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1. INS-1E cellule: manutenzione e placcatura
<ol><li>Mantenere cellule INS-1E in un incubatore 37 ˚C/5% CO2 e coltivate con medium RPMI 1640 completati con 5% (v/v) inattivati siero bovino fetale, 2 mM L-Glutammina, 10 mM HEPES, piruvato di sodio di 1 mM, 100 U/mL di penicillina/streptomicina soluzione, 50 µM β-mercaptoetanolo. Della coltura di cellule in 10 mL di terreno completo RPMI 1640 (per piastra), finché non raggiungono la confluenza di 80-90%, quando possono essere tripsinizzate e attraversate o utilizzati per il dosaggio di secrezione di insulina.</li>
	<li>
1 ° giorno: Media di aspirare e lavare le cellule una volta con 5 mL di PBS preriscaldata. Aggiungere 0,5 mL di tripsina (0.025%) diluito 1:1 in 0,5 mL di PBS tripsinizzano le cellule e incubare per 3-4 min a 37 ° c. Disattivare tripsina aggiungendo 9 mL completo media e cellule di trasferimento di una provetta da centrifuga da 15 mL pipettando.</li>
	<li>Pellet di cellule mediante centrifugazione e risospendere il pellet cellulare nei mezzi di comunicazione fresco di circa 5 mL.</li>
	<li>Prendere 10 µ l di cellule ri-sospensione e mescolare con 10 µ l di colorante vitale tripan blu per verificare la vitalità cellulare. Contare le celle vivi e morte in 10 µ l di questa miscela utilizzando un emocitometro. Livello di redditività dovrebbe essere superiore al 90%. Diluire le cellule risospese in media fresco per 1 milione di cellule per mL.</li>
	<li>Le cellule di INS-1E 0,5 mL di seme per pozzetto in una poli-L-lisina prerivestiti piastra a 24 pozzetti, ad una densità di 500.000 cellule per pozzetto.</li>
	<li>
2 ° giorno: Rimuovere media 18-24 h dopo la placcatura e aggiungere 500 µ l/pozzetto di fresco RPMI 1640 media. Incubare le cellule per altre 24 ore consentire alle cellule di recuperare completamente dal passaggio precedente e passaging. Questo tempo aggiuntivo permette alle cellule di diffondere sulla piastra di coltura del tessuto.</li>
</ol>2. insulina secrezione Assay (giorno 3)
<ol><li>Preparare il tampone di KRB: 132,2 mM NaCl, 3,6 mM KCl, 5mm NaHCO3, 0,5 mM NaH2PO4, 0,5 mM MgCl2, 1,5 mM CaCl2e 0,001 g/mL di sieroalbumina bovina (BSA), pH 7,4.</li>
	<li>Media da cellule di aspirare e lavare due volte con PBS preriscaldata.</li>
	<li>Per il passaggio di fame di glucosio, aggiungere 450 µ l/pozzetto KRB (contenente BSA) senza glucosio per 1 h a 37 ˚C/5% CO2.</li>
	<li>Durante la fase di inedia di glucosio, preparare diluizioni seriali delle droghe in KRB contenente 200 mM glucosio (10x concentrazione).<ol>
<li>Preparare droga a 10x la concentrazione finale in KRB completati con 200 millimetri di glucosio (anche 10 volte la concentrazione di glucosio finale del dosaggio). Se lo stock di droga (plus aggiunto 10 x glucosio) è in DMSO, assicurarsi che la percentuale di DMSO è mantenuto costante durante tutto il test (percentuale idealmente finale inferiore a 0.1% DMSO).</li>
		<li>Per i trattamenti di dopamina e quinpirole, utilizzare una gamma di concentrazione di farmaco dosaggio finale di 100 µM a 100 pM (dalla più alta alla più bassa concentrazione), con l'ultimo punto della dose risposta non contenente nessuna droga. Per la bromocriptina, utilizzare una gamma di concentrazione finale del dosaggio di 10 µM a 22, con l'ultimo punto della dose risposta essendo il controllo droga-libero.</li>
	</ol>
</li>
	<li>Dopo aver fame di glucosio, aggiungere diluizioni seriali di droga per il dosaggio di produrre una reazione al dosaggio.</li>
	<li>Aggiungere 50 µ l/pozzetto di ciascuna diluizione seriale nei pozzetti corrispondenti (dosaggio totale volume 500 µ l).</li>
	<li>Per la fase di stimolazione del glucosio, incubare le cellule con le diluizioni seriali di rispettivi droga (in presenza di glucosio 20 mM) 90 min a 37 ˚C/5% CO2. Comprendono una serie di pozzetti di controllo: (1) stimolazione con 20 millimetri di glucosio da solo in assenza di qualsiasi farmaco aggiuntivo e (2) le cellule che vengono stimolate né droga né glucosio (che fornisce un tasso basale di secrezione).</li>
	<li>Dopo la fase di stimolazione, rimuovere con cautela i surnatanti (uso direttamente o conservare a 4 ° c). Nota: Un passo di centrifugazione delicata di ulteriori 5 minuti (600 x g, 1 min) può essere introdotti a questo punto per rimuovere tutte le cellule rimanenti nei surnatanti dosaggio.</li>
</ol>3. HTRF per misura la secrezione dell'insulina
