Name: Forfatter Spotlight: Vurdering af mitofagiflux i β-celler i bugspytkirtlen ved hjælp af effektive og robuste komplementære tilgange
Uploaded: 2023-09-15T14:40:00
Description: Watch this Scientific Journal Video about Assessment of Mitophagy Flux in Pancreatic β-Cells Using Effective and Robust Complementary Approaches at JoVE.com

E.L-D. anerkender støtte fra NIH (T32-AI007413 og T32-AG000114). SAS anerkender støtte fra JDRF (COE-2019-861), NIH (R01 DK135268, R01 DK108921, R01 DK135032, R01 DK136547, U01 DK127747), Department of Veterans Affairs (I01 BX004444), Brehm-familien og Anthony-familien.

De dyreforsøg, der præsenteres i denne protokol, blev gennemgået og godkendt af University of Michigan Institutional Animal Care and Use Committee. Tyve uger gamle mandlige C57BL / 6J mus, på enten en 15-ugers regelmæssig fedtdiæt (RFD) eller fedtfattig diæt (HFD), blev brugt til denne undersøgelse.
1. Vurdering af mitofagi, via den farvestofbaserede MtPhagy-tilgang (metode 1)
<ol><li>Forberedelse og behandling af museøen<ol><li>Udfør økultur og valinomycinekspone...

Kvantitativ analyse af mitofagi i β-celler fra murine bugspytkirtel

<ol>
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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=An+intrinsically+disordered+protein+region+encoded+by+the+human+disease+gene+CLEC16A+regulates+mitophagy.">An intrinsically disordered protein region encoded by the human disease gene CLEC16A regulates mitophagy.</a> Autophagy. 19 (2), 525-543 (2023).</li><li>Sun, N., Malide, D., Liu, J., Rovira, I. I., Combs, C. A., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-based+imaging+method+to+measure+in+vitro+and+in+vivo+mitophagy+using+mt-Keima.">A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima.</a> Nature Protocols. 12 (8), 1576-1587 (2017).</li><li>Zhao, J., Bertoglio, B. A., Gee, K. R., Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zinc+indicator+FluoZin-3+is+not+perturbed+significantly+by+physiological+levels+of+calcium+or+magnesium.">The zinc indicator FluoZin-3 is not perturbed significantly by physiological levels of calcium or magnesium.</a> Cell Calcium. 44 (4), 422-426 (2008).</li><li>Da Silva Xavier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cells+of+the+Islets+of+Langerhans.">The Cells of the Islets of Langerhans.</a> Journal of Clinical Medicine. 7 (3), 54 (2018).</li><li>Thapa, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+&#946;-cell+function+using+a+zinc-responsive+MRI+contrast+agent+may+identify+first+responder+islets.">Imaging &#946;-cell function using a zinc-responsive MRI contrast agent may identify first responder islets.</a> Frontiers in Endocrinology. 12, 809867 (2022).</li><li>Iwashita, H., 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=Live+cell+imaging+of+mitochondrial+autophagy+with+a+novel+fluorescent+small+molecule.">Live cell imaging of mitochondrial autophagy with a novel fluorescent small molecule.</a> ACS Chemical Biology. 12 (10), 2546-2551 (2017).</li><li>Crowley, L. C., Christensen, M. E., Waterhouse, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+transmembrane+potential+by+TMRE+staining.">Measuring mitochondrial transmembrane potential by TMRE staining.</a> Cold Spring Harbor Protocols. 2016 (12), 087361 (2016).</li><li>Klein, B., W&#246;rndl, K., L&#252;tz-Meindl, U., Kerschbaum, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation+of+intracellular+K(+)+homeostasis+with+valinomycin+promotes+cell+death+by+mitochondrial+swelling+and+autophagic+processes.">Perturbation of intracellular K(+) homeostasis with valinomycin promotes cell death by mitochondrial swelling and autophagic processes.</a> Apoptosis. 16 (11), 1101-1117 (2011).</li><li>Ji, Y., 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=Toll-like+receptors+TLR2+and+TLR4+block+the+replication+of+pancreatic+&#946;+cells+in+diet-induced+obesity.">Toll-like receptors TLR2 and TLR4 block the replication of pancreatic &#946; cells in diet-induced obesity.</a> Nature Immunology. 20 (6), 677-686 (2019).</li><li>Sauvat, 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=Quantification+of+cellular+viability+by+automated+microscopy+and+flow+cytometry.">Quantification of cellular viability by automated microscopy and flow cytometry.</a> Oncotarget. 6 (11), 9467-9475 (2015).</li><li>Liu, Y. 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=Mt-Keima+detects+PINK1-PRKN+mitophagy+in+vivo+with+greater+sensitivity+than+mito-QC.">Mt-Keima detects PINK1-PRKN mitophagy in vivo with greater sensitivity than mito-QC.</a> Autophagy. 17 (11), 3753-3762 (2021).</li><li>Mauro-Lizcano, M., 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=New+method+to+assess+mitophagy+flux+by+flow+cytometry.">New method to assess mitophagy flux by flow cytometry.</a> Autophagy. 11 (5), 833-843 (2015).</li></ol>

