Organ donors and their families are appreciated for their invaluable donations, and the International Institute for Organ Procurement Organizations, Advancement of Medicine (IIAM) and the National Disease Research Exchange (NDRI) are acknowledged for their partnership in making human pancreatic tissue accessible for research. This work was supported by the Human Islet Research Network (RRID:SCR_014393), the Human Pancreas Analysis Program (RRID:SCR_016202), DK106755, DK123716, DK123743, DK120456, DK104211, DK108120, DK112232, DK117147, DK112217, EY032442, and DK20593 (Vanderbilt Diabetes Research and Training Center), The Leona M. and Harry B. Helmsley Charitable Trust, JDRF, the U.S. Department of Veterans Affairs (BX000666), the NIGMS of the National Institutes of Health (T32GM007347), F30DK134041, F30DK118830, and the National Science Foundation Graduate Research Fellowship (1937963).

Intracellular Signaling Events and Hormone Secretion in Human Pseudoislets

<ol>
	<li>Tokarz, V. L., MacDonald, P. E., Klip, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+systemic+insulin+function.">The cell biology of systemic insulin function.</a> The Journal of Cell Biology. 217 (7), 2273-2289 (2018).</li><li>Yu, Q., Shuai, H., Ahooghalandari, P., Gylfe, E., Tengholm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+controls+glucagon+secretion+by+directly+modulating+cAMP+in+alpha+cells.">Glucose controls glucagon secretion by directly modulating cAMP in alpha cells.</a> Diabetologia. 62 (7), 1212-1224 (2019).</li><li>Hughes, J. W., Ustione, A., Lavagnino, Z., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+islet+glucagon+secretion:+Beyond+calcium.">Regulation of islet glucagon secretion: Beyond calcium.</a> Diabetes, Obesity and Metabolism. 20, 127-136 (2018).</li><li>Chen, C., Cohrs, C. M., Stertmann, J., Bozsak, R., Speier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+beta+cell+mass+and+function+in+diabetes:+Recent+advances+in+knowledge+and+technologies+to+understand+disease+pathogenesis.">Human beta cell mass and function in diabetes: Recent advances in knowledge and technologies to understand disease pathogenesis.</a> Molecular Metabolism. 6 (9), 943-957 (2017).</li><li>Halban, P. 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=&#946;-cell+failure+in+type+2+diabetes:+Postulated+mechanisms+and+prospects+for+prevention+and+treatment.">&#946;-cell failure in type 2 diabetes: Postulated mechanisms and prospects for prevention and treatment.</a> Journal of Clinical Endocrinology and Metabolism. 99 (6), 1983-1992 (2014).</li><li>Brissova, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;+cell+function+and+gene+expression+are+compromised+in+type+1+diabetes.">&#945; cell function and gene expression are compromised in type 1 diabetes.</a> Cell Reports. 22 (10), 2601-2614 (2018).</li><li>Walker, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+human+pseudoislet+system+and+microfluidic+platform+demonstrate+differences+in+GPCR+signaling+in+islet+cells.">Integrated human pseudoislet system and microfluidic platform demonstrate differences in GPCR signaling in islet cells.</a> JCI Insight. 5 (10), e06990 (2020).</li><li>Giannoukakis, 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=Infection+of+intact+human+islets+by+a+lentiviral+vector.">Infection of intact human islets by a lentiviral vector.</a> Gene Therapy. 6 (9), 1545-1551 (1999).</li><li>Curran, M. 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=Efficient+transduction+of+pancreatic+islets+by+feline+immunodeficiency+virus+vectors1.">Efficient transduction of pancreatic islets by feline immunodeficiency virus vectors1.</a> Transplantation. 74 (3), 299-306 (2002).</li><li>Kayton, N. S., 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=Human+islet+preparations+distributed+for+research+exhibit+a+variety+of+insulin-secretory+profiles.">Human islet preparations distributed for research exhibit a variety of insulin-secretory profiles.</a> American Journal of Physiology-Endocrinology and Metabolism. 308 (7), E592-E602 (2015).</li><li>Cabrera, O., 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=high-throughput+assays+for+evaluation+of+human+pancreatic+islet+function.">high-throughput assays for evaluation of human pancreatic islet function.</a> Cell Transplantation. 16 (10), 1039-1048 (2007).</li><li>Lenguito, 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=Resealable,+optically+accessible,+PDMS-free+fluidic+platform+for+ex+vivo+interrogation+of+pancreatic+islets.">Resealable, optically accessible, PDMS-free fluidic platform for ex vivo interrogation of pancreatic islets.</a> Lab on a Chip. 17 (5), 772-781 (2017).</li><li>Tewson, P. H., Martinka, S., Shaner, N. C., Hughes, T. E., Quinn, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+DAG+and+cAMP+sensors+optimized+for+live-cell+assays+in+automated+laboratories.">New DAG and cAMP sensors optimized for live-cell assays in automated laboratories.</a> Journal of Biomolecular Screening. 21 (3), 298-305 (2015).</li><li>Tengholm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+dynamics+in+the+pancreatic+&#946;-cell.">Cyclic AMP dynamics in the pancreatic &#946;-cell.</a> Upsala Journal of Medical Sciences. 117 (4), 355-369 (2012).</li><li>Klemen, M. S., Dolen&#353;ek, J., Rupnik, M. S., Sto&#382;er, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+triggering+pathway+to+insulin+secretion:+Functional+similarities+and+differences+between+the+human+and+the+mouse+&#946;+cells+and+their+translational+relevance.">The triggering pathway to insulin secretion: Functional similarities and differences between the human and the mouse &#946; cells and their translational relevance.</a> Islets. 9 (6), 109-139 (2017).</li></ol>

