Die hier beschriebene Arbeit wurde durch das US Department of Veterans Affairs Biomedical Laboratory Forschung und Entwicklung BX001686-1A1 und VA Rehabilitation Forschung und Entwicklung finanziert.

1. Überzug und Pflege von Zellkultur <ol><li> Warme Dulbecco modifiziertem Eagle Medium (DMEM), Kulturmedien (DMEM + 10% FBS + 1% Penicillin / Streptomycin / Neomycin) bis 37 ° C </li><li> Erhalten Lager von gefrorenen Zellen A12 von -80 ° C </li><li> Schnell auftauen Zellen in 37 ° C Wasserbad; einmal aufgetaut, sanft Pipette Zellen auf eine 75 cm 2 entlüftet Kulturflasche übertragen und 10 ml der Kulturmedien. </li><li> Inkubieren Kolben über Nacht in 5% CO 2 Inkubator bei 37 ° C. Saugen Medien nach 24 Stunden und ersetzen mit frischen Medien. Weiterhin wachsende Zellen, bis 70-80% Konfluenz erreicht ist (2-3 Tagen Wachstum). </li></ol> 2. Zählen und Überzug Cells <ol><li> Warmen DMEM und 1x-Trypsin-EDTA-Lösung auf 37 ° C </li><li> Aspirat Medien von Zellen und Waschen zweimal mit steriler phosphatgepufferter Salzlösung (PBS). </li><li> Gib 500 ul Trypsin-Lösung und Inkubation bei 37 ° C für 2-5 min. </li><li> TrennenZellen von der Flasche mit einem Schaber und Zellen zu suspendieren in 5 ml Medium. Pass-Zellen durch Sieb und durch Zell Sieb in einen sauberen 50-ml-Tube. Möglicherweise müssen Sie Zellen durch Sieb mehr als einmal passieren, aber nicht mehr als drei Mal zu wiederholen. </li><li> Zählen von Zellen mit einer Zählkammer nach Menge der Zellen zu bestimmen, die Kultur in einer 96-well klar unten schwarz oder weiß ummauerten Platte in einem Gesamtvolumen von 100 ul Medium Saatgut 6.000 Zellen / gut. Inkubieren Platte in 5% CO 2 bei 37 ° C für 24 Stunden. </li></ol> 3. Bestimmung Zelllebensfähigkeit und Caspase-3/7 Aktivität <ol><li> PA übertragen, um eine sterile 5 ml Glasfläschchen und löslich in 100% Dimethylsulfoxid (DMSO). Verdünnen Lager PA-Lösung 1:10 in DMSO und fügen Sie es DMEM vorgewärmt, um eine endgültige Arbeitskonzentration von 0,1 mM PA erhalten. Für Kontrollmedien, fügen die gleiche Konzentration an DMSO, die Medien, die PA hinzugefügt. </li><li> Entfernen Sie die Medien in jede Vertiefung und ersetzen mit 50 ul Medien + / -PA. Inkubiere die Platte für 2 Stunden in 5% CO 2 bei 37 ° C. Daraufhin 5 ul Resazurin Reagenz und Inkubation für 10 min bei Raumtemperatur. </li><li> Mit einem Multimode-Mikroplatten-Reader, Platten Fluoreszenz (560 EX / 590 EM) auf die Lebensfähigkeit der Zellen zu messen. Die Ergebnisse sind als relative Fluoreszenzeinheiten (RFU) dargestellt. </li><li> Auf die gleiche Platte, fügen Sie 55 ul von Caspase-Reagenz und bei Raumtemperatur für 2 Stunden. </li><li> Auch mit Hilfe der Mikroplatten-Reader, Platten Lumineszenz Caspase-3/7 Aktivität zu messen. Die Ergebnisse sind als relative Lumineszenz-Einheiten (RLU) dargestellt, mit Lumineszenz direkt proportional zur Caspase-3/7 Aktivität. </li><li> Um normalisieren Caspase-3/7 Aktivität Zellzahl, teilen die Lebensfähigkeit der Zellen (RFU) durch Caspase-3/7 Aktivität (RLU). </li></ol>

