E.L-D. acknowledges support from the NIH (T32-AI007413 and T32-AG000114). SAS acknowledges support from the JDRF (COE-2019-861), the NIH (R01 DK135268, R01 DK108921, R01 DK135032, R01 DK136547, U01 DK127747), the Department of Veterans Affairs (I01 BX004444), the Brehm family, and the Anthony family.

The animal studies presented in this protocol were reviewed and approved by the University of Michigan Institutional Animal Care and Use Committee. Twenty-week-old male C57BL/6J mice, on either a 15-week regular fat diet (RFD) or high-fat diet (HFD), were used for this study.
1. Assessing mitophagy&#160;via the dye-based MtPhagy approach (Method 1)
<ol>
	<li>Mouse islet preparation and treatment
	<ol>
		<li>Perform islet culture and valinomycin exposure following the steps below.
		<ol>
			<li>Isolate islets from either regular fat diet (RFD) or high-fat diet (HFD, 60 kcal% Fat19) mice, following the previously described methods2,10.</li>
			<li>Culture islet samples overnight at 37 &#176;C in RPMI medium supplemented with 100 units/mL antimycotic-antibiotic, 50 units/mL penicillin-streptomycin, 1 mM sodium pyruvate, 10 mM HEPES and 10% fetal bovine serum (FBS) (see Table of Materials). This medium will be referred to as &#34;islet medium&#34;.</li>
			<li>Using a pipette, pick 100 islets per condition into 6 cm Petri dishes in 2 mL of islet medium. 
			NOTE: Conditions used for this protocol: (1) Unstained control: unstained RFD islets; (2) DAPI only control: RFD islets stained with DAPI; (3) Fluozin-3-AM only control: RFD islets stained with Fluozin-3-AM; (4) MtPhagy only control: RFD islets stained with MtPhagy; (5) TMRE only control: RFD islets stained with TMRE; (6) RFD untreated: RFD islets stained with MtPhagy, TMRE, Fluozin-3-AM, and DAPI; (7) Valinomycin-exposed RFD islets: RFD islets exposed to valinomycin and stained with MtPhagy, TMRE, Fluozin-3-AM, and DAPI; (8) HFD untreated: HFD islets stained with MtPhagy, TMRE, Fluozin-3-AM, and DAPI; (9) Valinomycin-exposed HFD islets: HFD islets exposed to valinomycin and stained with MtPhagy, TMRE, Fluozin-3-AM, and DAPI.</li>
			<li>For conditions (7) and (9) (see NOTE above) exposed to valinomycin, add 2 &#181;L of 250 n&#924; valinomycin stock (see Table of Materials) to islets in the Petri dish for 3 h to induce mitophagy.</li>
		</ol>
		</li>
		<li>Perform single cell dissociation.
		<ol>
			<li>After 3 h valinomycin exposure, pick 100 islets from each condition into separate microcentrifuge tubes using a pipette.</li>
			<li>Spin islets at 350 x g for 1 min at 10 &#176;C and discard the supernatant using a pipette.</li>
			<li>Wash samples 2x with 1 mL 1x phosphate buffered saline (PBS), with spin steps (350 x g/1 min, 10 &#176;C) between each wash. Discard supernatant with a pipette after each wash.</li>
			<li>To dissociate islets into single cells, add 500 &#181;L of 0.05% trypsin (pre-warmed to 37 &#176;C) to the microcentrifuge tube of one sample to prevent over-digestion of islets. Pipette up and down repeatedly until islets are visibly dispersed.</li>
			<li>Immediately add 1 mL of pre-warmed islet medium to neutralize trypsin. Repeat trypsinization and neutralization for each sample, one at a time.</li>
