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(1) INS-1E Zellen: Wartung und Beschichtung
<ol><li>Pflegen Sie, INS-1E Zellen in einem befeuchteten 37 ˚C/5% CO2 Inkubator und kultivierten mit RPMI 1640 Medium mit 5 % (V/V) Hitze-inaktivierten fötalen Rinderserum, 2 mM L-Glutamin, 10 mM HEPES, 1 mM Natrium Pyruvat, 100 U/mL Penicillin/Streptomycin ergänzt Lösung, 50 µM β-Mercaptoethanol. Kulturzellen Sie in 10 mL komplette RPMI 1640 Medium (pro Platte), bis zum Zusammenfluss von 80-90 % erreichen, wenn sie trypsiniert und passagiert oder verwendet werden können für die Insulin-Sekretion-Assay.</li>
	<li>
Tag 1: Aspirieren Sie Medien und waschen Sie Zellen einmal mit 5 mL vorgewärmten PBS. Fügen Sie 0,5 mL Trypsin (0,025 %) verdünnt 1:1 in 0,5 mL PBS trypsinize Zellen und 3-4 min bei 37 ° c inkubieren. Deaktivieren Sie Trypsin, indem eine 15 mL Zentrifugenröhrchen durch pipettieren 9 mL komplette Medien und Transfer Zellen hinzufügen.</li>
	<li>Pellet-Zellen durch Zentrifugation und erneut aussetzen der Zelle Pellet in etwa 5 mL frische Medien.</li>
	<li>Nehmen Sie 10 µL der neu suspendierten Zellen und mischen mit 10 µL Trypan blau vital Farbstoff für Zellviabilität zu überprüfen. Lebende und tote Zellen in 10 µL dieser Mischung mit einer Hemocytometer zu zählen. Lebensfähigkeit sollte über 90 % liegen. Verdünnen Sie die neu suspendierten Zellen in frische Medien auf 1 Million Zellen pro mL.</li>
	<li>0,5 mL INS-1E Samenzellen pro Bohrloch in einer Poly-L-Lysin vorbeschichtet 24-Well-Platte bei einer Dichte von 500.000 Zellen/gut.</li>
	<li>
Tag 2: Entfernen Sie die Medien 18-24 h nach der Beschichtung und 500 µL/Well frische RPMI 1640 Medien hinzufügen. Inkubieren Sie die Zellen für eine weitere 24 h damit Zellen vollständig von der vorherigen passaging Schritt zu erholen. Diese zusätzliche Zeit erlaubt die Zellen auf der Gewebekultur Platte verteilen.</li>
</ol>2. Insulin-Sekretion Assay (Tag 3)
<ol><li>KRB Puffer vorzubereiten: 132,2 mM NaCl, 3,6 mM KCl, 5 mM Nahco33, 0,5 mM NaH2PO4, 0,5 mM MgCl2, 1,5 mM CaCl2und 0,001 g/mL Rinderserumalbumin (BSA), pH 7.4.</li>
	<li>Aspirieren Sie Medien aus Zellen und zweimal mit vorgewärmten PBS waschen.</li>
	<li>Fügen Sie für den Glukose-Hunger-Schritt 450 µL/Well KRB (mit BSA) ohne Glukose für 1 h bei 37 ˚C/5% CO2.</li>
	<li>Während der Glukose-Hunger-Schritt vorzubereiten Verdünnungsreihen Gefäßsystem im KRB mit 200 mM Glukose (10 X-Konzentration).<ol>
<li>Bereiten Sie Medikament bei 10 x die Endkonzentration im KRB ergänzt mit 200 mM Glukose (auch 10 x die endgültige Assay-Glukosekonzentration vor). Wenn die Droge-Lager (plus die zusätzlichen 10 x Glukose) ist in DMSO, stellen Sie sicher, DMSO Prozentsatz Assay (idealerweise endgültige Prozentsatz von weniger als 0,1 % DMSO) konstant gehalten wird.</li>