<ol>
	<li>Akil, L., Ahmad, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+obesity+and+cardiovascular+diseases+in+four+southern+states+and+Colorado.">Relationships between obesity and cardiovascular diseases in four southern states and Colorado.</a> J. Health Care Poor Underserved. 22, 61-72 (2011).</li><li>Posey, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+proinflammatory+lipid+accumulation,+inflammation,+and+insulin+resistance+in+rats+fed+a+high-fat+diet.">Hypothalamic proinflammatory lipid accumulation, inflammation, and insulin resistance in rats fed a high-fat diet.</a> Am. J. Physiol. Endocrinol. Metab. 296, 1003-1012 (2009).</li><li>Moraes, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-fat+diet+induces+apoptosis+of+hypothalamic+neurons.">High-fat diet induces apoptosis of hypothalamic neurons.</a> PloS one. 4, (2009).</li><li>Mayer, C. M., Belsham, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitate+attenuates+insulin+signaling+and+induces+endoplasmic+reticulum+stress+and+apoptosis+in+hypothalamic+neurons:+rescue+of+resistance+and+apoptosis+through+adenosine+5'+monophosphate-activated+protein+kinase+activation.">Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation.</a> Endocrinology. 151, 576-585 (2010).</li><li>Benoit, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitic+acid+mediates+hypothalamic+insulin+resistance+by+altering+PKC-theta+subcellular+localization+in+rodents.">Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents.</a> J. Clin. Invest. 119, 2577-2589 (2009).</li><li>Thaler, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+is+associated+with+hypothalamic+injury+in+rodents+and+humans.">Obesity is associated with hypothalamic injury in rodents and humans.</a> J. Clin. Invest. 122, 153-162 (2012).</li><li>Williams, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+dysfunction+in+obesity.">Hypothalamic dysfunction in obesity.</a> Proc. Nutr. Soc. 71, 521-533 (2012).</li><li>Belsham, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+phenotypic+array+of+hypothalamic+neuronal+cell+models+to+study+complex+neuroendocrine+disorders.">Generation of a phenotypic array of hypothalamic neuronal cell models to study complex neuroendocrine disorders.</a> Endocrinology. 145, 393-400 (2004).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+cell+viability+and+proliferation+in+hydrogel-encapsulated+system+by+resazurin+assay.">Monitoring of cell viability and proliferation in hydrogel-encapsulated system by resazurin assay.</a> Appl. Biochem. Biotechnol. 162, 1996-2007 (2010).</li><li>Butterick, T. A., Nixon, J. P., Billington, C. J., Kotz, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orexin+A+decreases+lipid+peroxidation+and+apoptosis+in+a+novel+hypothalamic+cell+model.">Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model.</a> Neurosci. Lett. 524, 30-34 (2012).</li><li>Riss, T. L., Moravec, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+multiple+assay+endpoints+to+investigate+the+effects+of+incubation+time,+dose+of+toxin,+and+plating+density+in+cell-based+cytotoxicity+assays.+Assay+Drug+Dev.">Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev.</a> Technol. 2, 51-62 (2004).</li><li>Niles, A. L., Moravec, R. A., Riss, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+viability+and+cytotoxicity+testing+and+same-well+multi-parametric+combinations+for+high+throughput+screening.">In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening.</a> Curr. Chem. Genomics. 3, 33-41 (2009).</li><li>Steffen, W., Linck, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+immunoblot:+a+sensitive+technique+to+stain+proteins+and+detect+multiple+antigens+on+a+single+two-dimensional+replica.">Multiple immunoblot: a sensitive technique to stain proteins and detect multiple antigens on a single two-dimensional replica.</a> Electrophoresis. 10, 714-718 (1989).</li><li>Gingrich, J. C., Davis, D. R., Nguyen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+and+quantitation+of+proteins+on+western+blots+using+fluorescent+probes.">Multiplex detection and quantitation of proteins on western blots using fluorescent probes.</a> Biotechniques. 29, 636-642 (2000).</li><li>Riss, T. L., Moravec, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+multiple+assay+endpoints+to+investigate+the+effects+of+incubation+time,+dose+of+toxin,+and+plating+density+in+cell-based+cytotoxicity+assays.">Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays.</a> Assay Drug Dev. Technol. 2, 51-62 (2004).</li><li>Smyth, P. G., Berman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+of+apoptosis:+methods+for+elucidating+the+mechanism+of+apoptotic+cell+death+from+the+nervous+system.">Markers of apoptosis: methods for elucidating the mechanism of apoptotic cell death from the nervous system.</a> Biotechniques. 32, 648-650 (2002).</li><li>Lavrik, I. N., Golks, A., Krammer, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases:+pharmacological+manipulation+of+cell+death.">Caspases: pharmacological manipulation of cell death.</a> J. Clin. Invest. 115, 2665-2672 (2005).</li></ol>

