Name: Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices (Video) | JoVE Journal
Uploaded: 2021-04-12T13:30:00.000+00:00
Duration: 5 min 51 s
Description: Learn live pancreatic tissue slicing to study islet function and immune cell interactions in their native microenvironment. This method preserves physiological context for investigating diabetes and pancreatic disease mechanisms.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was funded by NIH grants R01 DK123292, T32 DK108736, UC4 DK104194, UG3 DK122638, and P01 AI042288. This research was performed with the support of the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project sponsored by JDRF (nPOD: 5-SRA-2018-557-Q-R), and The Leona M. &#38; Harry B. Helmsley Charitable Trust (Grant #2018PG-T1D053). The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at http://www.jdrfnpod.org/for-partners/npod-partners/. Thank you to Dr. Kevin Otto, University of Florida, for providing the vibratome used to generate mouse slices.&#160;

<ol><li>Uc, A., Fishman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pancreatic+disorders%5BTitle%5D)+AND+%22Pediatric+Clinics+of+North+America%22%5BJournal%5D)">Pancreatic disorders.</a> Pediatric Clinics of North America. 64 (3), 685-706 (2017).</li>
<li>Bluestone, J. A., Herold, K., Eisenbarth, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics%2C+pathogenesis+and+clinical+interventions+in+type+1+diabetes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Genetics, pathogenesis and clinical interventions in type 1 diabetes.</a> Nature. 464 (7293), 1293-1300 (2010).</li>
<li>Taylor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type+2+diabetes%3A+etiology+and+reversibility%5BTitle%5D)+AND+%22Diabetes+Care%22%5BJournal%5D)">Type 2 diabetes: etiology and reversibility.</a> Diabetes Care. 36 (4), 1047-1055 (2013).</li>
<li>Meier, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Islet+of+Langerhans+isolation+from+pediatric+and+juvenile+donor+pancreases%5BTitle%5D)+AND+%22Transplant+International%22%5BJournal%5D)">Islet of Langerhans isolation from pediatric and juvenile donor pancreases.</a> Transplant International. 27 (9), 949-955 (2014).</li>
<li>Marciniak, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+pancreas+tissue+slices+for+in+situ+studies+of+islet+of+Langerhans+and+acinar+cell+biology%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology.</a> Nature Protocols. 9 (12), 2809-2822 (2014).</li>
<li>Panzer, J. K., Cohrs, C. M., Speier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+pancreas+tissue+slices+for+the+study+of+islet+physiology%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Using pancreas tissue slices for the study of islet physiology.</a> Methods in Molecular Biology. 2128, 301-312 (2020).</li>
<li>Speier, S., Rupnik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+approach+to+in+situ+characterization+of+pancreatic+beta-cells%5BTitle%5D)+AND+%22Pflugers+Archive%22%5BJournal%5D)">A novel approach to in situ characterization of pancreatic beta-cells.</a> Pflugers Archive. 446 (5), 553-558 (2003).</li>
<li>Panzer, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pancreas+tissue+slices+from+organ+donors+enable+in+situ+analysis+of+type+1+diabetes+pathogenesis%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Pancreas tissue slices from organ donors enable in situ analysis of type 1 diabetes pathogenesis.</a> JCI Insight. 5 (8), 134525(2020).</li>
<li>Dolai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pancreatitis-induced+depletion+of+syntaxin+2+promotes+autophagy+and+increases+basolateral+exocytosis%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Pancreatitis-induced depletion of syntaxin 2 promotes autophagy and increases basolateral exocytosis.</a> Gastroenterology. 154 (6), 1805-1821 (2018).</li>
<li>Dolai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pancreas-specific+SNAP23+depletion+prevents+pancreatitis+by+attenuating+pathological+basolateral+exocytosis+and+formation+of+trypsin-activating+autolysosomes%5BTitle%5D)+AND+%22Autophagy%22%5BJournal%5D)">Pancreas-specific SNAP23 depletion prevents pancreatitis by attenuating pathological basolateral exocytosis and formation of trypsin-activating autolysosomes.</a> Autophagy. , 1-14 (2020).</li>
<li>Qadir, M. M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+culture+of+human+pancreatic+slices+as+a+model+to+study+real-time+islet+regeneration%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Long-term culture of human pancreatic slices as a model to study real-time islet regeneration.</a> Nature Communications. 11 (1), 3265(2020).</li>
<li>Cohrs, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dysfunction+of+persisting+%CE%B2+cells+is+a+key+feature+of+early+type+2+diabetes+pathogenesis%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Dysfunction of persisting β cells is a key feature of early type 2 diabetes pathogenesis.</a> Cell Reports. 31 (1), 107469(2020).</li>