			<li>Spin samples at 500 x g for 5 min at 10 &#176;C. Remove supernatant with a pipette, careful not to disturb the pellet. 
			NOTE: Pellet will be delicate for samples exposed to valinomycin.</li>
			<li>Wash samples 2x with RPMI medium, no phenol red, supplemented with 100 units/mL antimycotic-antibiotic, 50 units/mL penicillin-streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, and 10% bovine serum albumin (BSA). This medium will be referred to as &#34;islet flow medium&#34;. Include spin steps (350 x g/1 min, 10 &#176;C) between each wash. Discard supernatant with a pipette after each wash.</li>
		</ol>
		</li>
		<li>Stain cells with MtPhagy, TMRE, Fluozin-3-AM, and DAPI to prepare for flow cytometry.
		<ol>
			<li>Resuspend islet samples in 500 &#181;L of islet flow medium.</li>
			<li>Add 0.25 &#181;L of 100 &#181;M MtPhagy stock (see Table of Materials) to tubes receiving MtPhagy dye (conditions 4, 6, 7, 8, and 9, NOTE to step 1.1.1).</li>
			<li>Add 0.25 &#181;L of 100 &#181;M TMRE stock (see Table of Materials) to tubes receiving TMRE dye (conditions 5, 6, 7, 8, and 9).</li>
			<li>Add 0.25 &#181;L of 1 mM Fluozin-3-AM stock (see Table of Materials) to tubes receiving Fluozin-3-AM dye (conditions 3, 6, 7, 8, and 9).</li>
			<li>Vortex tubes at low speed for 5 s to mix.</li>
			<li>Wrap microcentrifuge tubes in aluminum foil to protect from light and incubate at 37 &#176;C for 30 min. Halfway through incubation, vortex tubes at low speed for 5-10 s to mix.</li>
			<li>After incubation, centrifuge samples at 350 x g for 1 min at 10 &#176;C. Discard supernatant using a pipette.</li>
			<li>Resuspend samples in 500 &#181;L islet flow medium. Add 0.2 &#181;g/mL DAPI (see&#160;Table of Materials) to conditions 2, 6, 7, 8, and 9.</li>
			<li>Spin samples at 350 x g for 1 min at 10 &#176;C. Discard supernatant using a pipette and resuspend in 500 &#181;L of islet flow medium.</li>
			<li>Place samples on ice.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Startup the instrument (see Table of Materials). 
		NOTE: Any flow cytometer with the appropriate filters will work. The following filters were used in this study: (1) VL1 for DAPI: Excitation/Emission - 405 nm/440 nm (50 nm); (2) BL1 for Fluozin-3-AM: Excitation/Emission - 488 nm/530 nm (30 nm); (3) BL2 for MtPhagy dye: Excitation/Emission - 488 nm/590 nm (40 nm); (4) YL1 for TMRE: Excitation/Emission - 561 nm/585 nm (16 nm).</li>
		<li>Adjust the voltages for forward (FSC) and side scatter (SSC) to ensure cell populations are at the center of the scatter plot. For this protocol, voltages used were 120 V for FSC and 260 V for SSC to ensure cells fall evenly within the FSC-A vs. SSC-A plot (Figure 1A).</li>
		<li>To exclude non-single cells, add a rectangular gate on FSC-H vs. FSC-W followed by SSC-H vs. SSC-W (Figure 1B,C).</li>
		<li>Adjust voltages and compensation for DAPI to filter for live &#946;-cells. Set fluorescence gates for each fluorophore used based on the unstained RFD sample (Figures 1D-F). 
		NOTE: Voltages used for each channel: (1) VL1: 320-360 V; (2) BL1: 300-340 V; (3) BL2: 260-300 V; (4) YL1: 300-340 V.</li>
		<li>Once gates are established, collect 10,000 events per sample. 
		NOTE: Utilizing this gating approach, mitophagy levels under basal conditions and upon valinomycin induction were assessed in both RFD and HFD islets (Figure 2).</li>
