Name: leprdb mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets
Uploaded: 2015-05-11T14:00:00
Description: Watch this Scientific Journal Video about Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets at JoVE.com

This work was supported by an Australian Research Council Grant DP110100642 (to PT) and National Health and Medical Research Council Grants APP1002520 and APP1059426 (to PT and HYG).

BEMÆRK: Alle nuværende eksperimenter blev udført i henhold til lokale dyr etik procedurer University of Queensland (godkendt af University of Queensland, Anatomisk Biosciences etiske komité). 1. Islet Isolation <ol><li> Reagens forberedelse <ol><li> Blanding af enzymer <ol><li> For bugspytkirtlen fordøjelse, skal du bruge en blanding af Liberase og collagenase type IV. Fortynd Liberase TL (thermolysin lav) 1 hætteglas med 5 mg med 26 ml DMEM (Dulbeccos Modified Eagle Medium). </li><l...

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	<li>Guariguata, L., 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=Global+estimates+of+diabetes+prevalence+for+2013+and+projections+for+2035.">Global estimates of diabetes prevalence for 2013 and projections for 2035.</a> Diabetes Research and Clinical Practice. 103, 137-149 (2014).</li><li>Ashcroft, F. M., Rorsman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+mellitus+and+the+beta+cell:+The+last+ten+years.">Diabetes mellitus and the beta cell: The last ten years.</a> Cell. 148, 1160-1171 (2012).</li><li>Weir, G. C., Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+stages+of+evolving+beta-cell+dysfunction+during+progression+to+diabetes.">Five stages of evolving beta-cell dysfunction during progression to diabetes.</a> Diabetes. 53, S16-S21 (2004).</li><li>Kjorholt, C., Akerfeldt, M. C., Biden, T. J., Laybutt, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hyperglycemia,+independent+of+plasma+lipid+levels,+is+sufficient+for+the+loss+of+beta-cell+differentiation+and+secretory+function+in+the+db/db+mouse+model+of+diabetes.">Chronic hyperglycemia, independent of plasma lipid levels, is sufficient for the loss of beta-cell differentiation and secretory function in the db/db mouse model of diabetes.</a> Diabetes. 54, 2755-2763 (2005).</li><li>Wang, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+type+2+diabetic+rodent+models.">Spontaneous type 2 diabetic rodent models.</a> Journal of Diabetes Research. , (2013).</li><li>Hummel, K. P., Dickie, M. M., Coleman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+a+new+mutation+in+mouse.">Diabetes a new mutation in mouse.</a> Science. 153, 1127-1128 (1966).</li><li>Do, O. H., Low, J. T., Gaisano, H. Y., Thorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretory+deficit+in+islets+from+db/db+mice+is+mainly+due+to+a+loss+of+responding+beta+cells.">The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells.</a> Diabetologia. 57, 1400-1409 (2014).</li><li>Low, J. T., 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=Glucose+principally+regulates+insulin+secretion+in+mouse+islets+by+controlling+the+numbers+of+granule+fusion+events+per+cell.">Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell.</a> Diabetologia. 56, 2629-2637 (2013).</li><li>Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T., Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+pore+dynamics+and+insulin+granule+exocytosis+in+the+pancreatic+islet.">Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet.</a> Science. 297, 1349-1352 (2002).</li><li>Low, J. T., 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=Insulin+secretion+from+beta+cells+in+intact+mouse+islets+is+targeted+towards+the+vasculature.">Insulin secretion from beta cells in intact mouse islets is targeted towards the vasculature.</a> Diabetologia. 57, 1655-1663 (2014).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomedical Engineering Online. 2, 13 (2003).</li></ol>

Den mest kritiske faktor i islet isolation er den oprindelige perfusion af pancreas; en under perfunderet bugspytkirtel resulterer i et betydeligt lavere ø-udbytte. Andre faktorer påvirker også isolation kvalitet, såsom fordøjelse tid og niveauet for omrystning som dels kunne kompensere for niveauet af perfusion. For eksempel vil en fuldt perfunderet pancreas skal inkuberes ved 37 ° C i ~ 18 min 30 sek mens forsigtig omrystning i modsætning en under fordøjet bugspytkirtlen kan kræve ~ 20 min fordøjelse med hå...