<li>Liang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+human+pancreatic+slice+preparations+offer+a+valuable+model+for+studying+pancreatic+exocrine+biology%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Ex vivo human pancreatic slice preparations offer a valuable model for studying pancreatic exocrine biology.</a> Journal of Biological Chemistry. 292 (14), 5957-5969 (2017).</li>
<li>Shultz, L. D., Ishikawa, F., Greiner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Humanized+mice+in+translational+biomedical+research%5BTitle%5D)+AND+%22Nat+Reviews.+Immunology%22%5BJournal%5D)">Humanized mice in translational biomedical research.</a> Nat Reviews. Immunology. 7 (2), 118-130 (2007).</li>
<li>Lamont, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compensatory+mechanisms+allow+undersized+anchor-deficient+class+I+MHC+ligands+to+mediate+pathogenic+autoreactive+T+cell+responses%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Compensatory mechanisms allow undersized anchor-deficient class I MHC ligands to mediate pathogenic autoreactive T cell responses.</a> Journal of Immunology. 193 (5), 2135-2146 (2014).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Anesthesia+and+analgesia+in+laboratory+animals%22">Anesthesia and analgesia in laboratory animals</a>. Fish, R., Danneman, P. J., Brown, M., Karas, A. , Academic Press. (2011).</li>
<li>Clark, S. A., Borland, K. M., Sherman, S. D., Rusack, T. C., Chick, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staining+and+in+vitro+toxicity+of+dithizone+with+canine%2C+porcine%2C+and+bovine+islets%5BTitle%5D)+AND+%22Cell+Transplantation%22%5BJournal%5D)">Staining and in vitro toxicity of dithizone with canine, porcine, and bovine islets.</a> Cell Transplantation. 3 (4), 299-306 (1994).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li>
<li>Monette, R., Small, D. L., Mealing, G., Morley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+fluorescence+confocal+assay+to+assess+neuronal+viability+in+brain+slices%5BTitle%5D)+AND+%22Brain+Research+Protocols%22%5BJournal%5D)">A fluorescence confocal assay to assess neuronal viability in brain slices.</a> Brain Research Protocols. 2 (2), 99-108 (1998).</li>
<li>Gál, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+in+situ+approach+to+studying+pancreatic+ducts+in+mice%5BTitle%5D)+AND+%22Frontiers+in+Physiology%22%5BJournal%5D)">A novel in situ approach to studying pancreatic ducts in mice.</a> Frontiers in Physiology. 10, 938(2019).</li>
<li>Stožer, A., Dolenšek, J., Rupnik, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose-stimulated+calcium+dynamics+in+islets+of+Langerhans+in+acute+mouse+pancreas+tissue+slices%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Glucose-stimulated calcium dynamics in islets of Langerhans in acute mouse pancreas tissue slices.</a> PloS One. 8 (1), 54638(2013).</li>
<li>Stožer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+connectivity+in+islets+of+Langerhans+from+mouse+pancreas+tissue+slices%5BTitle%5D)+AND+%22PLoS+Computational+Biology%22%5BJournal%5D)">Functional connectivity in islets of Langerhans from mouse pancreas tissue slices.</a> PLoS Computational Biology. 9 (2), 1002923(2013).</li>
<li>Früh, E., Elgert, C., Eggert, F., Scherneck, S., Rustenbeck, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucagonotropic+and+glucagonostatic+effects+of+KATP+channel+closure+and+potassium+depolarization%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Glucagonotropic and glucagonostatic effects of KATP channel closure and potassium depolarization.</a> Endocrinology. 162 (1), 136(2021).</li>
<li>Satin, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+mechanisms+for+sulfonylurea+control+of+insulin+secretion%5BTitle%5D)+AND+%22Endocrine%22%5BJournal%5D)">New mechanisms for sulfonylurea control of insulin secretion.</a> Endocrine. 4 (3), 191-198 (1996).</li>
<li>Ren, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slow+oscillations+of+KATP+conductance+in+mouse+pancreatic+islets+provide+support+for+electrical+bursting+driven+by+metabolic+oscillations%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Endocrinology+and+Metabolism%22%5BJournal%5D)">Slow oscillations of KATP conductance in mouse pancreatic islets provide support for electrical bursting driven by metabolic oscillations.</a> American Journal of Physiology-Endocrinology and Metabolism. 305 (7), 805-817 (2013).</li>
<li>Marciniak, A., Selck, C., Friedrich, B., Speier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+pancreas+tissue+slice+culture+facilitates+long-term+studies+of+exocrine+and+endocrine+cell+physiology+in+situ%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.</a> PLoS One. 8 (11), 78706(2013).</li>
<li>Dzhagalov, I. L., Melichar, H. J., Ross, J. O., Herzmark, P., Robey, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-photon+imaging+of+the+immune+system%5BTitle%5D)+AND+%22Current+Protocols+in+Cytometry%22%5BJournal%5D)">Two-photon imaging of the immune system.</a> Current Protocols in Cytometry. , Chapter 12, Unit 12.26 (2012).</li>
</ol>