Denne protokol beskrev to komplementære metoder til kvantificering af mitofagiflux i dissocierede primære museøer. Ved hjælp af mt-Keima-metoden blev en stigning i mitofagien kvantificeret som et øget forhold mellem sure (561 nm) / neutrale (405 nm) celler, mens i MtFagy-metoden blev øget mitofagiflux kvantificeret som en stigning i FluozinhøjMtPhagyhøjTMRElavcellepopulation . Disse metoder giver mulighed for hurtige, kvantitative og β-cellespecifikke vurderinger af mitofagiflux....

Pancreas β-celler producerer og udskiller insulin for at imødekomme metaboliske krav, og β-celle dysfunktion er ansvarlig for hyperglykæmi og diabetes debut i både type 1 og type 2 diabetes. β-celler kobler glukosemetabolisme med insulinsekretion via mitokondriel energetik og metabolisk output, som afhænger af en reserve af funktionel mitokondriemasse 1,2,3. For at opretholde optimal β-cellefunktion er β-celler afhængige af mitokondrie kvalitetskontrolmekanismer for at fjerne gamle eller beskadigede mitokondrier og bevare funktionel mitokondriemasse4. Selektiv mitokondriel autofagi, også kendt som mitofagi, er en nøglekomponent i mitokondriekvalitetskontrolvejen.
Vurderinger af mitofagi, i levende celler, er ofte afhængige af ændringer i mitokondriel pH, der opstår under mitofagi. Mitokondrier har en let alkalisk pH, og sunde mitokondrier befinder sig normalt i den pH-neutrale cytosol. Under mitofagi, beskadigede eller dysfunktionelle mitokondrier er selektivt indarbejdet i autofagosomer og til sidst ryddet inden for sure lysosomer5. Flere in vivo transgene mitofagi-reportermusemodeller, såsom mt-Keima6, mitoQC7 og CMMR8, samt transfektable mitofagiprober, såsom Cox8-EGFP-mCherry-plasmid9, bruger denne pH-ændring til at give kvantitative vurderinger af mitofagi. Anvendelse af transgene mus, der udtrykker mt-Keima pH-følsom dual-excitation ratiometrisk sonde, er blevet optimeret til mitofagi-vurderinger i øer og β-celler via flowcytometri10,11. Forholdet mellem sure og neutrale mt-Keima-bølgelængdeemissioner (forholdet mellem sur 561 nm og neutral 480 nm excitation) kan bruges til robust kvantificering af mitofagien 6,12.
Denne protokol beskriver en optimeret tilgang til vurdering af mitofagiflux i primære øer og β-celler isoleret fra mt-Keima transgene mus10,11. Mens mt-Keima er en meget følsom sonde, kræver den enten komplicerede dyreavlsordninger eller transfektion af celler, hvilket ofte kan være udfordrende, når man arbejder i kombination med andre genetiske modeller eller med primære menneskelige øer. Derudover kan brugen af flere fluorescenslasere og detektorer til at identificere neutrale og sure cellepopulationer begrænse den kombinatoriske brug af andre fluorescerende reportere.
For at overvinde disse udfordringer beskriver denne protokol også en komplementær, enkelt fluorescerende kanal, farvestofbaseret metode til robust påvisning af mitofagi, i β-celler fra isolerede museøer. Denne tilgang, kaldet Mtfagy-metoden, anvender en kombination af tre cellegennemtrængelige farvestoffer til at vælge β-celler, kvantificere cellepopulationerne, der aktivt gennemgår mitofagi, og vurdere mitokondriemembranpotentiale (MMP eller Δψm) samtidigt.
Det første af disse farvestoffer er Fluozin-3-AM, en cellegennemtrængelig Zn2+ indikator med en Ex/Em 494/516 nm13. Museøer omfatter en heterogen population af funktionelt forskellige celler, herunder α-, β-, δ- og PP-celler. β-celler udgør ca. 80% af cellerne i museøen og kan skelnes fra andre øcelletyper på grund af deres høje Zn2+ koncentration inden for insulingranulat14,15, hvilket muliggør identifikation af β-celler som Fluozin-3-AMhøj population. MtPhagy-farvestoffet, et pH-følsomt farvestof, der immobiliseres på mitokondrier via en kemisk binding og udsender svag fluorescens, anvendes også i denne protokol16. Ved mitofaginduktion inkorporeres beskadigede mitokondrier i det sure lysosom, og Mtfagy-farvestoffet øger dets fluorescensintensitet inden for det lave pH-miljø (Ex/Em 561/570-700 nm).