<ol><li>Diluire il dosaggio surnatanti 01:10 in KRB (senza BSA), preferibilmente in piastre da 96 pozzetti chiaro.</li>
	<li>Preparare la curva standard di insulina per il dosaggio di insulina HTRF (tabella 1).</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Soluzione standard stock 500 ng/ml</td>
			<td>Diluizioni seriali</td>
			<td>Lavoro [insulina] ng/ml</td>
		</tr>
<tr>
<td>STD 7</td>
			<td>30 µ l stock + 140 µ l KRB</td>
			<td>150</td>
		</tr>
<tr>
<td>STD 6</td>
			<td>30 µ l STD 7 + 45 µ l KRB</td>
			<td>60</td>
		</tr>
<tr>
<td>STD 5</td>
			<td>30 µ l STD 6 + 45 µ l KRB</td>
			<td>24</td>
		</tr>
<tr>
<td>STD 4</td>
			<td>30 µ l STD 5 + 45 µ l KRB</td>
			<td>9.6</td>
		</tr>
<tr>
<td>STD 3</td>
			<td>30 µ l STD 4 + 45 µ l KRB</td>
			<td>3.84</td>
		</tr>
<tr>
<td>STD 2</td>
			<td>30 µ l STD 3 + 45 µ l KRB</td>
			<td>1.54</td>
		</tr>
<tr>
<td>STD 1</td>
			<td>30 µ l STD 2 + 45 µ l KRB</td>
			<td>0.61</td>
		</tr>
<tr>
<td>STD 0</td>
			<td>KRB 45 µ l</td>
			<td>0</td>
		</tr>
<tr>
<td colspan="3">Nota: STD Stock è 500 ng/ml</td>
		</tr>
</tbody></table>Tabella 1. Diluizioni seriali per rendere la curva standard di insulina.
<ol start="3"><li>Aggiungere i campioni di curva standard ed i surnatanti di dosaggio diluito alla piastra HTRF. La misurazione di insulina secreta da HTRF può avvenire in un formato di piastra 384 pozzetti, tenendo presente che il volume di dosaggio deve essere ri-regolato o un 96 pozzetti. Uso 10 µ l/pozzetto campione in bianchi 96 pozzetti metà-zona lastre o 5 µ l/pozzetto in un piatto bianco 384 pozzetti di basso volume, turno-fondo (Vedi Tabella materiali).</li>
	<li>Preparare la miscela di anticorpi nel buffer di rilevamento (Vedi Tabella materiali) in un donatore di 1:2 (criptato) / rapporto accettore (XL-665). Nota: Ulteriori dettagli specifici circa l'analisi HTRF sono disponibili presso il produttore.</li>
	<li>Aggiungi mix di anticorpo per test a 30 µ l/pozzetto (per un formato di 96 pozzetti saggio) o 15 µ l/pozzetto (per un formato di test piastra 384 pozzetti).</li>
	<li>La piastra ed incubare a temperatura ambiente.</li>
	<li>Leggere la piastra dopo 2h, 4h, e/o pernottamento incubazione con anticorpi usando il lettore di piastre e il modulo di ottica HTRF appropriato (337 665 620 nm) (Vedi Tabella dei materiali e le istruzioni del produttore). Impostare l'inizio di integrazione alle 60 µs e il tempo d'integrazione alle 400 µs. uso 200 lampeggia per pozzetto. Nota: Questi parametri sono stati basati sull'uso del nostro lettore particolare. La lettura a 620 nm e 665 nm possono variare tra i diversi strumenti. Questo è uno dei motivi che si consiglia di utilizzare il rapporto di 665/620. Nel calcolo di questo rapporto, eventuali differenze di potenziale dal lettore al lettore saranno normalizzati e forniscono valori coerenti indipendentemente lo strumento utilizzato per misurare HTRF.</li>
</ol>4. normalizzazione e analisi dei dati
<ol><li>Calcolare le concentrazioni di insulina nei pozzetti di saggio tramite estrapolazione di raziometrici letture di fluorescenza (665 nm/620 nm) a una secondo ordine polinomiale curva quadratica (Figura 2).</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57531/57531fig2.jpg"> Figura 2 : Curva standard insulina. Stock di insulina umana di concentrazioni note è stato utilizzato per generare la curva standard di insulina. I rapporti HTRF risultanti (665 nm / 620 nm) sono state tracciate contro le concentrazioni di insulina. I dati erano più adatta a una curva polinomiale quadratica di secondo ordine (R2 = 0.99996). Si tratta di una curva standard rappresentativa. Barre di errore = SEM. <a href="/files/ftp_upload/57531/57531fig2large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<ol start="2"><li>Con i dati estrapolati come ng/mL di insulina secernuta, normalizzare (insulina secernuta in risposta all'aumento delle concentrazioni di ligando) al valore medio dei pozzi % insulina massima secrezione assay (stato solo del glucosio di 20 mM).</li>
	<li>Utilizzare la curva adatta (R2) da un singolo esperimento per calcolare la variazione di intraplacca. All'interno dell'esperimento, derivato i singoli valori di R2 dai duplicati intra-sperimentali, che consentano di calcolare l'errore standard della media per la curva.</li>
	<li>Per la determinazione della variazione interplacca, utilizzare i dati da almeno tre singoli esperimenti per calcolare l'errore standard della media per il valore di2 R della curva del collettivo.</li>
</ol>