The authors declare no competing interests.

The objective of this protocol is to explicate the generation of pancreas slices and the procedures needed to employ the slices in functional and immunological studies. There are many benefits to using live pancreatic slices. However, there are several critical steps that are essential for the tissue to remain viable and useful during the described experiment protocols. It is imperative to work quickly. The length of time between injecting the pancreas and generating the slices on the vibratome should be minimized to mai...

Involvement of the pancreas is pathognomonic to diseases such as pancreatitis, T1D, and T2D1,2,3. The study of function in isolated islets usually involves removal of the islets from their surrounding environment4. The live pancreatic tissue slice method was developed to allow for the study of pancreatic tissue while maintaining intact islet microenvironments and avoiding the use of stressful islet isolation procedures5,6,7. Pancreatic tissue slices from human donor tissue have been successfully used to study T1D and have demonstrated processes of beta cell loss and dysfunction in addition to immune cell infiltration8,9,10,11,12,13. The live pancreatic tissue slice method can be applied to both mouse and human pancreatic tissue5,6,8. Human pancreatic tissue slices from organ donor tissues are obtained through a collaboration with the Network for Pancreatic Organ Donors with Diabetes (nPOD). Mouse slices can be generated from a variety of different mouse strains.
This protocol will focus on non-obese diabetic-recombination activating gene-1-null (NOD.Rag1-/-) and T cell receptor transgenic (AI4) (NOD.Rag1-/-.AI4&#945;/&#946;) mouse strains. NOD.Rag1-/- mice are unable to develop T and B cells due to a disruption in the recombination-activating gene 1 (Rag1)14. NOD.Rag1-/-.AI4&#945;/&#946; mice are used as a model for accelerated type 1 diabetes because they produce a single T cell clone that targets an epitope of insulin, resulting in consistent islet infiltration and rapid disease development15. The protocol featured here describes procedures for functional and immunological studies using live human and mouse pancreatic slices through the application of confocal microscopy approaches. The techniques described herein include viability assessments, islet identification and location, cytosolic Ca2+ recordings, as well as staining and identification of immune cell populations.