Den rolle, som beta-celle dysfunktion i sygdom er blevet bredt anerkendt 3,4. Cellelinjer såsom MIN6 og INS-1 er nyttige værktøjer til at forstå biologi beta celle adfærd. Men den fysiologiske kontrol af insulinsekretion finder sted inden for de Langerhanske øer. Disse øer indeholder tusinder af tætpakket betaceller, samt blodkar og andre endokrine celletyper. Dette miljø inden holmen påvirker insulinsekretion og vil sandsynligvis være vigtigt i diabetes. Derfor, for at forstå den fysiologiske kontrol af insulinsekretion, og patofysiologien af ​​sygdom, er det vigtigt at studere intakte øer. Islet isolation Levende menneskelige holme og især humane øer fra type 2-diabetikere er vanskelige at opnå. Desuden humane øer har begrænsede muligheder for eksperimentel molekylær manipulation. Forskere har derfor anvendes, erlader fra dyr og dyremodeller af type 2-diabetes. En sådan sygdomsmodel er db / db mus. Dette er en spontan mutation, modeller type 2-diabetes med en fænotype progression, der nøje paralleller human sygdom 5,6. Protokollen præsenteres her for holm isolation fra diabetiske db / db-mus har mange trin til fælles med andre grupper med nogle justeringer for bedre udbytte, rensning og forbedret ø overlevelse. 2 foton billedbehandling Den levende celle 2-foton-assayet beskrevet her giver forskere at kvantificere antallet og evaluere egenskaberne af enkelt insulin-holdige granuler fra mange celler af diabetisk 7 og vildtype holme 8,9.

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liberase TL 5mg</td>
			<td style="width:173px;">Roche</td>
			<td style="width:119px;">-5401020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase type IV-1g</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Histopaque 1077 500ml</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:119px;">10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 Medium (10X) 1L</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:119px;">R1383</td>
			<td>to prepare isolation media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 (1X) 500ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">21870-076</td>
			<td>to prepare cultured media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin 100ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>to prepare cultured media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum 500ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">10099-141</td>
			<td>to prepare cultured media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">11966-025</td>
			<td>to dilute the liberase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metamorph program</td>
			<td style="width:173px;">Molecular Devices, USA</td>
			<td style="width:119px;"></td>
			<td>to analyze the 2-photon images</td>
		</tr>
	</tbody>
</table>

leprdb mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets

Type 2-diabetes er en kronisk sygdom, der påvirker 382 mio mennesker i 2013, og forventes at stige til 592 millioner i 2035 1. I løbet af de sidste 2 årtier, rolle beta-celle dysfunktion hos type 2-diabetes har klart fastslået 2. Forskning fremskridt har krævet metoder til isolering af Langerhanske øer. Protokollen af holmen isolation præsenteres her deler mange fælles skridt med protokoller fra andre grupper, med nogle ændringer for at forbedre udbyttet og kvaliteten af isolerede øer fra både vildtype og diabetisk Lepr db (db / db) mus. En live-cell 2-foton imaging metode er da præsenteret som kan anvendes til at undersøge kontrollen med insulinsekretion inden øer.

Ø-udbytte og oprensning For en normal vildtype mus, er omkring 200 øer forventet. Sunde holme ser lyse, rund og har en glat kant. En over-fordøjet isolation batch sædvanligvis har små og fuzzy øer, mens en under-spaltet parti har færre øer og acinære celler knyttet øer (figur 3). For diabetiske db / db-mus, ø-udbytte og udseende afhænger af sygdomsprogression med bedre glykæmisk mus har større, lysere og mere øer (op til 300 øer) sammenlignet med dårligere g...

Watch this Scientific Journal Video about Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets at JoVE.com

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Mus Model af type 2 diabetes: Pancreas Islet Isolering og live-cell 2-Photon Imaging af intakte Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

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Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

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