Name: imaging calcium dynamics in subpopulations of mouse pancreatic islet cells
Uploaded: 2019-11-26T12:00:00
Description: Watch this Scientific Journal Video about Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells at JoVE.com

AH was a recipient of a Diabetes UK PhD Studentship, EV was supported by the OXION-Wellcome Trust Training Programme, AIT held an Oxford Biomedical Research Council postdoctoral fellowship.

All methods described here were developed in accordance with the United Kingdom Animals (Scientific Procedures) Act (1986) and the University of Oxford ethical guidelines.
1. Isolate mouse pancreatic islets
<ol>
	<li>Prepare the culture medium and the isolation solution.
	<ol>
		<li>Make up the culture medium: RMPI1640 (see Table of Materials), supplemented with 10% of fetal calf serum, 100 units/mL penicillin, 100 &#956;g/mL streptomycin. Dispense the medium into two 60 mm ...

<ol>
	<li>Elayat, A. A., el-Naggar, M. M., Tahir, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunocytochemical+and+morphometric+study+of+the+rat+pancreatic+islets.">An immunocytochemical and morphometric study of the rat pancreatic islets.</a> Journal of Anatomy. 186, 629 (1995).</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=The+unique+cytoarchitecture+of+human+pancreatic+islets+has+implications+for+islet+cell+function.">The unique cytoarchitecture of human pancreatic islets has implications for islet cell function.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (7), 2334-2339 (2006).</li><li>Brereton, M. F., Vergari, E., Zhang, Q., Clark, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-,+delta-and+PP-cells:+are+they+the+architectural+cornerstones+of+islet+structure+and+co-ordination.">Alpha-, delta-and PP-cells: are they the architectural cornerstones of islet structure and co-ordination.</a> Journal of Histochemistry &amp; Cytochemistry. 63 (8), 575-591 (2015).</li><li>F&#230;rch, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+is+accompanied+by+increased+fasting+glucagon+and+delayed+glucagon+suppression+in+individuals+with+normal+and+impaired+glucose+regulation.">Insulin resistance is accompanied by increased fasting glucagon and delayed glucagon suppression in individuals with normal and impaired glucose regulation.</a> Diabetes. 65 (11), 3473-3481 (2016).</li><li>Dabrowski, M., Tarasov, A., Ashcroft, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+architecture+of+the+ATP-binding+site+of+the+KATP+channel+subunit+Kir6+2.">Mapping the architecture of the ATP-binding site of the KATP channel subunit Kir6 2.</a> The Journal of Physiology. 557 (2), 347-354 (2004).</li><li>Liu, Y. J., Vieira, E., Gylfe, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+store-operated+mechanism+determines+the+activity+of+the+electrically+excitable+glucagon-secreting+pancreatic+alpha-cell.">A store-operated mechanism determines the activity of the electrically excitable glucagon-secreting pancreatic alpha-cell.</a> Cell Calcium. 35 (4), 357-365 (2004).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+Ca+2++probe+composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise Ca 2+ probe composed of a single green fluorescent protein.</a> Nature Biotechnology. 19 (2), 137 (2001).</li><li>Nagai, T., Sawano, A., Park, E. S., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circularly+permuted+green+fluorescent+proteins+engineered+to+sense+Ca2.">Circularly permuted green fluorescent proteins engineered to sense Ca2.</a> Proceedings of the National Academy of Sciences. 98 (6), 3197-3202 (2001).</li><li>Broussard, G. J., Liang, R., Tian, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+activity+in+neural+circuits+with+genetically+encoded+indicators.">Monitoring activity in neural circuits with genetically encoded indicators.</a> Frontiers in Molecular Neuroscience. 7, 97 (2014).</li><li>Akerboom, J., 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=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> Journal of Neuroscience. 32 (40), 13819-13840 (2012).</li><li>Rodriguez, E. 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+growing+and+glowing+toolbox+of+fluorescent+and+photoactive+proteins.">The growing and glowing toolbox of fluorescent and photoactive proteins.</a> Trends in Biochemical Sciences. 42 (2), 111-129 (2017).</li><li>Tarasov, A. I., Rutter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in enzymology. 542, 289-311 (2014).</li><li>Tarasov, A. I., 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=Frequency-dependent+mitochondrial+Ca(2+)+accumulation+regulates+ATP+synthesis+in+pancreatic+&#946;+cells.">Frequency-dependent mitochondrial Ca(2+) accumulation regulates ATP synthesis in pancreatic &#946; cells.</a> Pflugers Archiv: European Journal of Physiology. 465 (4), 543-554 (2013).</li><li>Smith, N. 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=Fluorescent+Ca2++indicators+directly+inhibit+the+Na,+K-ATPase+and+disrupt+cellular+functions.">Fluorescent Ca2+ indicators directly inhibit the Na, K-ATPase and disrupt cellular functions.</a> Science Signal. 11 (515), 2039 (2018).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expanded+palette+of+genetically+encoded+Ca2++indicators.">An expanded palette of genetically encoded Ca2+ indicators.</a> Science. 333 (6051), 1888-1891 (2011).</li><li>Dana, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+red+protein+calcium+indicators+for+imaging+neural+activity.">Sensitive red protein calcium indicators for imaging neural activity.</a> Elife. 5, 12727 (2016).