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Ringraziamo Nicolas Pierre (Cisbio analisi biologiche) per consigli utili e Dr. Pierre Maechler (Università di Ginevra) per fornire generosamente alle cellule INS-1E. Questo lavoro è stato sostenuto dal finanziamento dal dipartimento della difesa (grant PR141292 a Z.F.), John F. e Nancy A. Emmerling Fund della Fondazione Pittsburgh (a Z.F.).

Il dosaggio di insulina HTRF descritto qui offre un sistema rapido, efficiente per misurare la secrezione di insulina da un sistema basato su cellule coltivata. Tra i suoi più importanti vantaggi, questo saggio offre un segnale basso fondo dovuto l'alto rapporto segnale-rumore. Inoltre, abbiamo confermato che il segnale HTRF è stabile per lunghi periodi di tempo (> 24 h)7. Tuttavia, poiché gli anticorpi monoclonali di insulina-associazione raggiungere velocemente la saturazione di associazione ...

La regolazione del metabolismo energetico è messo a punto da un importante ormone anabolizzante, insulina. L'insulina viene sintetizzata e rilasciato da cellule beta pancreatiche in risposta ai livelli aumentati del glucosio extracellulare. L'insulina rilasciata innesca l'assorbimento di glucosio da tessuti insulino sensibili1,2. Fisiologicamente, questo è legato all'elevazione della concentrazione di glucosio dopo un pasto, seguita dalla secrezione di insulina per regolare l'assorbimento del glucosio. Perturbazioni nell'omeostasi del glucosio conducono ai danni metabolici che culmina nell'insulino-resistenza e, infine, nell'insorgenza del diabete di tipo 22,3,4.
Anche se la secrezione dell'insulina è stata studiata estesamente, suoi meccanismi regolatori rimangono poco compresi. Un'area critica di indagine è stata l'identificazione di nuovi modulatori della secrezione di insulina dalle cellule beta5,6,7,8. Questi studi richiedono una migliore comprensione del rapporto accoppiamento tra stimolazione del glucosio e la secrezione di insulina. Di conseguenza, la possibilità di monitorare accuratamente e quantificare i livelli di secrezione insulinica glucosio-mediata (GSIS) è stato essenziale. Fin qui, tuttavia, solo un numero limitato di metodi era disponibile per consentire la quantificazione di GSIS utilizzando linee di cellule secernenti insulina e/o isole pancreatiche. Uno è la radioimmunoanalisi (RIA), che utilizza il radioisotopo-etichetta dell'insulina e gli anticorpi. Le principali limitazioni di questo approccio includono problemi di sicurezza dovuto la manipolazione e lo smaltimento di materiali radioattivi. Inoltre, questo metodo è laborioso, che coinvolgono più lunga lavaggio e le fasi di incubazione. Analisi enzima-collegata dell'immunosorbente (ELISA) è un altro approccio costoso e laborioso che utilizza anticorpi per il rilevamento di insulina. Variazione di affinità dell'anticorpo e dell'efficienza di riconoscere insulina stanno limitando i fattori di questo metodo e può influenzare la riproducibilità dei risultati. ELISA, né RIA è stato progettato per esperimenti di alto-rendimento. AlphaScreen è un dosaggio omogeneo utilizzato per il rilevamento e la misurazione dei livelli di secrezione dell'insulina. AlphaScreen tecnologia si basa sulla conversione dell'ossigeno ambientale in uno stato di singoletto eccitato ossigeno che possa reagire con specie a chemiluminescenza, conseguente generazione di chemiluminescenza. Perché il test sia omogeneo, molti i passaggi di lavaggio associato a RIA ed ELISA sono eliminati. Tuttavia, l'instabilità del segnale a causa della natura della reazione è un fattore limitante che può influenzare la lettura del test. (TR-tenaglia, sviluppato da Heyduk e colleghi9, è un altro approccio omogeneo alla misura dell'insulina basata sul legame degli anticorpi separati due epitopi diversi della molecola di insulina. Gli anticorpi sono ogni chimicamente legata al doppio DNA incagliato con strapiombi di DNA incagliati