The authors have nothing to disclose.

The integration of a microperifusion system, biosensor-expressing pseudoislets, and laser-scanning confocal microscopy allows for the synchronous assessment of intracellular signaling events and dynamic hormone secretory profiles. The dynamic microperifusion system can deliver a series of well-defined stimuli to the pseudoislets and allows for the collection of the effluent, in which the insulin and glucagon concentrations can be measured by commercially available ELISA. Concurrently, live-cell imaging of the biosensor-e...

The islets of Langerhans are mini organs scattered throughout the pancreas whose function is crucial for the maintenance of glucose homeostasis. Insulin is secreted from beta cells following the metabolism of glucose, an increase in the ATP/ADP ratio, the closure of ATP-sensitive potassium channels, depolarization of the plasma membrane, and the influx of extracellular calcium1. Glucagon secretion from alpha cells is less understood, but it has been postulated that intracellular and paracrine pathways contribute to glucagon granule exocytosis2,3,4. Both type 1 and type 2 diabetes are associated with islet cell dysfunction5,6,7. Therefore, elucidating the intracellular signaling pathways mediating islet hormone secretion is essential for understanding physiologic and pathologic mechanisms in pancreatic islets.
The spherical architecture of islets presents certain obstacles to experimentation. These challenges include islet size variation and the 3D nature of islets, which reduces viral transduction within the islet core8,9. To overcome these challenges, a pseudoislet system was developed, in which primary human islets are dispersed into single cells, adenovirally transduced with constructs encoding targets of interest, and reaggregated to form size-controlled, islet-like structures termed pseudoislets7. Compared to native islets from the same donor that have been cultured in parallel, these pseudoislets are similar in morphology, endocrine cell composition, and hormone secretion7. This method allows for the expression of constructs throughout the pseudoislet, meaning it overcomes a previous barrier to the uniform genetic manipulation of primary human islets7,8,9.
In this protocol, the pseudoislet system is integrated with a microfluidic device to express biosensors in primary human islet cells and gain temporal resolution of&#160;pseudoislet hormone secretion during dynamic perifusion10,11,12. The pseudoislets are placed in a microchip and exposed to a steady flow of different secretagogues via a peristaltic pump12. The microchip has a transparent glass bottom and is mounted on a confocal microscope to record the intracellular signaling dynamics via changes in the biosensor fluorescence intensity. Biosensor imaging is synchronized with the collection of microperifusion effluent for the subsequent analysis of insulin and glucagon secretion7. Compared to macroperifusion, this microperifusion approach allows for fewer pseudoislets to be used due to the smaller volume of the microfluidic device compared to the macroperifusion chamber7.
To harness the utility of this system, the cyclic adenosine monophosphate (cAMP) difference detector in situ (cADDis) biosensor was expressed in human pseudoislets to assess cAMP dynamics and hormone secretion. The cADDis biosensor is composed of a circularly permuted green fluorescent protein (cpGFP) positioned in the hinge region of an exchange protein activated by cAMP 2 (EPAC2), connecting its regulatory and catalytic regions. The binding of cAMP to the regulatory region of EPAC2 elicits a conformational change in the hinge region that increases fluorescence from the cpGFP13. Intracellular messengers such as cAMP elicit insulin and glucagon secretion after the upstream activation of G-protein coupled receptors14. Live-cell imaging coupled with microperifusion helps to connect the intracellular cAMP dynamics with islet hormone secretion. Specifically, in this protocol, cADDis-expressing pseudoislets are generated to monitor cAMP responses in alpha and beta cells to various stimuli: low glucose (2 mM glucose; G 2), high glucose plus isobutylmethylxanthine (IBMX; 20 mM glucose + 100 &#181;M IBMX; G 20 + IBMX), and low glucose plus epinephrine (Epi; 2 mM glucose + 1 &#181;M Epi; G 2 + Epi). This treatment workflow allows for the assessment of the intracellular cAMP dynamics directly via 1) IBMX-mediated phosphodiesterase inhibition, which enhances intracellular cAMP levels by preventing its degradation, and 2) epinephrine, a known cAMP-dependent stimulator of alpha cell glucagon secretion mediated by &#946;-adrenergic receptor activation. The steps for setting up the microperifusion apparatus for live-cell imaging experiments, the loading of the pseudoislets into the microchip, synchronous live-cell imaging and microperifusion, and the analysis of the biosensor traces and hormone secretion by microplate-based hormone assays are detailed below.