Derudover anvendes tetramethylrhodamin, ethylester (TMRE), til vurdering af MMP. TMRE er et cellegennemtrængeligt positivt ladet farvestof (Ex/Em 552/575 nm), der bindes af sunde mitokondrier på grund af den relative negative ladning, der opretholdes af deres membranpotentiale17. Beskadigede eller usunde mitokondrier spreder deres membranpotentiale, hvilket resulterer i nedsat evne til at sekvestre TMRE. Ved hjælp af disse farvestoffer sammen kan β-celler, der gennemgår mitofagi, identificeres som FluozinhøjMtPhagyhøjTMRElav befolkning via flowcytometri. Da mitofagien er en dynamisk snarere end statisk proces, blev denne protokol optimeret til at vurdere mitofagiflux ved anvendelse af valinomycin, en K +-ionofor, der inducerer mitofagi, efter spredning af MMP18. Sammenligning af mitofagi, i nærvær og fravær af valinomycin, muliggør vurdering af mitofagiflux i forskellige prøvegrupper.
Den farvestofbaserede karakter af den nuværende tilgang gør det muligt at ekstrapolere den til menneskelige øer og andre vanskeligt transfekterede celletyper og omgår behovet for komplicerede dyreavlsordninger, i modsætning til mt-Keima-protokollen. Det overordnede mål med denne protokol er at kvantificere mitofagien i β-celler på enkeltcelleniveau via to uafhængige flowcytometribaserede metoder. Samlet set beskriver denne protokol to kraftfulde og komplementære metoder, der giver mulighed for både præcision og fleksibilitet i den kvantitative undersøgelse af mitokondriel kvalitetskontrol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune NxT Flow Cytometer</td>
			<td>Thermofisher Scientific</td>
			<td>A24858</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermofisher Scientific</td>
			<td>D1306</td>
			<td>DAPI reconstituted in ddH2O to reach 0.2 &#181;g/mL stock</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>317275</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatty Acid Free heat shock BSA powder</td>
			<td>Equitech</td>
			<td>BAH66</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gemini Bio</td>
			<td>900-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluozin-3AM&#160;</td>
			<td>Thermofisher Scientific&#160;</td>
			<td>F24195</td>
			<td>100 &#956;g Fluozin-3AM powder reconstituted in 51 &#956;L DMSO and 51 &#956;L Pluronic F-127 to reach 1 mM stock.&#160;</td>
		</tr>
		<tr>
			<td>Gibco&#160;RPMI 1640 Medium</td>
			<td>Fisher Scientific</td>
			<td>11-875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>MtPhagy dye</td>
			<td>Dojindo</td>
			<td>MT02-10</td>
			<td>5 &#956;g MtPhagy powder reconstituted with 50 &#956;L DMSO to reach 100 &#956;M stock.&#160;</td>
		</tr>
		<tr>
			<td>MtPhagy dye</td>
			<td>Dojindo</td>
			<td>MT02-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100x)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>1x Solution used in procotol by diluting 1:10 in ddH2O</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10x</td>
			<td>Fisher Scientific</td>
			<td>BP399-20</td>
			<td>1x Solution used in procotol by diluting 1:10 in ddH2O</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100x)</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td>5 &#956;g MtPhagy powder reconstituted with 50 &#956;L DMSO to reach 100 &#956;M stock.&#160;</td>
		</tr>
		<tr>
			<td>TMRE [Tetramethylrhodamine, ethyl ester, perchlorate]</td>
			<td>Anaspec</td>
			<td>AS-88061</td>
			<td>TMRE powder reconstituted in DMSO to reach 100 &#956;M stock.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermofisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Valinomycin</td>
			<td>Sigma</td>
			<td>V0627</td>
			<td>Valinomycin powder reconsituted in DMSO to reach 250 nM stock.</td>
		</tr>
	</tbody>
</table>