		<li>Save data as FCS files for analysis.</li>
	</ol>
	</li>
</ol>
2. Assessing mitophagy using the genetically encoded mt-Keima reporter (Method 2)
<ol>
	<li>Mouse islet preparation and single-cell dissociation
	<ol>
		<li>Perform islet culture and valinomycin exposure following the steps below.
		<ol>
			<li>Isolate pancreatic islets from wild-type (WT) and mt-Keima/+ (mt-Keima) mice6. In this method, 20-week-old male WT or mt-Keima/+ fed RFD mice were used.</li>
			<li>Culture islet samples overnight at 37 &#176;C in islet medium.</li>
			<li>Using a pipette, pick 100 islets per condition into 6 cm Petri dishes with 2 mL of islet medium. 
			NOTE: Conditions used for this protocol: (1) Unstained control: Unstained WT islets; (2) DAPI only control: WT islets stained with DAPI; (3) Fluozin-3-AM only control: WT islets stained with Fluozin-3-AM; (4) mt-Keima only control: Unstained mt-Keima/+ islets; (5) Untreated: mt-Keima/+ islets stained with Fluozin-3-AM and DAPI; (6) Valinomycin: mt-Keima/+ islets exposed to valinomycin and stained with Fluozin-3-AM and DAPI.</li>
			<li>For condition (6) with valinomycin exposure, add 2 &#181;L of 250 nM valinomycin stock to islets in Petri dish for 3 h to induce mitophagy.</li>
		</ol>
		</li>
		<li>Perform single-cell dissociation.
		<ol>
			<li>Perform single cell dissociation steps, as described in step 1.1.2.</li>
		</ol>
		</li>
		<li>Stain cells with Fluozin-3-AM and DAPI.
		<ol>
			<li>Resuspend islet samples in 500 &#181;L of islet flow medium.</li>
			<li>Add 0.25 &#181;L of 1 mM Fluozin-3-AM stock to tubes receiving Fluozin-3-AM dye (conditions 3, 5, and 6).</li>
			<li>Incubate cells at 37 &#176;C and proceed to DAPI treatment, as described in steps 1.1.3.5-1.3.10.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Startup the instrument. Any flow cytometer with the appropriate filters will work. 
		NOTE: The following filters were used in this study: (1) VL1 for DAPI: Excitation/Emission - 405 nm/440 nm (50 nm); (2) BL1 for Fluozin-3-AM: Excitation/Emission - 488 nm/530 nm (30 nm); (3) VL3 for mt-Keima neutral: Excitation/Emission - 405 nm/603 nm (48 nm); (4) YL2 for mt-Keima acid: Excitation/Emission - 561 nm/620 nm (15 nm).</li>
		<li>Adjust FSC and SSC voltages and exclude non-single cells as described in steps 1.2.2-1.2.3 (Figures 3A-C).</li>
		<li>Adjust voltages and compensation for DAPI, Fluozin-3-AM and mt-Keima using unstained and single-positive controls (conditions 1-4) to ensure that fluorescence-positive cell populations are distinguishable from unstained cells. Once appropriate compensation has been applied to each channel, set up DAPI negative gate and Fluozin-3-AM positive gates to filter for live &#946;-cells (Figures 3D-E).</li>
		<li>Set up a triangle gating scheme using the mt-Keima positive sample (condition 4) to identify acidic and neutral cell populations (Figure 3F). 
		NOTE: Voltages typically used for each channel: (1) VL1: 320-340 V; (2) BL2: 260-280 V; (3) VL3: 280-300 V; (4) YL2: 280-300 V.</li>
		<li>Once gates are established, collect 10,000 events per sample. Gating schemes for conditions 5 and 6 are illustrated in Figure 3A-G.</li>
		<li>Save data as FCS files for analysis.</li>
	</ol>
	</li>
</ol>