		<li>Für Dopamin und Quinpirole Behandlungen, verwenden Sie einen abschließenden Test Medikament Konzentrationsbereich von 100 µM bis 100 pM (vom höchsten zum niedrigsten Konzentration), mit dem letzten Punkt der Dosis-Antwort enthält kein Medikament. Verwenden Sie für Bromocriptin einen abschließenden Test Konzentrationsbereich von 10 µM bis 22:00, mit dem letzten Punkt der Dosis-Antwort wird die wirkstofffreie Kontrolle.</li>
	</ol>
</li>
	<li>Fügen Sie nach Glukose Hunger Medikament Verdünnungsreihen Assay, eine Dosis-Antwort zu produzieren hinzu.</li>
	<li>Fügen Sie 50 µL/Well jedes serielle Verdünnung in die entsprechenden Vertiefungen (total Assay Volume 500 µL).</li>
	<li>Zellen mit der jeweiligen Droge Verdünnungsreihen (in Anwesenheit von 20 mM Glukose) inkubieren Glukose Stimulation Schritt 90 min bei 37 ˚C/5% CO2. Eine Reihe von Kontroll-Vertiefungen enthalten: (1) Stimulation mit 20 mM Glukose allein in Ermangelung jeder zusätzliche medikamentöse und (2) Zellen, die weder mit Medikamenten noch Glukose (bietet eine basale Rate der Sekretion) stimuliert werden.</li>
	<li>Entfernen Sie nach der Stimulation Schritt vorsichtig die Überstände (Verwendung direkt oder bei 4 ° c). Hinweis: Eine zusätzliche 5 min. sanfte Zentrifugationsschritt (600 X g, 1 min) kann an dieser Stelle eingeführt werden, um alle verbleibenden Zellen in der Assay-Überstände entfernen.</li>
</ol>3. HTRF, Maßnahme Insulin-Sekretion
<ol><li>Verdünnen Sie Assay Überstände 01:10 in KRB (ohne BSA), vorzugsweise in klaren 96-Well Platten.</li>
	<li>Bereiten Sie der Insulin-Standardkurve für HTRF Insulin Assay (Tabelle 1).</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Standard-Stammlösung 500 ng/ml</td>
			<td>Verdünnungsreihen</td>
			<td>Arbeiten [Insulin] ng/ml</td>
		</tr>
<tr>
<td>STD 7</td>
			<td>30 µl Lager + 140 µl KRB</td>
			<td>150</td>
		</tr>
<tr>
<td>STD 6</td>
			<td>30 µl STD 7 + 45 µl KRB</td>
			<td>60</td>
		</tr>
<tr>
<td>5 STD</td>
			<td>30 µl STD 6 + 45 µl KRB</td>
			<td>24</td>
		</tr>
<tr>
<td>4 STD</td>
			<td>30 µl STD 5 + 45 µl KRB</td>
			<td>9.6</td>
		</tr>
<tr>
<td>3 STD</td>
			<td>30 µl STD 4 + 45 µl KRB</td>
			<td>3.84</td>
		</tr>
<tr>
<td>2 STD</td>
			<td>30 µl STD 3 + 45 µl KRB</td>
			<td>1,54</td>
		</tr>
<tr>
<td>1 STD</td>
			<td>30 µl STD 2 + 45 µl KRB</td>
			<td>0,61</td>
		</tr>
<tr>
<td>STD 0</td>
			<td>45 µl KRB</td>
			<td>0</td>
		</tr>
<tr>
<td colspan="3">Hinweis: STD Lager ist 500 ng/ml</td>
		</tr>
</tbody></table>Tabelle 1. Verdünnungsreihen die Standardkurve Insulin zu machen.
<ol start="3"><li>Die HTRF-Platte die Standardkurve Proben und der verdünnten Probe Überstände hinzufügen. Die Messung des ausgeschiedenen Insulin durch HTRF kann erfolgen, in eine 96-Well oder einem 384-Well-Plattenformat, unter Berücksichtigung, die das Assay-Volumen muss neu eingestellt werden. Einsatz 10 µL/Well Probe in 96-Well weiße Hälfte-Bereich Platten oder 5 µL/Well in einem weißen 384-Well geringem Volumen, Rundboden Teller (siehe Tabelle der Materialien).</li>