complementary approaches to interrogate mitophagy flux in pancreatic &#946;-cells

Mitofagi er en kvalitetskontrolmekanisme, der er nødvendig for at opretholde optimal mitokondriefunktion. Dysfunktionel β-celle mitofagi, resulterer i utilstrækkelig insulinfrigivelse. Avancerede kvantitative vurderinger af mitofagi, kræver ofte brug af genetiske reportere. mt-Keima-musemodellen, der udtrykker en mitokondrie-målrettet pH-følsom dual-excitation ratiometrisk sonde til kvantificering af mitofagien via flowcytometri, er blevet optimeret i β-celler. Forholdet mellem sure og neutrale mt-Keima-bølgelængdeemissioner kan bruges til robust kvantificering af mitofagi. Brug af genetiske mitofagireportere kan dog være udfordrende, når man arbejder med komplekse genetiske musemodeller eller celler, der er vanskelige at transfektere, såsom primære menneskelige øer. Denne protokol beskriver en ny komplementær farvestofbaseret metode til kvantificering af β-celle mitofagi, i primære øer ved hjælp af MtPhagy. MtPhagy er et pH-følsomt, cellegennemtrængeligt farvestof, der akkumuleres i mitokondrierne og øger dets fluorescensintensitet, når mitokondrier er i miljøer med lav pH, såsom lysosomer under mitofagi. Ved at kombinere MtPhagy-farvestoffet med Fluozin-3-AM, en Zn2+ -indikator, der vælger β-celler, og tetramethylrhodamin, ethylester (TMRE) til vurdering af mitokondriemembranpotentiale, kan mitofagiflux kvantificeres specifikt i β-celler via flowcytometri. Disse to tilgange er meget komplementære, hvilket giver mulighed for fleksibilitet og præcision i vurderingen af mitokondriel kvalitetskontrol i adskillige β-cellemodeller.