<table><tbody><tr><td>#3 Style Scalpel Handle</td><td>Fisherbrand</td><td>12-000-163</td><td></td></tr><tr><td>1 M HEPES</td><td>Fisher Scientific</td><td>BP299-100</td><td>HEPES Buffer, 1M Solution</td></tr><tr><td>10 cm Untreated Culture Dish</td><td>Corning</td><td>430591</td><td></td></tr><tr><td>10 mL Luer-Lok Syringe</td><td>BD</td><td>301029</td><td>BD Syringe with Luer-Lok Tips</td></tr><tr><td>27 G Needle</td><td>BD</td><td>BD 305109</td><td>BD General Use and PrecisionGlide Hypodermic Needles</td></tr><tr><td>35 mm coverglass-bottom Petri dish</td><td>Ibidi</td><td>81156</td><td>&amp;micro;-Dish 35 mm, high</td></tr><tr><td>50 mL syringe</td><td>BD</td><td>309653</td><td></td></tr><tr><td>8-well chambered coverglass</td><td>Ibidi</td><td>80826</td><td>&amp;micro;-Slide 8 Well</td></tr><tr><td>APC anti-mouse CD8a antibody</td><td>Biolegend</td><td>100712</td><td></td></tr><tr><td>BSA</td><td>Fisher Scientific</td><td>199898</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5670</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma</td><td>C7902</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (dihydrate)</td></tr><tr><td>Compact Digital Rocker</td><td>Thermo Fisher Scientific</td><td>88880020</td><td></td></tr><tr><td>Confocal laser-scanning microscope</td><td>Leica</td><td>SP8</td><td>Pinhole&amp;thinsp;= 1.5-2 airy units; acquired with 10x/0.40 numerical aperture HC PL APO CS2 dry and 20x/0.75 numerical aperture HC PL APO CS2 dry objectives at 512&amp;thinsp;&amp;times;&amp;thinsp;512 pixel resolution</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;</td></tr><tr><td>ddiH2O</td><td></td><td></td><td></td></tr><tr><td>Dithizone</td><td>Sigma-Aldrich</td><td>D5130-10G</td><td></td></tr><tr><td>DMSO</td><td>Invitrogen</td><td>D12345</td><td>Dimethyl sulfoxide</td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>2805</td><td></td></tr><tr><td>Falcon 35 mm tissue culture dish</td><td>Corning</td><td>353001</td><td>Falcon Easy-Grip Tissue Culture Dishes</td></tr><tr><td>FBS</td><td>Gibco</td><td>10082147</td><td></td></tr><tr><td>Feather No. 10 Surgical Blade</td><td>Electron Microscopy Sciences</td><td>7204410</td><td></td></tr><tr><td>fluo-4-AM</td><td>Invitrogen</td><td>F14201</td><td>cell-permeable Ca&lt;sup&gt;2+&lt;/sup&gt; indicator</td></tr><tr><td>Gel Control Super Glue</td><td>Loctite</td><td>45198</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11049-10</td><td></td></tr><tr><td>Hardened Fine Scissors</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025092</td><td>Hanks Balanced Salt Solution</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td>C&lt;sub&gt;8&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;S</td></tr><tr><td>Ice bucket</td><td>Fisherbrand</td><td>03-395-150</td><td></td></tr><tr><td>Isoflurane</td><td>Patterson Veterinary</td><td>NDC 14043-704-05</td><td></td></tr><tr><td>Johns Hopkins Bulldog Clamp</td><td>Roboz Surgical Store</td><td>RS-7440</td><td>&amp;nbsp;Straight; 500-900 Grams Pressure; 1.5&quot; Length</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional</td><td>34705</td><td>Kimtech Science&amp;trade; Kimwipes&amp;trade; Delicate Task Wipers, 2-Ply</td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td>This kit contains the calcein-AM live cell dye.</td></tr><tr><td>Low glucose DMEM</td><td>Corning</td><td>10-014-CV</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma</td><td>M9272</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (hexahydrate)</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>M2773</td><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (heptahydrate)</td></tr><tr><td>Magnetic Heated Platform</td><td>Warner Instruments</td><td>PM-1</td><td>Platform for imaging chamber for dynamic stimulation recordings</td></tr><tr><td>Microwave</td><td>GE</td><td>JES1460DSWW</td><td></td></tr><tr><td>Nalgene Syringe Filter</td><td>Thermo Fisher Scientific</td><td>726-2520</td><td></td></tr><tr><td>No.4 Paintbrush</td><td>Michaels</td><td>10269140</td><td></td></tr><tr><td>Open Diamond Bath Imaging Chamber</td><td>Warner Instruments</td><td>RC-26</td><td>Imaging chamber for dynamic stimulation recordings</td></tr><tr><td>Oregon Green 488 BAPTA-1-AM</td><td>Invitrogen</td><td>O6807</td><td>cell-permeable