<table><tbody><tr><td>Ad-CMV-cADDis</td><td>Welgen</td><td>Not applicable</td><td></td></tr><tr><td>&amp;nbsp;0.01&amp;rdquo; FEP tubing</td><td>IDEX</td><td>1527L</td><td></td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630-080</td><td>Enriched-CMRL Media Component</td></tr><tr><td>1.5 mL and conical tubes</td><td>Any</td><td>Any</td><td></td></tr><tr><td>10 &amp;mu;m PTFE filter</td><td>Cole-Parmer</td><td>SK-21940-41</td><td>Change every 8-10 runs</td></tr><tr><td>100 mM Sodium Pyruvate</td><td>Thermo Scientific</td><td>11360070</td><td>Enriched-CMRL Media Component</td></tr><tr><td>190 proof Ethanol</td><td>Decon labs</td><td>2816</td><td>Acid Ethanol Component</td></tr><tr><td>200 mM GlutaMAX-I Supplement</td><td>Gibco</td><td>35050061</td><td>Enriched-CMRL Media Component</td></tr><tr><td>Ascorbate</td><td>Sigma</td><td>A5960</td><td>DMEM Perifusion Buffer Component</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A7888</td><td>DMEM Perifusion Buffer Component</td></tr><tr><td>Bubble trap&amp;nbsp;</td><td>Omnifit</td><td>006BT</td><td></td></tr><tr><td>CellCarrier ULA&amp;nbsp;96-well Microplates</td><td>Perkin Elmer</td><td>6055330</td><td></td></tr><tr><td>cellSens analysis software</td><td>Olympus</td><td>v3.1</td><td>Software used for data analysis</td></tr><tr><td>CMRL 1066</td><td>MediaTech&amp;nbsp;</td><td>15-110-CV</td><td>Enriched-CMRL Media Component</td></tr><tr><td>Conical adapter (IDEX, P-794)</td><td>IDEX</td><td>P-794</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7528</td><td>Glucose Buffer Component</td></tr><tr><td>DMEM&amp;nbsp;</td><td>Sigma</td><td>D5030</td><td>DMEM Perifusion Buffer Component</td></tr><tr><td>Environmental chamber</td><td>okolab</td><td>IX83</td><td></td></tr><tr><td>Epinepherine (Epi)</td><td>Sigma</td><td>E4250</td><td>Stimulation Buffer Component</td></tr><tr><td>Fetal Bovine Serum (FBS), Heat Inactivated</td><td>Sigma</td><td>12306C</td><td>Enriched-CMRL Media Component</td></tr><tr><td>Glucagon ELISA</td><td>Mercodia</td><td>10-1281-01</td><td></td></tr><tr><td>Glucagon Kit HTRF</td><td>Cisbio</td><td>62CGLPEH</td><td></td></tr><tr><td>HCl (12N)</td><td>Any</td><td>Any</td><td>Acid Ethanol Component</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H7523</td><td>DMEM Perifusion Buffer Component</td></tr><tr><td>iCell Endothelial Cells Medium Supplement</td><td>Cell Dynamics</td><td>M1019</td><td>iEC Media Component</td></tr><tr><td>Idex Derlin nut &amp;amp; ferrule 1/4-24</td><td>Cole-Parmer</td><td>EW-00414-LW</td><td></td></tr><tr><td>Insulin ELISA</td><td>Mercodia</td><td>10-1113-01</td><td></td></tr><tr><td>Isobutylmethylonine (IBMX)</td><td>Sigma</td><td>I5879</td><td>Stimulation Buffer Component</td></tr><tr><td>Laser scanning confocal microscope</td><td>Olympus</td><td>FV3000</td><td></td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G8540</td><td>DMEM Perifusion Buffer Component</td></tr><tr><td>Microchip (University of Miami, FP-3W)</td><td>University of Miami</td><td>FP-3W</td><td></td></tr><tr><td>Microchip holder&amp;nbsp;</td><td>Micronit Microfluidics</td><td>FC_PRO_CH4525</td><td></td></tr><tr><td>Model 2110 Fraction Collector</td><td>Biorad</td><td>7318122</td><td></td></tr><tr><td>P10, P200, and P1000 pipets and tips</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Enriched-CMRL Media Component</td></tr><tr><td>Peristaltic pump&amp;nbsp;</td><td>Instech</td><td>P720</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Gibco</td><td>14190-144</td><td>Wash Islets</td></tr><tr><td>Sarstedt dishes</td><td>Sarstedt</td><td>depends on dish diameter</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S6014</td><td>DMEM Perifusion Buffer Component</td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>P2256</td><td>&amp;nbsp;DMEM Perifusion Buffer Component</td></tr><tr><td>Stereoscope</td><td>Olympus</td><td>SZX12</td><td></td></tr><tr><td>Steriflip Filter (0.22 &amp;mu;m)</td><td>Millipore</td><td>SCGP00525</td><td>Filter all buffers twice</td></tr><tr><td>VascuLife VEGF Medium Complete Kit</td><td>LifeLine Cell Technology</td><td>LL-0003</td><td>iEC Media Component</td></tr></tbody></table>