Pancreatic &#946;-cells produce and secrete insulin to meet metabolic demands, and &#946;-cell dysfunction is responsible for hyperglycemia and diabetes onset in both type 1 and type 2 diabetes. &#946;-Cells couple glucose metabolism with insulin secretion via mitochondrial energetics and metabolic output, which depend on a reserve of functional mitochondrial mass1,2,3. To maintain optimal &#946;-cell function, &#946;-cells rely on mitochondrial quality control mechanisms to remove aged or damaged mitochondria and preserve functional mitochondrial mass4. Selective mitochondrial autophagy, also known as mitophagy, is a key component of the mitochondrial quality control pathway.
Assessments of mitophagy in live cells often rely on changes in mitochondrial pH that occur during mitophagy. Mitochondria have a slightly alkaline pH, and healthy mitochondria normally reside in the pH-neutral cytosol. During mitophagy, damaged or dysfunctional mitochondria are selectively incorporated into autophagosomes and eventually cleared within acidic lysosomes5. Several in vivo transgenic mitophagy reporter mouse models, such as mt-Keima6, mitoQC7, and CMMR8, as well as transfectable mitophagy probes, such as the Cox8-EGFP-mCherry plasmid9, utilize this pH change to provide quantitative assessments of mitophagy. Use of transgenic mice expressing the mt-Keima pH-sensitive dual-excitation ratiometric probe has been optimized for mitophagy assessments in islets and &#946;-cells via flow cytometry10,11. The ratio of acidic-to-neutral mt-Keima wavelength emissions (the ratio of acidic 561 nm to neutral 480 nm excitation) can be used to robustly quantify mitophagy6,12.
This protocol describes an optimized approach to assess mitophagy flux in primary islets and &#946;-cells isolated from mt-Keima transgenic mice10,11. While mt-Keima is a highly sensitive probe, it requires either complicated animal breeding schemes or the transfection of cells, which can often be challenging when working in combination with other genetic models or with primary human islets. Additionally, the use of multiple fluorescence lasers and detectors to identify neutral and acidic cell populations can limit the combinatorial use of other fluorescent reporters.
To overcome these challenges, this protocol also describes a complementary, single fluorescent channel, dye-based method for robust detection of mitophagy in &#946;-cells from isolated mouse islets. This approach, referred to as the MtPhagy method, utilizes a combination of three cell-permeable dyes to select for &#946;-cells, quantify the cell populations actively undergoing mitophagy, and assess mitochondrial membrane potential (MMP or &#916;&#968;m) simultaneously.
The first of these dyes is Fluozin-3-AM, a cell-permeable Zn2+ indicator with an Ex/Em 494/516 nm13. Mouse islets comprise a heterogeneous population of functionally distinct cells, including &#945;-, &#946;-, &#948;-, and PP cells. &#946;-Cells comprise approximately 80% of cells within the mouse islet and can be distinguished from other islet cell types due to their high Zn2+ concentration within insulin granules14,15, allowing for identification of &#946;-cells as the Fluozin-3-AMhigh population. The MtPhagy dye, a pH-sensitive dye that is immobilized on mitochondria via a chemical bond and emits weak fluorescence, is also utilized in this protocol16. Upon mitophagy induction, damaged mitochondria are incorporated into the acidic lysosome, and the MtPhagy dye increases its fluorescence intensity within the low pH environment (Ex/Em 561/570-700 nm).
Additionally, Tetramethylrhodamine, ethyl ester (TMRE), is used to assess MMP. TMRE is a cell-permeable positively charged dye (Ex/Em 552/575 nm) that is sequestered by healthy mitochondria due to the relative negative charge upheld by their membrane potential17. Damaged or unhealthy mitochondria dissipate their membrane potential, resulting in decreased ability to sequester TMRE. Utilizing these dyes together, &#946;-cells undergoing mitophagy can be identified as the FluozinhighMtPhagyhighTMRElow population via flow cytometry. Since mitophagy is a dynamic rather than static process, this protocol was optimized to assess mitophagy flux using valinomycin, a K+-ionophore that induces mitophagy following dissipation of MMP18. Comparison of mitophagy in the presence and absence of valinomycin allows for assessment of mitophagy flux in different sample groups.
The dye-based nature of the current approach allows it to be extrapolated to human islets and other difficult-to-transfect cell types and circumvents the need for complicated animal breeding schemes, unlike the mt-Keima protocol. The overarching goal of this protocol is to quantify mitophagy in &#946;-cells at the single-cell level via two independent flow cytometry-based methods. Taken together, this protocol describes two powerful and complementary methods that allow for both precision and flexibility in the quantitative study of mitochondrial quality control.