Die Autoren haben keine Konflikte offen zu legen.

Das Multiplex-Assay ist ein gut anerkanntes Verfahren, die von Wissenschaftlern in zahlreichen Anwendungen wie PCR-Mikroarrays, Immun und andere Protein-basierten Nachweismethoden 13, 14 verwendet wurde. Kürzlich, Multiplexing-Tests haben sich in den in-vitro-Platte-basierte Experimente zunehmend genutzt und haben als eine genaue Methode zur Bewertung der Zytotoxizität validiert und Lebensfähigkeit 12. In dem obigen Protokoll zeigen wir die Wirksamkeit der Multiplex-Fluoreszenz-und Lumi...

Ernährung reich an gesättigten Fettsäuren, wie Palmitinsäure (PA) haben, Fettleibigkeit und andere Begleiterkrankungen, einschließlich Herz-Kreislauf-Erkrankungen und Diabetes 1, 2 verbunden worden. Hohe Fett-Diäten haben auch gezeigt, dass oxidativer Stress, Apoptose und neuronale Degeneration im Hypothalamus, einem wichtigen Regulator der Appetit und Energieaufwand 3-7 erhöhen. Das Verständnis der Mechanismus, durch den hohen Fett-Diät-Exposition induziert Hypothalamus Dysregulation ist somit wichtig für die Entwicklung von pharmakologischen Behandlungen für Übergewicht. , Die zellulären Mechanismen, durch die Nahrungsfett beeinflusst die neuronale Funktion bleibt jedoch unklar. Ein besseres Verständnis, wie Fette, wie PA könnte Einsetzen der Hypothalamus-apoptotische Signalwege auslösen, ist ein notwendiger erster Schritt hin zu diesem Ziel. Das Ziel dieses Artikels ist es, ein Multiplex-Assays für in-vitro-Tests der neuronalen Antwort auf PA Exposition, für den Einsatz in Studien von h entwickelt beschreibenypothalamic Neurodegeneration. Wir liefern eine detaillierte Beschreibung eines in vitro 96-well Format Multiplex-Assay zur Messung der Caspase 3/7 Aktivität pro Gesamtzahl der Zellen in einem differenzierten verewigt erwachsenen Maus Hypothalamus-Zelllinie (A12 bezeichnet) nach oxidativer Herausforderung mit PA-8. Kurz gesagt, wird die Lebensfähigkeit der Zellen nach PA Herausforderung über ein Resazurin basierte Assays bestimmt. Resazurin ist eine Zelle durchlässige Verbindung, die enzymatische Reduktion in metabolisch aktiven Zellen unterzogen, um ein Verfahren, dachte über die Mitochondrien 9 auftreten. Lebensfähige Zellen kontinuierlich Resazurin zu Resorufin zu konvertieren, wodurch ein Fluoreszenzsignal proportional zur Anzahl der lebensfähigen Zellen. Caspase-3/7 Aktivität wird dann unter Verwendung eines DEVD-basierten Assay lumogenic. DEVD eine Aminosäure-Sequenz (Asp-Glu-Val-Asp) von Caspase-3 gespalten wird. Wenn diese Sequenz mit einer lumogenic Substanz bei Aktivierung des intrazellulären Caspase-3/7 und anschließende Spaltung gekoppelt ist,DEVD von Substrat wird die lumineszierende Produkt freigesetzt. Diese Reaktion ist proportional zu Caspase-Aktivität und damit zur Induktion von Apoptose. Da tote Zellen nicht Caspase zu erzeugen, ist Caspase-3/7 Aktivität von Natur aus vergänglich; Analyse daher sollte zwischen 30 min bis 4 Stunden nach der Herausforderung, je nach Wirksamkeit der Zell Stressor abgeschlossen werden. Zelllebensfähigkeit ist umgekehrt proportional zu der Caspase-3/7 Aktivität und kann verwendet werden, um zu bestimmen Mechanismen des Zelltods. Beispielsweise wurde dieses Verfahren bisher verwendet worden, um diese Vorbehandlung mit dem Peptidhormon Orexin verringert die Apoptose in Zellen des Hypothalamus mit Wasserstoffperoxid in Frage, was darauf hindeutet, dass die Mechanismen durch diese Behandlung nicht betroffen sind beim Schutz gegen oxidative Schädigung 10 wichtig zu zeigen. Es ist wichtig zu beachten, diese Tests sind Zelllinie-und gewebeabhängig, wie sie verlassen sich auf die mitochondriale Aktivität Assay-Reagenzien zu reduzieren. Dieses Protokoll wurde für erwachsene Maus Hypothalamus (A12) Zellen optimiert; jedochbeschriebenen Verfahren geändert werden, um im Rahmen der Forschung ähnlich zu passen. Multiplexing-Tests aus einer einzigen Kultur und bietet einen Vorteil gegenüber der traditionellen Methode der Durchführung einzelner Tests aus mehreren Gründen. Neben der Zeitersparnis, Zellproben, Reagenzien und Kultur kann auch Multiplex-Assays Wissen des Zellüberlebens und Tod, stellen interne Kontrollen, und die Notwendigkeit für Wiederholungsversuche 11, 12. Alternative Methoden haben sich auf Western-Blots oder ELISAs, die zuverlässig sind Tests verlassen, sind aber teuer und zeitaufwändig (1-2 Tage), insbesondere bei der Verwendung eines 96-Well-Format. Außer der Zeit, die zur Kultivierung von Zellen für die Multiplexassay, ist die Gesamtzeit von weniger als 3 Stunden. Während dieses Protokoll wurde für die Verwendung mit A12-Zellen optimiert wurde, kann es für den Einsatz in anderen Modellen verändert werden, ohne dass dabei Faktoren, die die Integrität der Zelle beeinflussen können. AbschreckenBergbau, wenn dieses Protokoll für eine Reihe von Experimenten funktionieren würde, hängt von der Anzahl der Proben oder experimentellen Bedingungen und über geplante nachgeschalteten Experimenten.