	<li>Antikörper-Mix in detektionspuffer vorzubereiten (siehe Tabelle der Materialien) in einem 1:2 Spender (Cryptate) / Akzeptor (XL-665) Verhältnis. Hinweis: Weitere spezifische Details über die HTRF Probe sind beim Hersteller erhältlich.</li>
	<li>Der Test auf 30 µL/Well (für eine 96-Well-Platte-Assay-Format) oder 15 µL/Well (für ein 384-Well-Platte-Assay-Format) Antikörper-Mischung hinzufügen.</li>
	<li>Die Dichtplatte und bei Raumtemperatur inkubieren.</li>
	<li>Platte nach 2 h, 4 h und/oder Übernachtung Inkubation mit Antikörpern, die mit der Platte-Leser und die entsprechenden HTRF optische Modul zu lesen (337 665 620 nm) (siehe Tabelle der Werkstoffe und Anweisungen des Herstellers). Die Integration Start-bei 60 µs und die Integrationszeit bei 400 µs. Einsatz 200 blinkt pro Bohrloch. Hinweis: Diese Parameter beruhten auf den Einsatz unserer speziellen Leser. Das Auslesen bei 620 nm und 665 nm variieren zwischen verschiedenen Instrumenten. Dies ist einer der Gründe, warum wir empfehlen die Verwendung der 665/620-Verhältnis. Bei der Berechnung dieses Verhältnis, Potentialdifferenzen von Leser zu Leser werden normalisiert und konsistente Werte unabhängig von dem Instrument zur Messung der HTRF bieten.</li>
</ol>(4) Datenanalyse und Normalisierung
<ol><li>Berechnen Sie die Insulin-Konzentrationen der Assay-Brunnen durch Extrapolation der ratiometrischen Fluoreszenz Lesungen (665 nm/620 nm), eine zweite Bestellung quadratische polynomische Kurve (Abbildung 2).</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57531/57531fig2.jpg"> Abbildung 2 : Insulin Standardkurve. Humaninsulin Lager der bekannten Konzentrationen wurde verwendet, um die Standardkurve Insulin zu generieren. Die daraus resultierende HTRF Verhältnisse (665 nm / 620 nm) wurden gegen die Insulin-Konzentrationen aufgetragen. Die Daten am besten passte, eine zweite Bestellung quadratische polynomische Kurve (R2 = 0.99996). Dies ist eine repräsentative Standardkurve. Fehlerbalken = SEM <a href="/files/ftp_upload/57531/57531fig2large.jpg" target="_blank">Klicken Sie bitte hier, um eine größere Version dieser Figur.</a>
<ol start="2"><li>Normalisieren Sie mit den hochgerechneten Daten wie ng/mL Insulin sezerniert (Insulin abgesondert in Reaktion auf die steigende Konzentrationen Liganden) auf den Mittelwert der % maximale Insulin-Sekretion Assay Brunnen (20 mM Glukose allein Zustand).</li>
	<li>Verwenden Sie die Kurve Passform (R2) aus einem einzigen Experiment um Intraplate Variation zu berechnen. Im Rahmen des Experiments R2 Einzelwerte von Intra experimentelle Duplikate, so dass die Berechnung der Standardfehler des Mittelwerts für die Kurvenanpassung abgeleitet.</li>
	<li>Verwenden Sie für die Bestimmung der interplate Variation die Daten aus mindestens drei einzelnen Experimente um den Standardfehler des Mittelwerts für die R-2 -Wert der kollektiven Kurve berechnen.</li>
</ol>