Ca&lt;sup&gt;2+&lt;/sup&gt; indicator</td></tr><tr><td>Overnight imaging chamber</td><td>Okolab</td><td>H201-LG</td><td></td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>20012050</td><td>To make agarose for slice generation</td></tr><tr><td>PE-labeled insulin tetramer</td><td>Emory Tetramer Research Core</td><td>sequence YAIENYLEL</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P5405</td><td>KCl</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P5655</td><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Razor Blades</td><td>Electron Microscopy Sciences</td><td>71998</td><td>For Vibratome; Double Edge Stainless Steel, uncoated</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>SeaPlaque low melting-point agarose</td><td>Lonza</td><td>50101</td><td>To make agarose for slice generation</td></tr><tr><td>Slice anchor</td><td>Warner Instruments</td><td>64-1421</td><td></td></tr><tr><td>Slice anchor (dynamic imaging)</td><td>Warner Instruments</td><td>640253</td><td>Slice anchor for dynamic imaging chamber</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S5886</td><td>NaCl</td></tr><tr><td>Sodium phosphate monohydrate</td><td>Sigma</td><td>S9638</td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;(monohydrate)</td></tr><tr><td>Soybean Trypsin Inhibitor</td><td>Sigma</td><td>T6522-1G</td><td>Trypsin inhibitor from Glycine max (soybean)</td></tr><tr><td>Stage Adapter</td><td>Warner Instruments</td><td>SA-20MW-AL</td><td>To fit imaging chamber for dynamic stimulation recordings on the microscope stage</td></tr><tr><td>Stage-top incubator</td><td>Okolab</td><td>H201</td><td></td></tr><tr><td>Stereoscope</td><td>Leica</td><td>IC90 E MSV266</td><td></td></tr><tr><td>SYTOX Blue Dead Cell Stain</td><td>Invitrogen</td><td>S34857</td><td>blue-fluorescent nucleic acid stain</td></tr><tr><td>Transfer Pipet</td><td>Falcon</td><td>357575</td><td>Falcon&amp;trade; Plastic Disposable Transfer Pipets</td></tr><tr><td>Valve Control System</td><td>Warner Instruments</td><td>VCS-8</td><td>System for dynamic stimulation recordings</td></tr><tr><td>Vibratome VT1000 S</td><td>Leica</td><td>VT1000 S</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>FSGPD02</td><td>Fisherbrand Isotemp General Purpose Deluxe Water Bath GPD 02</td></tr></tbody></table>

observing islet function and islet-immune cell interactions in live pancreatic tissue slices

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This protocol will yield live pancreatic tissue slices suitable for both functionality studies and immune cell recordings. Slice appearance in both brightfield and under reflected light are shown in Figure 1A,B. As discussed, islets can be found in slices using reflected light due to their increased granularity that occurs because of their insulin content (Figure 1C) and are clearly observed compared to the background tissue when reflected light...

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

observing-islet-function-islet-immune-cell-interactions-live

Research

JoVE Journal

Immunology and Infection

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

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

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

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250–350 high quality and fully functional islets per mouse.

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

Pancreatic Islet Embedding for Paraffin Sections

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.

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

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

This protocol describes how to generate human pancreas slices from deceased organ donors to study cell function under near-physiological conditions. This innovative approach enables the investigation of normal and structurally damaged islets and the intricate interplay between endocrine and exocrine compartments.

Generating Human Pancreatic Tissue Slices to Study Endocrine and Exocrine Pancreas Physiology

Learn live pancreatic tissue slicing to study islet function and immune cell interactions in their native microenvironment. This method preserves physiological context for investigating diabetes and pancreatic disease mechanisms.

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Explore More Videos