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Adult Mouse Hypothalamus Cell Line mHypoA-1/2&#160;</td>
			<td style="width:137px;">Cellutions Biosystems Inc.</td>
			<td style="width:151px;">CLU172</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">10313-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Fetal bovine serum&#160;</td>
			<td style="width:137px;">PAA Labs</td>
			<td style="width:151px;">A15-751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Penicillin/Streptomycin</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Palmitic Acid</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">P0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dimethy sulfoxide&#160;</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;"> 
			PrestoBlue Cell Viability Reagent</td>
			<td style="width:137px;">Invitrogen</td>
			<td>A13262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Caspase-Glo 3/7 Assay Systems</td>
			<td style="width:137px;">Promega</td>
			<td style="width:151px;">G8091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Equipment</td>
			<td style="width:137px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:268px;">96W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile</td>
			<td style="width:137px;">Thermo Fisher Scientific</td>
			<td style="width:151px;">165305</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">SpectraMax M5 Multi-Mode Microplate</td>
			<td style="width:137px;">Molecular Devices</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model

Die Fähigkeit, Tests in Studien von komplexen zellulären Mechanismen Multiplexen beseitigt die Notwendigkeit für wiederholte Versuche, stellt die internen Kontrollen und verringert Abfall Kosten und Reagenzien. Hier beschreiben wir die Optimierung eines Multiplex-Assays, um die Apoptose zu beurteilen Ein Palmitinsäure (PA) Herausforderung in einem in vitro-Modell des Hypothalamus, unter Verwendung von sowohl Fluoreszenz-und Lumineszenz-basierten Assays, um lebensfähige Zellzahlen und Caspase-3/7-Aktivität in einer 96-Well-Messung Mikrotiterplatten-Format. Nach PA Challenge wurden lebensfähigen Zellen durch eine Resazurin-basierte Fluoreszenztest bestimmt. Caspase-3/7 Aktivität wurde dann unter Verwendung eines luminogenen Substrat, DEVD und normalisiert auf die Zellzahl. Dieses Multiplexing Assay ist eine nützliche Technik zum Bestimmen Änderung Caspase-Aktivität nach einer apoptotischen Stimulus, wie z. B. gesättigte Fettsäure Herausforderung. Die gesättigten Fettsäuren PA Hypothalamus oxidativen Stress und Apoptose zu erhöhen, was die potenziellen Einfuhrnz der Assays wie hier beschrieben in die Untersuchung der Beziehung zwischen gesättigten Fettsäuren und neuronalen Funktion.