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Wir danken Nicolas Pierre (Cisbio Bioassays) für hilfreiche Ratschläge und Dr. Pierre Maechler (Universität Genf) für die großzügige Bereitstellung von INS-1E Zellen. Diese Arbeit wurde unterstützt durch die Finanzierung aus dem Department of Defense (Grant PR141292, Z.F.), John F. und Nancy A. Emmerling Fund of The Pittsburgh Foundation (Z.F.).

Die hier beschriebenen HTRF Insulin Assay bietet eine schnelle, effiziente System zur Messung der Insulin-Sekretion aus einem kultivierten Zell-basierte System. Einer der wichtigsten Vorteile bietet dieser Test eine geringe Hintergrundsignal aufgrund der hohen Signal-Rausch-Verhältnis. Darüber hinaus haben wir bestätigt, dass das HTRF-Signal über einen längeren Zeitraum hinweg stabil ist (> 24 h)7. Dennoch, da die Insulin-Bindung monoklonaler Antikörper schnell bindende Sättigung nach Zugab...

Die Verordnung des Energiestoffwechsels ist durch ein großen anabole Hormon Insulin verfeinert. Insulin ist synthetisiert und von pankreatischen Betazellen in Reaktion auf erhöhte extrazelluläre Blutzuckerwerte veröffentlicht. Das freigesetzte Insulin löst die Aufnahme von Glukose durch Insulin-empfindlichen Gewebe1,2. Physiologisch ist dies auf die Höhe der Glukosekonzentration nach einer Mahlzeit, gefolgt von Sekretion von Insulin, Glukoseaufnahme Regeln verbunden. Störungen in Glukose Homöostase zu metabolische Beeinträchtigungen bis hin zu Insulinresistenz führen und letztlich bei der Entstehung von Typ-2 Diabetes2,3,4.
Obwohl die Insulinsekretion ausgiebig untersucht wurde, bleiben seine Regulationsmechanismen schlecht verstanden. Ein kritischer Bereich Untersuchung wurde Identifikation von neuartigen Modulatoren der Insulin-Sekretion von Beta-Zellen5,6,7,8. Diese Studien erfordern ein besseres Verständnis der Kupplung Beziehung zwischen Stimulation von Glukose und Insulin-Sekretion. Daher ist die Fähigkeit, genau zu überwachen und zu quantifizieren, das Niveau der Glukose-stimulierten Insulinsekretion (GSIS) unerlässlich gewesen. Bisher wurden jedoch nur eine begrenzte Anzahl von Methoden zur Quantifizierung der GSIS mit Insulin-sezernierenden Zelllinien und/oder pankreatischen kleinen Inseln. Einer ist Radioimmunoassay (RIA), das Radioisotop-Tags Insulin und Antikörper verwendet. Die wichtigsten Einschränkungen dieses Ansatzes umfassen Fragen der Sicherheit durch die Handhabung und Entsorgung von radioaktiven Stoffen. Darüber hinaus ist diese Methode arbeitsintensiv, mit mehreren langen waschen und inkubationsschritte. Enzym-linked Immunosorbentprobe Assay (ELISA) ist ein weiterer Kosten- und arbeitsintensive Ansatz, der Antikörper gegen Insulin-Erkennung nutzt. Veränderung des Antikörpers Affinitäten und der Effizienz von Insulin zu erkennen sind limitierende Faktoren dieser Methode und die Reproduzierbarkeit der Ergebnisse beeinflussen können. ELISA weder RIA wurde für Hochdurchsatz-Experimente entwickelt. AlphaScreen ist ein homogenes Assay verwendet für die Erkennung und Messung der Insulin-Sekretion. AlphaScreen Technologie basiert auf der Umwandlung von ambient Sauerstoff in einem aufgeregt Sauerstoff Singulett-Zustand, die mit Chemilumineszenz Arten, was die Generation der Chemilumineszenz reagieren kann. Da die Probe homogen ist, entfallen viele der Waschschritte RIA und ELISA zugeordnet. Die Instabilität des Signals aufgrund der Art der Reaktion ist jedoch ein limitierender Faktor, der die Ablesung des Tests beeinflussen kann. () TR-Zange, entwickelt von Heyduk und Kollegen9, ist ein weiterer homogenen Ansatz zur Insulin-Messung basiert auf der Bindung von zwei separaten Antikörper gegen unterschiedliche Epitope auf die Insulin-Molekül. Die Antikörper sind jedes chemisch mit Doppel stranded DNA mit kurze ergänzende einzelne stranded DNA Überhänge. Bindung von Antikörpern an Insulin