Forfatter Spotlight: Vurdering af mitofagiflux i β-celler i bugspytkirtlen ved hjælp af effektive og robuste komplementære tilgange 

Komplementære tilgange til at forhøre mitofagiflux i β-celler i bugspytkirtlen

Our protocol describes two complementary methods to study beta-cell mitophagy flux at the single-cell resolution via flow cytometry. The mt-Keima protocol utilizes a genetic mitophagy reporter, whereas the MtPhagy protocol combines three fluorescent dyes, which makes it easy to extrapolate to difficult-to-transfect cells in human samples. Assessments of mitophagy using traditional biochemical approaches and imaging approaches are both time consuming and challenging.Thus, it's important to develop new, robust, and effective methods to study mitophagy flux in beta-cells. Both the mt-Keima and MtPhagy method are quantitative assessments that are efficient for studying mitophagy flux in pancreatic beta cells. So, the Soleimanpour lab focuses on the different mechanisms that drive pancreatic beta-cell failure and diabetes.And we specifically focus on the different aspects of mitochondrial lifecycle, including mitophagy, to understand how defects in mitochondrial function drive the development of diabetes.

Vurdering af mitofagi: via den farvestofbaserede MtPhagy-tilgang Denne farvestofbaserede tilgang blev optimeret til at analysere mitofagiflux inden for primære muse-β-celler uden behov for en genetisk reporter ved hjælp af Fluozin-3-AM, TMRE og MtPhagy samt DAPI for at udelukke døde celler. Ved at parre disse farvestoffer med valinomycin for at inducere mitofagi, skitserer denne protokol en farvestofbaseret metode til selektivt at måle mitofagiflux i primære muse-β-celler18</s...

Watch this Scientific Journal Video about Assessment of Mitophagy Flux in Pancreatic β-Cells Using Effective and Robust Complementary Approaches at JoVE.com

Denne protokol skitserer to metoder til kvantitativ analyse af mitofagia i bugspytkirtlen β-celler: for det første en kombination af cellegennemtrængelige mitokondriespecifikke farvestoffer og for det andet en genetisk kodet mitofagireporter. Disse to teknikker supplerer hinanden og kan anvendes baseret på specifikke behov, hvilket giver mulighed for fleksibilitet og præcision i kvantitativ håndtering af mitokondriel kvalitetskontrol.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

Vores protokol beskriver to komplementære metoder til at studere beta-celle mitofagiflux ved enkeltcelleopløsningen via flowcytometri. mt-Keima-protokollen bruger en genetisk mitofagireporter, mens MtPhagy-protokollen kombinerer tre fluorescerende farvestoffer, hvilket gør det let at ekstrapolere til celler, der er vanskelige at transfektere i humane prøver. Vurderinger af mitofagi, ved hjælp af traditionelle biokemiske tilgange og billeddannelsesmetoder, er både tidskrævende og udfordrende.Derfor er det vigtigt at udvikle nye, robuste og effektive metoder til at studere mitofagiflux i betaceller. Både mt-Keima og MtPhagy metoden er kvantitative vurderinger, der er effektive til at studere mitofagiflux i pancreas beta-celler. Så Soleimanpour-laboratoriet fokuserer på de forskellige mekanismer, der driver betacellesvigt i bugspytkirtlen og diabetes.Og vi fokuserer specifikt på de forskellige aspekter af mitokondrielivscyklus, herunder mitofagi, for at forstå, hvordan defekter i mitokondriefunktionen driver udviklingen af diabetes.

complementary-approaches-to-interrogate-mitophagy-flux-pancreatic

Research

JoVE Journal

Biology

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Single-Cell Dissociation of Murine Islets for Flow Cytometric Analysis of &#946;-Cell Mitophagy

Begin by culturing the eyelet samples isolated from regular fat diet or high fat diet mice overnight at 37 degrees Celsius. Design the desired experimental conditions for the study. For each experimental condition, using a pipette, pick 100 eyelets into a six centimeter Petri dish containing two milliliters of eyelet medium.To induce Mitophagy, treat the eyelets in the appropriate Petri dishes with two microliters of 250 nano molar Valinomycin stock for three hours. Isolate the eyelets and using a pipette, transfer the eyelets from each Petri dish to a separate micro centrifuge tube. Then, spin the eyelets at 350 G for one minute at 10 degrees Celsius and using a pipette, discard the supernatant.Wash the obtained sample twice with one milliliter of PBS. Between the washes, spin the sample and discard the supernatant as demonstrated previously. To dissociate eyelets into single cells, add 500 microliters of 0.05%Trypsin prewarmed to 37 degrees celsius, to one sample tube.Pipette the contents of the tube up and down repeatedly until the eyelets are visibly dispersed. Immediately add one milliliter of prewarmed eyelet medium to neutralize trypsin. Spin the samples at 500 G for five minutes at 10 degrees Celsius.Remove the supernatant carefully without disturbing the pellet. Then, wash the samples two times with eyelet flow medium. The cell samples are now ready to be stained for flow cytometric analysis of beta cell mitophagy.