Das Protokoll oben beschrieben, führt Multiplexen zwei Assays zur Bestimmung Mechanismen des Zelltods. Fig. 1 zeigt eine Übersicht über das Protokoll zur Zelllebensfähigkeit und Caspase-3/7-Aktivität zu bestimmen. Caspase-Aktivität wurde in Zellen, die mit PA nach 2 h Inkubation (Fig. 2A und Tabelle 1) in Frage deutlich erhöht. Verlust der Zellmembranintegrität ist eine morphologische Veränderung mit der Induktion von Apoptose, die nach 2 Stunden Einwirkung PA ...

Watch this Scientific Journal Video about Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model at JoVE.com

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

Multiplex-Assays können nützliche Informationen für grundlegende zelluläre Mechanismen und beseitigen Abfälle von Reagenzien und unnötig wiederholende Experimenten. Wir beschreiben hier ein Multiplex-Caspase-3/7 Aktivitätstest unter Verwendung von fluoreszierenden und lumineszier-basierte Verfahren, um die Lebensfähigkeit der Zellen in einem in vitro Modell Hypothalamus folgenden oxidativen Herausforderung mit Palmitinsäure bestimmen.

Verwendung eines Caspase Multiplexing-Test zur Apoptose in einer Zelle Hypothalamus Modell ermitteln

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

use-caspase-multiplexing-assay-to-determine-apoptosis-hypothalamic

Research

JoVE Journal

Neuroscience

14.9K Aufrufe.  Minneapolis Veterans Affairs Health Care System.  Multiplex-Assays können nützliche Informationen für grundlegende zelluläre Mechanismen und beseitigen Abfälle von Reagenzien und unnötig wiederholende Experimenten. Wir beschreiben hier ein Multiplex-Caspase-3/7 Aktivitätstest unter Verwendung von fluoreszierenden und lumineszier-basierte Verfahren, um die Lebensfähigkeit der Zellen in einem in vitro Modell Hypothalamus folgenden oxidativen Herausforderung mit Palmitinsäure bestimmen. 

Serie: Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Optic Nerve Durchtrennung: Ein Modell des Adult Neuron Apoptose in das zentrale Nervensystem

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Forschung

Neurowissenschaften

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural-Colony Forming Zell-Assay: Ein Test zur Bona Fide neuralen Stammzellen aus neuralen Vorläuferzellen Discriminate

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

Überwachung Cleaved Caspase-3 Aktivität und Apoptose von Immortalized Oligodendrozyten mit Live-cell Imaging und Cleaveable Fluorogenic-Farbstoff-Substrate Nach Kalium-induzierten Membrandepolarisation

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

Funktionelle Abfrage der Erotik Hypothalamus Neurogenese mit Brennradiologische Hemmung

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

A Novel leichte Schäden Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafisch

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Mikroglia als Surrogate Biosensor Nanoparticle Neurotoxicity zu ermitteln

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Die Verwendung von Trace Eyeblink klassische Konditionierung hippocampale Funktionsstörung in einem Rattenmodell der Fetal Alcohol Spectrum Disorders bewerten

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

Leben-Zelle Messung der Geruchsstoff Rezeptor Aktivierung mit einem Echtzeit-cAMP-Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Ein Protokoll für die Messung von Cue Reaktivität in einem Rattenmodell der Kokain-Gebrauch-Störung

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Ein Neurosphäre Assay auszuwertende endogenen neurale Stammzellen Aktivierung in einem Mausmodell der minimalen Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...