singoli complementari breve. Associazione degli anticorpi all'insulina li riunisce e porta ad un duplex di DNA incagliato doppio. Ogni anticorpo inoltre è associato con un fluoroforo donatore o accettore rispettivo, e l'associazione del duplex DNA riunisce questi fluorofori per generare il trasferimento di energia per risonanza (FRET). Uno dei potenziale limiti di TR-tenaglia, spetta tuttavia con il tasto stesso. L'incapacità di dissipare rapidamente fluorescenza di fondo durante la reazione di FRET può portare a livelli relativamente alti di fluorescenza di fondo e un basso rapporto segnale-rumore all'interno del dosaggio. Pertanto, sia ancora necessario per un'analisi affidabile, robusta e conveniente per quantificare GSIS in un modo ad alta velocità.
Gli avanzamenti recenti nella biofisica sono culminati nello sviluppo di un test di trasferimento (HTRF) basata risonanza energia omogenea fluorescenza risolta in tempo. In particolare, mentre l'energia di trasferimento all'interno l'analisi può essere descritto come basato su FRET, più precisamente, che HTRF si basa sulla luminescenza energia risonanza trasferimento (LRET)10 che è il non-radiative trasferimento di energia tra il donatore e accettore specie11,12,13. Questa distinzione è importante, poiché i tempi di una fluorescenza o tempra base FRET interazione è molto diversa da quella per LRET, anche se gli stessi tipi di gating possono essere utilizzati per FRET e LRET. Inoltre, l'uso di terre rare lantanidi cryptate composti quali europio o terbio cryptate in HTRF produce fluorescenza lunga emivita12,14. Questo offre un vantaggio unico dell'introduzione di un tempo di ritardo (µ sec) tra eccitazione del donatore e la misurazione del livello di emissione l'accettore (cioè, risolta in tempo saggio). Questo ritardo consente per un tempo sufficiente per la fluorescenza di fondo dissipare prima misurazione della fluorescenza emissione accettore. Di conseguenza, la lettura è libera di fluorescenza aspecifica e così, si ottiene un elevato rapporto segnale-rumore (Figura 1). Inoltre, la natura omogenea di HTRF Elimina la necessità per i passaggi di lavaggio per lavare via le specie non associate, che rende l'analisi molto più rapido rispetto ELISA o metodi basati su RIA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57531/57531fig1.jpg"> Figura 1: schematica del meccanismo per il rilevamento di insulina HTRF. Due anticorpi monoclonali indipendentemente generati specificamente riconoscono e si legano all'insulina presso siti separati. Questi anticorpi sono coniugati per il donatore cryptate europio o l'accettore XL665. Eccitazione del donatore a 337 risultati nm in emissione a 620 nm. Il trasferimento di energia provoca XL665 a emettere ad una lunghezza d'onda, 665 nm. <a href="/files/ftp_upload/57531/57531fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
Qui, forniamo un protocollo dettagliato per l'utilizzo di un approccio basato su HTRF per determinare i livelli di GSIS dalle cellule INS-1E, una consolidata insulina-secrezione beta cellula-derivato ratto insulinoma cella linea15. Inoltre, questo dosaggio può essere usato per identificare il profilo farmacologico di molecolari regolatori della secrezione dell'insulina. Applichiamo questo dosaggio di insulina basata su HTRF per esaminare la dopamina D2-come regolazione del recettore di GSIS. Sempre più studi hanno rivelato che la dopamina del neurotrasmettitore è un regolatore importante di GSIS8,16,17,18,19,20, 21 , 22. dopamina colpisce GSIS in modo autocrino/paracrino negativo tramite azioni sulla dopamina D2-come i ricevitori (D2, D3, ricevitori4 D) espressi sulla superficie delle cellule pancreatiche beta8 , 16 , 19. usando questa analisi, abbiamo conferma del ruolo di dopamina come un regolatore negativo di GSIS e dimostrare che la dopamina D2-come bromocriptina agonisti del recettore e quinpirole ridurre anche GSIS.