Enkeltcelledissociation af murine øer til flowcytometrisk analyse af β-celle mitofagi;

Flow Cytometric Analysis of Mitophagy in Murine Pancreatic &#946;-Cells Using MtPhagy Dye

Proceed to perform flow cytometric analysis of beta cell mitophagy, with a single cell suspension of about 100 isolated mirroring islets prepared, corresponding to each desired experimental condition to be studied. First, resuspend the islet samples for each condition in 500 microliters of islet flow medium. Add 0.25 microliters of MtPhagy stock to tubes designated to receive the MtPhagy dye.Similarly, add 0.25 microliters of TMRE stock and fluozin-3-AM stock to the respective designated tubes. Wrap the tubes in aluminum foil and vortex each tube at low speed for five seconds to mix the contents. Then incubate the tubes at 37 degrees Celsius for 30 minutes.Halfway through incubation, vortex each sample at low speed for five to 10 seconds. After incubation, centrifuge the tubes at 350 G for one minute at 10 degrees Celsius. Discard the supernatant.And resuspend the samples in 500 microliters of islet flow medium. Add DAPI to the microcentrifuge tubes containing samples that require DAPI treatment. Centrifuge again and discard the supernatant as demonstrated previously.Resuspend the residue in 500 microliters of islet flow medium. Finally, place the samples on ice and then proceed with flow cytometry. Adjust the voltages for forward scatter or FSC and side scatter or SSC to ensure cell populations are at the center of the scatter plot.To exclude multiplets, at a rectangular gate on FSC height versus FSC width, followed by SSC height versus SSC width. Adjust voltages and compensation for DAPI to filter for live beta cells. Set fluorescence gates for each fluorophore used based on the unstained RFD sample.Once gates are established, collect 10, 000 events per sample. Beta cells with high utilization of mitophagy were defined as the fluozin high MtPhagy high TMRE low population in quadrant three or Q3.Basal and valinomycin-induced mitophagy levels were characterized in both RFD and HFD islets.

Flow cytometrisk analyse af mitofagi, i murin pancreas β-celler ved anvendelse af MtPhagy farvestof

Flow Cytometric Analysis of Mitophagy in Murine Pancreatic &#946;-Cells Using the Genetically Encoded mt-Keima Reporter

To assess mitophagy in murine pancreatic beta cells using the genetically encoded mitophagy reporter, culture pancreatic islets isolated from wild type and Mt-Keima mice overnight at 37 degrees Celsius. The following day, add 100 islets into a six centimeter Petri dish containing two milliliters of islet medium for each experimental condition used in this protocol. Next, to dissociate the eyelets into single cells and stain with FluoZin-3-AM and DAPI as demonstrated previously for the Mt-Phagy dye based approach.Then proceed with the flow cytometric analysis of the samples. Adjust the forward scatter or FSC and side scatter or SSC voltages to attain an even distribution of cells on an SSCA versus FSCA plot. To select single cells and exclude multiplets, add a rectangular gate on FSC height versus FSC width, followed by SSC height versus SSE width.The multiplets are excluded due to their higher width signal values. Then set up a DAPI negative gate to exclude dead cells. After that, set up FluoZin-3-AM positive gates to filter for live beta cells.Set up a triangle gating scheme using the Mt-Keima positive sample to identify acidic and neutral cell populations. Once the primary and fluorescence gates were established, assess mitophagy flux using basal and valinomycin-induced changes in Mt-Keima fluorescence.