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. 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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><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>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&amp;nbsp;</td><td>Thermofisher Scientific&amp;nbsp;</td><td>F24195</td><td>100 &amp;mu;g Fluozin-3AM powder reconstituted in 51 &amp;mu;L DMSO and 51 &amp;mu;L Pluronic F-127 to reach 1 mM stock.&amp;nbsp;</td></tr><tr><td>Gibco&amp;nbsp;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 &amp;mu;g MtPhagy powder reconstituted with 50 &amp;mu;L DMSO to reach 100 &amp;mu;M stock.&amp;nbsp;</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 ddH&lt;sub&gt;2&lt;/sub&gt;O</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 ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sodium Pyruvate (100x)</td><td>Life Technologies</td><td>11360-070</td><td>5 &amp;mu;g MtPhagy powder reconstituted with 50 &amp;mu;L DMSO to reach 100 &amp;mu;M stock.&amp;nbsp;</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 &amp;mu;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 β-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 ...

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

Research

JoVE Journal

Biology

1.1K Views.  University of Michigan, Ann Arbor. Ons protocol beschrijft twee complementaire methoden om bètacelmitofagieflux te bestuderen bij de resolutie van één cel via flowcytometrie. Het mt-Keima-protocol maakt gebruik van een genetische mitofagie-reporter, terwijl het MtPhagy-protocol drie fluorescerende kleurstoffen combineert, waardoor het gemakkelijk te extrapoleren is naar moeilijk te transfecteren cellen in menselijke monsters. Beoordelingen van mitofagie met behulp van traditionele biochemische benaderingen en beeldvormingsb...

Video: Auteur in de schijnwerpers: beoordeling van mitofagieflux in pancreas-β-cellen met behulp van effectieve en robuuste complementaire benaderingen 

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

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 ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Medicine

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

Immunology and Infection

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.

Mitophagy is the primary mechanism of mitochondrial quality control. However, the evaluation of mitophagy in vivo is hindered by the lack of reliable quantitative assays. Presented here is a protocol for the observation of mitophagy in living cells using a cell-permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye.

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. ...

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins.
Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the &#34;3D Objects Counter&#34; tool.
The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.

The goal of this protocol is to determine autophagic levels in pancreatic cancer and pancreatic acinar cells through LC3 immunofluorescence and LC3 dot quantification.

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows ...

Quantifying Mitophagy in C. elegans and a Liver Cancer Cell Line

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Author Spotlight: Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells

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