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			<td height="42" style="height:42px;width:216px;">96-well white half area plate</td>
			<td style="width:104px;">Greiner Bio-One</td>
			<td style="width:139px;">82050-042</td>
			<td style="width:315px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">384-well white low-volume, round-bottom plate</td>
			<td style="width:104px;">Corning</td>
			<td>15100157</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insulin High Range Kit</td>
			<td style="width:104px;">Cisbio Bioassays</td>
			<td style="width:139px;">62IN1PEH&#160;</td>
			<td>10,000 test HTRF-based insulin assay kit</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PHERAstar FSX plate reader</td>
			<td style="width:104px;">BMG Labtech</td>
			<td>FSX</td>
			<td>Plate reader adapted for HTRF-based assay readings</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plate sealers</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:139px;">DY992</td>
			<td>To seal plate while antibodies incubate</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hemocytometer</td>
			<td style="width:104px;">Hausser Scientific</td>
			<td style="width:139px;">3100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI Medium 1640 (1x)</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">11875-093</td>
			<td>[+] L-glutamine</td>
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			<td height="21" style="height:21px;width:216px;">Trypsin 0.05%&#160;</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">25-052-CI</td>
			<td>0.53 mM EDTA, (-) sodium bicarbonate</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DPBS (Dulbecco&#39;s phosphate buffered saline)</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">21-031-CV</td>
			<td>Without calcium and magnesium</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">35-010-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">156-30-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">11360070</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin/Streptomycin solution 100x</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">30-002 CT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">M1348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dopamine hydrochloride</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">8502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromocriptine mesylate</td>
			<td style="width:104px;">Tocris</td>
			<td style="width:139px;">427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">(-)-Quinpirole hydrochloride</td>
			<td style="width:104px;">Tocris</td>
			<td style="width:139px;">1061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:104px;">Calbiochem</td>
			<td style="width:139px;">12659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly-L-Lysine</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">P4832</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO (Dimethyl sulfoxide)</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">276855</td>
			<td></td>
		</tr>
	</tbody>
</table>

homogeneous time-resolved f&#246;rster resonance energy transfer-based assay for detection of insulin secretion

