Name: Forfatter Spotlight: Vurdering av mitofagifluks i bukspyttkjertelens β-celler ved bruk av effektive og robuste komplementære tilnærminger
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. anerkjenner støtte fra NIH (T32-AI007413 og T32-AG000114). SAS anerkjenner 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.

Dyrestudiene presentert i denne protokollen ble gjennomgått og godkjent av University of Michigan Institutional Animal Care and Use Committee. Tjue uker gamle mannlige C57BL / 6J-mus, på enten en 15-ukers vanlig fettdiett (RFD) eller fettfattig diett (HFD), ble brukt til denne studien.
1. Vurdering av mitofagi via fargestoffbasert MtPhagy tilnærming (metode 1)
<ol><li>Mus holme forberedelse og behandling<ol><li>Utfør holmekultur og valinomycineksponering ved å følge tr...

Kvantitativ analyse av mitofagi i β-celler i bukspyttkjertelen hos murine

<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. 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Denne protokollen beskrev to komplementære metoder for å kvantifisere mitofagifluks i dissosierte primære museøyer. Ved hjelp av mt-Keima-metoden ble en økning i mitofagi kvantifisert som et økt forhold mellom sure (561 nm) / nøytrale (405 nm) celler, mens i MtPhagy-metoden ble økt mitofagifluks kvantifisert som en økning i FluozinhøyMtPhagyhøyTMRElav cellepopulasjon. Disse metodene tillater raske, kvantitative og β-cellespesifikke vurderinger av mitofagifluks.
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Bukspyttkjertel β-celler produserer og utskiller insulin for å møte metabolske krav, og β-celle dysfunksjon er ansvarlig for hyperglykemi og diabetesutbrudd i både type 1 og type 2 diabetes. β-celler kobler glukosemetabolisme med insulinsekresjon via mitokondriell energetikk og metabolsk utgang, som avhenger av en reserve av funksjonell mitokondriemasse 1,2,3. For å opprettholde optimal β-cellefunksjon er β-cellene avhengige av mitokondrielle kvalitetskontrollmekanismer for å fjerne gamle eller skadede mitokondrier og bevare funksjonell mitokondriemasse4. Selektiv mitokondriell autofagi, også kjent som mitofagi, er en nøkkelkomponent i mitokondriell kvalitetskontrollvei.
Vurderinger av mitofagi i levende celler er ofte avhengige av endringer i mitokondriell pH som oppstår under mitofagi. Mitokondrier har en svakt alkalisk pH, og sunne mitokondrier ligger normalt i den pH-nøytrale cytosolen. Under mitofagi blir skadede eller dysfunksjonelle mitokondrier selektivt innlemmet i autofagosomer og til slutt ryddet i sure lysosomer5. Flere in vivo transgene mitophagy reportermusemodeller, som mt-Keima6, mitoQC7 og CMMR8, samt transfektable mitofagiprober, som Cox8-EGFP-mCherry plasmid9, bruker denne pH-endringen for å gi kvantitative vurderinger av mitofagi. Bruk av transgene mus som uttrykker den mt-Keima pH-sensitive dual-excitation ratiometric probe har blitt optimalisert for mitofagivurderinger i øyer og β-celler via flowcytometri10,11. Forholdet mellom sure og nøytrale mt-Keima bølgelengdeutslipp (forholdet mellom sur 561 nm og nøytral 480 nm eksitasjon) kan brukes til å kvantifisere mitofagi 6,12.
Denne protokollen beskriver en optimalisert tilnærming for å vurdere mitofagifluks i primære øyer og β-celler isolert fra mt-Keima transgene mus10,11. Mens mt-Keima er en svært følsom sonde, krever det enten kompliserte dyreavlsordninger eller transfeksjon av celler, noe som ofte kan være utfordrende når man arbeider i kombinasjon med andre genetiske modeller eller med primære menneskelige øyer. I tillegg kan bruk av flere fluorescenslasere og detektorer for å identifisere nøytrale og sure cellepopulasjoner begrense den kombinatoriske bruken av andre fluorescerende reportere.
For å overvinne disse utfordringene beskriver denne protokollen også en komplementær, enkelt fluorescerende kanal, fargestoffbasert metode for robust påvisning av mitofagi i β-celler fra isolerte museøyer. Denne tilnærmingen, referert til som MtPhagy-metoden, benytter en kombinasjon av tre cellepermeable fargestoffer for å velge for β-celler, kvantifisere cellepopulasjonene som aktivt gjennomgår mitofagi og vurdere mitokondriemembranpotensial (MMP eller Δψm) samtidig.
Den første av disse fargestoffene er Fluozin-3-AM, en cellegjennomtrengelig Zn2+ indikator med en Ex / Em 494/516 nm13. Museøyer omfatter en heterogen populasjon av funksjonelt distinkte celler, inkludert α-, β-, δ- og PP-celler. β-celler utgjør omtrent 80% av cellene i museøya og kan skilles fra andre øycelletyper på grunn av deres høye Zn2+ konsentrasjon i insulingranulat14,15, noe som muliggjør identifisering av β-celler som Fluozin-3-AMhøy populasjon. MtPhagy-fargestoffet, et pH-følsomt fargestoff som immobiliseres på mitokondrier via en kjemisk binding og avgir svak fluorescens, brukes også i denne protokollen16. Ved mitofaginduksjon inkorporeres skadede mitokondrier i det sure lysosomet, og MtPhagy-fargestoffet øker fluorescensintensiteten i det lave pH-miljøet (Ex / Em 561/570-700 nm).
I tillegg brukes tetrametylrhodamin, etylester (TMRE), til å vurdere MMP. TMRE er et cellegjennomtrengelig positivt ladet fargestoff (Ex / Em 552 / 575 nm) som er sekvestrert av sunne mitokondrier på grunn av den relative negative ladningen som opprettholdes av membranpotensialet17. Skadede eller usunne mitokondrier sprer membranpotensialet, noe som resulterer i redusert evne til å binde TMRE. Ved å bruke disse fargestoffene sammen, kan β-celler som gjennomgår mitofagi identifiseres som FluozinhøyMtPhagyhøyTMRElav populasjon via flowcytometri. Siden mitofagi er en dynamisk snarere enn statisk prosess, ble denne protokollen optimalisert for å vurdere mitofagifluks ved bruk av valinomycin, en K +-ionofor som induserer mitofagi etter spredning av MMP18. Sammenligning av mitofagi i nærvær og fravær av valinomycin muliggjør vurdering av mitofagifluks i forskjellige prøvegrupper.
Den fargebaserte naturen til den nåværende tilnærmingen gjør det mulig å ekstrapolere til menneskelige øyer og andre vanskelig å transfektere celletyper og omgå behovet for kompliserte dyreavlsordninger, i motsetning til mt-Keima-protokollen. Det overordnede målet med denne protokollen er å kvantifisere mitofagi i β-celler på encellenivå via to uavhengige flowcytometribaserte metoder. Til sammen beskriver denne protokollen to kraftige og komplementære metoder som muliggjør både presisjon og fleksibilitet i den kvantitative studien av mitokondriell kvalitetskontroll.