bringt sie zusammen und führt zu ein Doppel stranded DNA Duplex. Jeder Antikörper ist auch verbunden mit einer jeweiligen Spender oder Akzeptor Fluorophor, und der Verband der DNA Duplex vereint diese Fluorophore, Förster Resonanz Energietransfer (FRET) zu generieren. Eine mögliche Einschränkung der TR-Zange, liegt jedoch bei den Bund selbst. Die Unfähigkeit, schnell zerstreuen Hintergrundfluoreszenz während der Bund Reaktion führen zu relativ hohen Hintergrundfluoreszenz und ein low-Signal Rauschabstand innerhalb der Assay. Daher besteht ein Bedürfnis nach wie vor für eine zuverlässige, robuste und kostengünstige Assay zur Quantifizierung der GSIS in gewissem Sinne Hochdurchsatz.
Jüngste Fortschritte in der Biophysik gipfelten in der Entwicklung eines homogenen Zeitaufgelöste Fluoreszenz Energie Resonanz Transfer (HTRF) basierte Assays. Insbesondere während der Energietransfer innerhalb kann der Assay als FRET-basierte, genauer gesagt, bezeichnen HTRF Lumineszenz Energie Resonanz Transfer (LRET)10 abhängig von der nichtstrahlenden Übertragung von Energie zwischen Donor und Akzeptor Art11,12,13. Diese Unterscheidung ist wichtig, da der Zeitpunkt für eine Fluoreszenz oder Abschreckung basiert Bund Interaktion ist viel anders als bei LRET, obwohl die gleichen Arten von gating für Bund und LRET verwendet werden können. Darüber hinaus die Verwendung von seltenen Erden lanthanid Cryptate Verbindungen wie Europium oder Terbium Cryptate in HTRF produziert lange Fluoreszenz Halbwertszeiten12,14. Dies bietet den einzigartigen Vorteil der Einführung einer zeitlichen Verzögerung (µsec) zwischen Spender Erregung und die Messung der Emission von den Akzeptor (d. h. Zeitaufgelösten Assay). Diese zeitliche Verzögerung ermöglicht ausreichend Zeit für die Hintergrundfluoreszenz, vor der Messung der Akzeptor Emission Fluoreszenz zu zerstreuen. Infolgedessen das Auslesen ist frei von unspezifischen Fluoreszenz und damit ein hohes Signal-Rausch-Verhältnis erreicht ist (Abbildung 1). Darüber hinaus entfallen homogene Artder HTRF Waschschritten ungebundenen Arten, so dass des Tests viel schneller als ELISA oder RIA-basierten Methoden abwaschen.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57531/57531fig1.jpg"> Abbildung 1: Schematische des Mechanismus der Insulin-Erkennung HTRF. Zwei unabhängig generierten monoklonale Antikörper spezifisch erkennen und Binden an Insulin an verschiedenen Standorten. Diese Antikörper sind entweder dem Europium Cryptate Spender oder den XL665 Akzeptor konjugiert. Anregung des Spenders auf 337 nm Ergebnisse in Emission bei 620 nm. Die daraus resultierende Energieübertragung bewirkt XL665 emittieren bei einer längeren Wellenlänge, 665 nm. <a href="/files/ftp_upload/57531/57531fig1large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
Hier bieten wir ein detailliertes Protokoll für die Verwendung eines HTRF ausgerichteten Ansatzes an der GSIS aus INS-1E Zellen bestimmen, eine gut etablierte Insulin-sezernierenden Beta-Zellen abgeleitet Ratte Insulinom Zelle Zeile15. Darüber hinaus kann dieser Assay verwendet werden, für die Ermittlung der pharmakologischen Profils der molekularen Regulatoren der Insulin-Sekretion. Wir wenden dieses HTRF-basierte Insulin-Assay untersuchen Dopamin D2-Rezeptor Regulierung der GSIS mag. Immer mehr Studien haben gezeigt, dass der Neurotransmitter Dopamin ein wichtiger Regulator der GSIS8,16,17,18,19,20, ist 21 , 22. Dopamin wirkt GSIS in gewissem negativen Autokrine/Parakrine über Aktionen auf dem Dopamin D-2-Rezeptoren (D2, D3, D-4 -Rezeptoren) an der Oberfläche der Beta-Zellen der Bauchspeicheldrüse8 zum Ausdruck gebracht, wie , 16 , 19. mit dieser Assay, wir bestätigen Dopamin die Rolle als negativer Regulator der GSIS und nachweisen, dass die Dopamin D2-wie Rezeptor-Agonisten Bromocriptin und Quinpirole auch GSIS reduzieren.