La rilevazione della secrezione dell'insulina è fondamentale per illuminante meccanismi di secrezione regolata anche negli studi di metabolismo. Anche se numerosi saggi di insulina sono esistite per decenni, il recente avvento di tecnologia tempo-risolta omogenea del Förster Resonance Energy Transfer (HTRF) ha notevolmente semplificato queste misurazioni. Questo è un saggio di ottico rapido, conveniente, riproducibile e robusto affidamento anticorpi coniugati a fluorofori luminose con emissione di lunga durata che facilita risolta in tempo Förster Resonance Energy Transfer. Inoltre, rilevamento di insulina HTRF è favorevole per lo sviluppo di saggi di screening ad alta resa. Qui usiamo HTRF per rilevare la secrezione dell'insulina in cellule INS-1E, una linea cellulare derivata da insulinoma del ratto. Questo ci permette di stimare i livelli basali di loro cambiamenti in risposta a stimolazione del glucosio e dell'insulina. Inoltre, utilizziamo questo sistema di rilevazione di insulina per confermare il ruolo della dopamina come un regolatore negativo della secrezione insulinica glucosio-mediata (GSIS). In modo simile, altri di dopamina D2-come agonisti del recettore, quinpirole e Bromocriptina, ridurre GSIS in un modo dipendente dalla concentrazione. I nostri risultati evidenziano l'utilità del formato di dosaggio di insulina HTRF nel determinare il ruolo dei numerosi farmaci in GSIS e loro profili farmacologici.

Abbiamo validato la nostra analisi HTRF insulina generando una curva standard di insulina utilizzando standard di insulina umana purificata delle concentrazioni predefiniti (Figura 2). Generazione della curva standard ha permesso di estrapolare le letture di fluorescenza raziometrici e quindi a determinare i livelli di insulina secreta in risposta ai trattamenti farmaco (Figura 2). Variazione di intraplacca per adattamento della ...

Watch this Scientific Journal Video about Homogeneous Time-resolved FÃƒÂ¶rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion at JoVE.com

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Qui, presentiamo omogeneo tempo risolto FRET (HTRF) come un metodo efficace per la rilevazione rapida di insulina secernuta dalle cellule.

Förster Time-resolved omogeneo risonanza energia basati su trasferimento test per la determinazione della secrezione dell'insulina

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

homogeneous-time-resolved-frster-resonance-energy-transfer-based

Research

JoVE Journal

Medicine

9.1K Views.  University of Pittsburgh. Qui, presentiamo omogeneo tempo risolto FRET (HTRF) come un metodo efficace per la rilevazione rapida di insulina secernuta dalle cellule.

Video: Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Produzione e di rilevamento di specie reattive dell&#39;ossigeno (ROS) in Cancro

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Ricerca

Medicina

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

Migliorare La somministrazione di insulina IV in un ospedale di comunità

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Rilevazione intraoperatoria di endometriosi sottile: un nuovo paradigma per la rilevazione e il trattamento del dolore pelvico associato con la perdita di integrità peritoneale

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Luminescenza Imaging Cerenkov (CLI) per il monitoraggio Cancer Therapy

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method&#x2014;adapted from a coculture model of synaptogenesis&#x2014;for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

Analisi co-coltura delle interazioni proteina extracellulare che influenzano secrezione di insulina da parte del pancreas cellule beta

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

Combinato di microscopia Intravital e l'ecografia contrapporre-aumentata del Hindlimb Mouse per studiare la perfusione del muscolo e la vasodilatazione indotta da insulina

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher magnetic field strengths and potentially provides improved signal contrast and spatial resolution. While promising results have been achieved, ultra-high field CMR is challenging due to energy deposition constraints and physical phenomena such as transmission field non-uniformities and magnetic field inhomogeneities. In addition, the magneto-hydrodynamic effect renders the synchronization of the data acquisition with the cardiac motion difficult. The challenges are currently addressed by explorations into novel magnetic resonance technology. If all impediments can be overcome, ultra-high field CMR may generate new opportunities for functional CMR, myocardial tissue characterization, microstructure imaging or metabolic imaging. Recognizing this potential, we show that multi-channel radio frequency (RF) coil technology tailored for CMR at 7 Tesla together with higher order B0 shimming and a backup signal for cardiac triggering facilitates high fidelity functional CMR. With the proposed setup, cardiac chamber quantification can be accomplished in examination times similar to those achieved at lower field strengths. To share this experience and to support the dissemination of this expertise, this work describes our setup and protocol tailored for functional CMR at 7 Tesla.

The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept.

Risonanza magnetica cardiaca a 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Studiando il segnale ipotalamico insulina ad intolleranza al glucosio periferico con un sistema di infusione del farmaco continuo nel cervello del Mouse

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

Analisi dell'attività dell'endotossina per il rilevamento dell'endotossiamia del sangue intero nei pazienti in malattia critica

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

Meccanismi di secrezione dell'ormone intestinale utilizzando l'isolato irrorati intestino tenue del ratto

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...