Quantitative Analysis of Mitophagy in Murine Pancreatic &#946;-Cells

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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=The+diabetes+susceptibility+gene+Clec16a+regulates+mitophagy.">The diabetes susceptibility gene Clec16a regulates mitophagy.</a> Cell. 157 (7), 1577-1590 (2014).</li><li>Pearson, G., 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=Clec16a,+Nrdp1,+and+USP8+form+a+ubiquitin-dependent+tripartite+complex+that+regulates+&#946;-cell+mitophagy.">Clec16a, Nrdp1, and USP8 form a ubiquitin-dependent tripartite complex that regulates &#946;-cell mitophagy.</a> Diabetes. 67 (2), 265-277 (2018).</li><li>Hattori, N., Saiki, S., Imai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+by+mitophagy.">Regulation by mitophagy.</a> International Journal of Biochemistry and Cell Biology. 53, 147-150 (2014).</li><li>Berezhnov, A. V., 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=Intracellular+pH+modulates+autophagy+and+mitophagy.">Intracellular pH modulates autophagy and mitophagy.</a> Journal of Biological Chemistry. 291 (16), 8701-8708 (2016).</li><li>Sun, N., 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=Measuring+in+vivo+mitophagy.">Measuring in vivo mitophagy.</a> Molecular Cell. 60 (4), 685-696 (2015).</li><li>McWilliams, T. G., 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=mito-QC+illuminates+mitophagy+and+mitochondrial+architecture+in+vivo.">mito-QC illuminates mitophagy and mitochondrial architecture in vivo.</a> Journal of Cell Biology. 214 (3), 333-345 (2016).</li><li>Aoyagi, K., 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=A+new+beta+cell-specific+mitophagy+reporter+mouse+shows+that+metabolic+stress+leads+to+accumulation+of+dysfunctional+mitochondria+despite+increased+mitophagy.">A new beta cell-specific mitophagy reporter mouse shows that metabolic stress leads to accumulation of dysfunctional mitochondria despite increased mitophagy.</a> Diabetologia. 66 (1), 147-162 (2023).</li><li>Ma, X., Ding, W. 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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. 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SAS has received grant funding from Ono Pharmaceutical Co., Ltd. and is a consultant for Novo Nordisk.

This protocol described two complementary methods to quantify mitophagy flux in dissociated primary mouse islets. Using the mt-Keima method, an increase in mitophagy was quantified as an increased ratio of acidic (561 nm)/neutral (405 nm) cells, whereas in the MtPhagy method, increased mitophagy flux was quantified as an increase in the FluozinhighMtPhagyhighTMRElow cell population. These methods allow for rapid, quantitative, and &#946;-cell-specific assessments of mitophagy flux.
<p...

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.

<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 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.

Assessing mitophagy via the dye-based MtPhagy approach 
This dye-based approach was optimized to analyze mitophagy flux within primary mouse &#946;-cells without the need for a genetic reporter, using Fluozin-3-AM, TMRE, and MtPhagy as well as DAPI to exclude dead cells. By pairing these dyes with valinomycin to induce mitophagy, this protocol outlines a dye-based method to selectively measure mitophagy flux in primary mouse &#946;-cells18. For the data shown using this MtPha...

Watch this Scientific Journal Video about Assessment of Mitophagy Flux in Pancreatic Î²-Cells Using Effective and Robust Complementary Approaches at JoVE.com

Author Spotlight: Assessment of Mitophagy Flux in Pancreatic &#946;-Cells Using Effective and Robust Complementary Approaches 

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.

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 ...

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Research

JoVE Journal

Biology

976 Views.  University of Michigan, Ann Arbor. This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic β-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.

Video: Author Spotlight: Assessment of Mitophagy Flux in Pancreatic β-Cells Using Effective and Robust Complementary Approaches 

Murine Pancreatic Islet Isolation

Experimental Approaches to Tissue Engineering

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main lysosome-dependent pathway is called autophagy, while the primary non-lysosomal method for protein catabolism is the ubiquitin-proteasome system. Recent studies in model organisms suggest that the activity of both autophagy and the ubiquitin-proteasome system is not constant across the day but instead varies according to a daily (circadian) rhythm. The ability to measure biological rhythms in protein turnover is important for understanding how cellular quality control is achieved and for understanding the dynamics of specific proteins of interest. Here we present a standardized protocol for quantifying autophagic and proteasomal flux in vivo that captures the circadian component of protein turnover. Our protocol includes details for mouse handling, tissue processing, fractionation, and autophagic flux quantification using mouse liver as the starting material.

We describe our protocol for measuring biological rhythms in protein catabolism via autophagy and the proteasome in mouse liver.

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...