come substrato coelenterazina sono meno facilmente inibito da piccole molecole 5. Il formato più comune per multiplazione luciferasi read-out comporta l&#39;uso di FL e RL largamente data la disponibilità di facile da usare kit con la capacità di misurare sequenzialmente attività enzimatiche da un singolo campione. Nella maggior parte kit, i campioni vengono prima esposti a luciferina per rivelare livelli di attività FL seguiti da un reagente tempra che viene contemporaneamente depositati presso coelenterazina per produrre una secondasegnale RL dary. Con l&#39;aggiunta di altri luciferases che possono essere secreti come GL o che utilizzano ancora un altro substrato quale Cypridina luciferasi (CL), un numero maggiore di possibilità per la generazione di dati di alta contenuti utilizzando luciferases sono emerse. Un esempio di un genoma schermo RNAi utilizzando questa tecnica può essere trovato qui 6. Questi progressi tecnologici hanno a loro volta stimolato lo sviluppo di meccanismi specifici per placare ogni tipo di enzima luciferasi per limitare diafonia 7. Nelle nostre mani, si nota una maggiore frequenza di &quot;effetti di bordo&quot; (tendenze inspiegabili in attività cellulare associato alla periferia di piastre di coltura ad alto titolo), con luciferases secrete come GL o CL. D&#39;altra parte, tali luciferases secrete sono utili per saggi cinetici quanto consentono di campionamento del segnale senza adulterare vitalità cellulare. Per il nostro studio qui presentato, avremo contemporaneamente monitorare l&#39;attività dei tre cancro rilevantiprocessi cellulari: il p53, Kras, e Wnt vie di trasduzione del segnale. I giornalisti incorporati nel nostro studio sono il pp53-TA-Luc FL giornalista Clontech (d&#39;ora in poi indicato come il giornalista p53-FL) 8, un sistema giornalista Elk1-GAL4/UAS-CL (d&#39;ora in poi Elk-1 giornalista, Agilent), e la 8X TCF RL giornalista che incorpora più elementi enhancer sintetici riconosciuti dalle Wnt pathway regolatori trascrizionali noti come fattori di cellule T (o TCF) 9-11.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

Interrogatorio genoma scala della funzione genica mediante RNA interference (RNAi) è di enorme speranza per l&#39;identificazione rapida delle chimicamente trattabili vulnerabilità delle cellule tumorali. Limitando il potenziale di questa tecnologia è l&#39;incapacità di delineare rapidamente la base meccanicistica dei risultati fenotipici e quindi informare lo sviluppo di strategie terapeutiche a bersaglio molecolare. Noi descriviamo qui i metodi di decostruire fenotipi cellulari indotti dal gene di RNAi mediato il targeting utilizzando sistemi reporter canalizzati che permettono il monitoraggio del cancro chiave processi associati alle cellule. Questa metodologia screening ad alto contenuto è versatile e può essere facilmente adattato per lo screening di altri tipi di grandi librerie molecolari.

Nonostante i progressi nella mappatura del paesaggio mutazionale di vari tipi di cancro che utilizzano enormi sforzi di sequenziamento del genoma 12-14, noi ancora non abbiamo in atto un approccio sistematico per la traduzione di queste osservazioni nelle strategie di intervento. Nel cancro del colon-retto (CRC), le mutazioni che si prevede di colpire i componenti di tre processi cellulari - Wnt / β-catenina, p53, e Kras segnalazione - si trovano nel 99% di tutti i tumori che suggeriscono che i fattori train...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Il raggiungimento di un livello di sistemi di comprensione dei processi cellulari è un obiettivo di biologia cellulare moderna. Descriviamo qui le strategie per multiplazione reporter luciferasi di varia funzione cellulare end-point per interrogare la funzione del gene utilizzando genoma scala librerie di RNAi.

Una piattaforma di screening Luciferase basata multiplex per interrogare il cancro-associata trasduzione del segnale in cellule in coltura

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

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11.0K Views.  UT Southwestern Medical Center. Il raggiungimento di un livello di sistemi di comprensione dei processi cellulari è un obiettivo di biologia cellulare moderna. Descriviamo qui le strategie per multiplazione reporter luciferasi di varia funzione cellulare end-point per interrogare la funzione del gene utilizzando genoma scala librerie di RNAi.

Video: A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Come misurare Folding corticale dalle immagini MR: uno step-by-step tutorial per calcolare indice locale Gyrification

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

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High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

Contenuto di screening ad alto contenuto di Malattie Neurodegenerative

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

L&#39;uso di cistometria in piccoli roditori: A Study of Chemosensation vescica

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Screening per Modificatori melanoma utilizzando un modello Zebrafish Tumore autoctone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

A High-contenuti<em&gt; In Vitro</em&gt; La replica della piattaforma Discovery isole pancreatiche β-cellule

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Screening delle nanoparticelle bioattive in cellule immunitarie fagocitiche per gli inibitori della segnalazione di ricetrasmettitori simili

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Routine di metodo di Screening per microparticelle in trasfusioni della piastrina

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

Hydra, una piattaforma basata su Computer per aiutare i clinici nella diagnosi e analisi cardiovascolare

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. 
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

In Vitro Metodo di controllo delle concentrazioni di gas alogenati in cellule epiteliali alveolari coltivate

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...