<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

Mitophagy er en kvalitetskontrollmekanisme som er nødvendig for å opprettholde optimal mitokondriell funksjon. Dysfunksjonell β-celle mitofagi resulterer i utilstrekkelig insulinfrigivelse. Avanserte kvantitative vurderinger av mitofagi krever ofte bruk av genetiske reportere. Mt-Keima-musemodellen, som uttrykker en mitokondrie-målrettet pH-sensitiv dual-excitasjonsratiometrisk sonde for kvantifisering av mitofagi via flowcytometri, er optimalisert i β-celler. Forholdet mellom sure og nøytrale mt-Keima bølgelengdeutslipp kan brukes til å kvantifisere mitofagi robust. Imidlertid kan bruk av genetiske mitofagireportere være utfordrende når du arbeider med komplekse genetiske musemodeller eller vanskelig å transfektere celler, for eksempel primære menneskelige øyer. Denne protokollen beskriver en ny komplementær fargestoffbasert metode for å kvantifisere β-celle mitofagi i primære øyer ved hjelp av MtPhagy. MtPhagy er et pH-følsomt, cellepermeabelt fargestoff som akkumuleres i mitokondriene og øker fluorescensintensiteten når mitokondrier er i lave pH-miljøer, som lysosomer under mitofagi. Ved å kombinere MtPhagy-fargestoffet med Fluozin-3-AM, en Zn2+ -indikator som selekterer for β-celler, og tetrametylrhodamin, etylester (TMRE) for å vurdere mitokondriemembranpotensialet, kan mitofagifluks kvantifiseres spesifikt i β-celler via flowcytometri. Disse to tilnærmingene er svært komplementære, noe som gir fleksibilitet og presisjon i vurderingen av mitokondriell kvalitetskontroll i mange 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 av mitofagifluks i bukspyttkjertelens β-celler ved bruk av effektive og robuste komplementære tilnærminger 

Komplementære tilnærminger for å forhøre mitofagifluks i bukspyttkjertelen β-celler

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 av mitofagi via fargestoffbasert MtPhagy-tilnærming Denne fargestoffbaserte tilnærmingen ble optimalisert for å analysere mitofagifluks i primære mus β-celler uten behov for en genetisk reporter, ved hjelp av Fluozin-3-AM, TMRE og MtPhagy samt DAPI for å utelukke døde celler. Ved å parre disse fargestoffene med valinomycin for å indusere mitofagi, skisserer denne protokollen en fargestoffbasert metode for selektivt å måle mitofagifluks i primære mus β-celler18<...

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

Denne protokollen skisserer to metoder for kvantitativ analyse av mitofagi i bukspyttkjertelen β-celler: For det første en kombinasjon av cellepermeable mitokondrie-spesifikke fargestoffer, og for det andre, en genetisk kodet mitofagi reporter. Disse to teknikkene er komplementære og kan distribueres basert på spesifikke behov, noe som gir fleksibilitet og presisjon i kvantitativt adressering av mitokondriell kvalitetskontroll.

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

Vår protokoll beskriver to komplementære metoder for å studere betacelle mitofagifluks ved encelleoppløsning via flowcytometri. Mt-Keima-protokollen benytter en genetisk mitophagy reporter, mens MtPhagy-protokollen kombinerer tre fluorescerende fargestoffer, noe som gjør det enkelt å ekstrapolere til vanskelige å transfektere celler i menneskelige prøver. Vurderinger av mitofagi ved hjelp av tradisjonelle biokjemiske tilnærminger og bildebehandlingsmetoder er både tidkrevende og utfordrende.Derfor er det viktig å utvikle nye, robuste og effektive metoder for å studere mitofagifluks i betaceller. Både mt-Keima og MtPhagy metoden er kvantitative vurderinger som er effektive for å studere mitofagifluks i betaceller i bukspyttkjertelen. Soleimanpour-laboratoriet fokuserer derfor på de forskjellige mekanismene som driver betacellesvikt i bukspyttkjertelen og diabetes.Og vi fokuserer spesielt på de ulike aspektene av mitokondriell livssyklus, inkludert mitofagi, for å forstå hvordan defekter i mitokondriell funksjon driver utviklingen av 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.

Encellet dissosiasjon av murinøyer for flowcytometrisk analyse av 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.

Flowcytometrisk analyse av mitofagi i Murine Pankreas β-celler ved bruk av MtPhagy Dye

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.