human pseudoislet system for synchronous assessment of fluorescent biosensor dynamics and hormone secretory profiles

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

Biosensor-expressing human pseudoislets were created via the adenoviral delivery of constructs encoding the cAMP biosensor cADDis (Figure 1A). Figure 1B shows the reaggregation of the transduced human islet cells over time, with fully formed pseudoislets observed after 6 days of culture. The cells began to show visible cADDis fluorescence within 48 h, and there was high biosensor expression in transduced cells by the end of the culture period. Using thi...

Watch this Scientific Journal Video about Investigating Islet Abnormalities and Function with a Pseudoislet Protocol at JoVE.com

Author Spotlight: Investigating Islet Abnormalities and Function with a Pseudoislet Protocol 

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, ...

human-pseudoislet-system-for-synchronous-assessment-fluorescent

Research

JoVE Journal

Biology

1.8K Views.  Vanderbilt University School of Medicine. This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system. 

Video: Author Spotlight: Investigating Islet Abnormalities and Function with a Pseudoislet Protocol 

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Genetics

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 &mu;m diameter and 150 &mu;m depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 &mu;m in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Medicine

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

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.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic &#946; cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.

This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes.

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, ...

Adult Pig Islet Isolation

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic &#946; cells, which results in little or no insulin production. Islet transplantation plays an important role in the treatment of T1DM, with the improved glycometabolic control, the reduced progression of complications, the reduction of hypoglycemic episodes when compared with traditional insulin therapy. The results of phase III clinical trial also demonstrated the safety and efficacy of islet allotransplantation in T1DM. However, the shortage of pancreas donors limits its widespread use. Animals as a source of islets such as the pig offer an alternative choice. Because the architecture of the pig pancreas is different from the islets of mice or humans, the pig islet isolation procedure is still challenging. Since the translation of alternative porcine islet sources (xenogeneic) to the clinical setting for treating T1DM through cellular transplantation is of great importance, a cost-effective, standardized, and reproducible protocol for isolating porcine islets is urgently needed. This manuscript describes a simplified and cost-effective method to isolate and purify adult porcine islets based on the previous protocols that have successfully transplanted porcine islets to non-human primates. This will be a beginners guide without the use of specialized equipment such as a COBE 2991 Cell Processor.

This detailed protocol covers the methodological steps of adult pig islet isolation from the digestion phase via purification to the final functional assessment of the islets. This outline can be used as a guideline for adult pig islet isolation in research institutions.

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic &#946; cells, which results in little or no insulin production. Islet ...

Slicing Human Pancreas for Islet Function Research

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

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the pancreas endocrine and exocrine physiology and interplay. Much has been learned from the study of isolated pancreatic islets, but this prevents examining their function and interactions in the context of the whole tissue. Pancreas slices provide a unique opportunity to explore the physiology of normal, inflamed, and structurally damaged islets within their native environment, in turn allowing the study of interactions between endocrine and exocrine compartments to better investigate the complex dynamics of pancreatic tissue. Thus, the adoption of the living pancreas slice platform represents a significant advancement in the field. This protocol describes how to generate living tissue slices from deceased organ donors by tissue embedding in agarose and vibratome slicing as well as their utilization to assess functional readouts such as dynamic secretion and live cell imaging.

Author Spotlight: Advancing Type 1 Diabetes Research Using Innovative Pancreatic Slice Platforms

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.

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the ...

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles

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Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

Adult Pig Islet Isolation

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