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		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<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

Die Erkennung der Insulin-Sekretion ist entscheidend für erhellendes Mechanismen der regulierten Sekretion sowie wie in Studien des Stoffwechsels. Obwohl zahlreiche Insulin-Assays für Dekaden bestanden haben, hat die jüngsten Aufkommen der homogenen Zeitaufgelösten Förster Resonance Energy Transfer (HTRF) Technologie diese Messungen deutlich vereinfacht. Dies ist eine schnelle, kostengünstige, reproduzierbare und robuste optische Assay abhängig, Antikörper konjugiert, helle Fluorophore mit lang anhaltende Emission, die Zeitaufgelöste Förster Resonance Energy Transfer erleichtert. Darüber hinaus ist HTRF Insulin Erkennung für die Entwicklung von Hochdurchsatz-Screening-Assays. Hier verwenden wir HTRF, um Insulin-Sekretion in INS-1E Zellen, eine Ratte Insulinom abgeleitet Zell-Linie zu erkennen. Dies ermöglicht es uns, basalen Ebenen von Insulin und deren Veränderungen in Reaktion auf die Stimulation der Glukose zu schätzen. Darüber hinaus verwenden wir diese Insulin-Detection-System, um die Rolle von Dopamin als negativer Regulator der Glukose-stimulierten Insulinsekretion (GSIS) zu bestätigen. In ähnlicher Weise, anderen Dopamin D2-wie-Rezeptor-Agonisten, Quinpirole und Bromocriptin, GSIS Konzentration abhängig zu senken. Unsere Ergebnisse zeigen das Dienstprogramm des Formates HTRF Insulin-Test bei der Bestimmung der Rolle von zahlreichen Medikamenten im GSIS und ihre pharmakologische Profile.

Wir validiert unsere Insulin HTRF Assay durch die Erzeugung eines Insulin-Standardkurve mit gereinigtem Humaninsulin Standards der vordefinierten Konzentrationen (Abbildung 2). Generation der Standardkurve erlaubt uns, die ratiometrischen Fluoreszenz Lesungen zu extrapolieren und somit bestimmen die sekretierten Insulinspiegel in Reaktion auf die medikamentöse Behandlungen (Abbildung 2). Intraplate Variante für Kurvenanpassung ...

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

Hier präsentieren wir homogene Zeitaufgelöste FRET (HTRF) als effiziente Methode zum Schnellnachweis von Insulin aus den Zellen abgesondert.

Homogene Zeitaufgelösten Förster Resonanz Energie Transfer-basierten Test zur Erkennung von Insulin-Sekretion

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 Aufrufe.  University of Pittsburgh. Hier präsentieren wir homogene Zeitaufgelöste FRET (HTRF) als effiziente Methode zum Schnellnachweis von Insulin aus den Zellen abgesondert.

Serie: 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.

Produktion und Nachweis von reaktiven Sauerstoffspezies (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 ...

Forschung

Medizin

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.

Die Verbesserung der IV-Verabreichung von Insulin in einem Community Hospital

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.

Intraoperative Detektion von Subtle Endometriosis: A Novel Paradigm for Detection und Behandlung von Pelvic Pain mit dem Loss of Peritoneal Integrity-Zustellbare

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.

Cerenkov Lumineszenz Imaging (CLI) for Cancer Therapy Überwachung

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.

Cokultur Analyse der extrazellulären Protein Wechselwirkungen, die Insulinsekretion durch Betazellen des Pankreas

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.

Kombiniert intravitalen Mikroskopie und Kontrast-verstärkte Sonographie der Maus Megalosauridae Insulin-induzierte Vasodilatation und Muskel-Durchblutung zu untersuchen

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.

Kardiale Magnetresonanztomographie bei 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.

Studium der Hypothalamus Insulin-Signal an peripheren Glukoseintoleranz mit einem kontinuierlichen Medikament Infusion System in das Gehirn der Maus

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.

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.

Mechanismen, die Hormonausschüttung der Darm mit der isolierten durchblutet Ratte Dünndarm

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