</li><li>Theis, L., 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=Benchmarking+Spike+Rate+Inference+in+Population+Calcium+Imaging.">Benchmarking Spike Rate Inference in Population Calcium Imaging.</a> Neuron. 90 (3), 471-482 (2016).</li><li>Hamilton, 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=Adrenaline+Stimulates+Glucagon+Secretion+by+Tpc2-Dependent+Ca(2+)+Mobilization+From+Acidic+Stores+in+Pancreatic+alpha-Cells.">Adrenaline Stimulates Glucagon Secretion by Tpc2-Dependent Ca(2+) Mobilization From Acidic Stores in Pancreatic alpha-Cells.</a> Diabetes. 67 (6), 1128-1139 (2018).</li><li>Adriaenssens, A. E., 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=Transcriptomic+profiling+of+pancreatic+alpha,+beta+and+delta+cell+populations+identifies+delta+cells+as+a+principal+target+for+ghrelin+in+mouse+islets.">Transcriptomic profiling of pancreatic alpha, beta and delta cell populations identifies delta cells as a principal target for ghrelin in mouse islets.</a> Diabetologia. 59 (10), 2156-2165 (2016).</li><li>DiGruccio, M. R., 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=Comprehensive+alpha,+beta+and+delta+cell+transcriptomes+reveal+that+ghrelin+selectively+activates+delta+cells+and+promotes+somatostatin+release+from+pancreatic+islets.">Comprehensive alpha, beta and delta cell transcriptomes reveal that ghrelin selectively activates delta cells and promotes somatostatin release from pancreatic islets.</a> Molecular Metabolism. 5 (7), 449-458 (2016).</li><li>Mourao, M., Satin, L., Schnell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+experimental+design+to+estimate+statistically+significant+periods+of+oscillations+in+time+course+data.">Optimal experimental design to estimate statistically significant periods of oscillations in time course data.</a> PloS One. 9 (4), 93826 (2014).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> Journal of Visualized Experiments. (50), (2011).</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=Glutamate+is+a+positive+autocrine+signal+for+glucagon+release.">Glutamate is a positive autocrine signal for glucagon release.</a> Cell Metabolism. 7 (6), 545-554 (2008).</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Transactions on Image Processing. 7 (1), 27-41 (1998).</li><li>Tarasov, A. I., 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=Monitoring+real-time+hormone+release+kinetics+via+high-content+3-D+imaging+of+compensatory+endocytosis.">Monitoring real-time hormone release kinetics via high-content 3-D imaging of compensatory endocytosis.</a> Lab on a Chip. 18 (18), 2838-2848 (2018).</li><li>Knudsen, J. 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=Dysregulation+of+Glucagon+Secretion+by+Hyperglycemia-Induced+Sodium-Dependent+Reduction+of+ATP+Production.">Dysregulation of Glucagon Secretion by Hyperglycemia-Induced Sodium-Dependent Reduction of ATP Production.</a> Cell Metabolism. , (2018).</li><li>Adam, J., 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=Fumarate+hydratase+deletion+in+pancreatic+&#946;+cells+leads+to+progressive+diabetes.">Fumarate hydratase deletion in pancreatic &#946; cells leads to progressive diabetes.</a> Cell Reports. 20 (13), 3135-3148 (2017).</li><li>Wills, Q. F., 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=Statistical+approaches+and+software+for+clustering+islet+cell+functional+heterogeneity.">Statistical approaches and software for clustering islet cell functional heterogeneity.</a> Islets. 8 (2), 48-56 (2016).</li><li>Berggren, P. O., Ostenson, C. G., Petersson, B., Hellman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+divergent+glucose+effects+on+calcium+metabolism+in+pancreatic+beta-+and+alpha+2-cells.">Evidence for divergent glucose effects on calcium metabolism in pancreatic beta- and alpha 2-cells.</a> Endocrinology. 105 (6), 1463-1468 (1979).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> The Journal of Biological Chemistry. 260 (6), 3440-3450 (1985).</li><li>Longo, E. 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=Oscillations+in+cytosolic+free+Ca2+,+oxygen+consumption,+and+insulin+secretion+in+glucose-stimulated+rat+pancreatic+islets.">Oscillations in cytosolic free Ca2+, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets.</a> The Journal of Biological Chemistry. 266 (14), 9314-9319 (1991).</li><li>Johansson, H., Gylfe, E., Hellman, B. <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+raises+cytoplasmic+calcium+in+pancreatic+alpha+2-cells+by+mobilizing+calcium+incorporated+in+response+to+glucose.">Cyclic AMP raises cytoplasmic calcium in pancreatic alpha 2-cells by mobilizing calcium incorporated in response to glucose.</a> Cell Calcium. 10 (4), 205-211 (1989).</li><li>Grapengiesser, E., Gylfe, E., Hellman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+types+of+cytoplasmic+Ca2++oscillations+in+stimulated+pancreatic+&#946;-cells.">Three types of cytoplasmic Ca2+ oscillations in stimulated pancreatic &#946;-cells.</a> Archives of Biochemistry and Biophysics. 268 (1), 404-407 (1989).</li><li>Okamoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cytosolic+Ca2++in+impaired+sensitivity+to+glucose+of+rat+pancreatic+islets+exposed+to+high+glucose+in+vitro.">Role of cytosolic Ca2+ in impaired sensitivity to glucose of rat pancreatic islets exposed to high glucose in vitro.</a> Diabetes. 41 (12), 1555-1561 (1992).</li><li>Gilon, P., Henquin, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+membrane+potential+changes+on+cytoplasmic+Ca2++concentration+in+an+electrically+excitable+cell,+the+insulin-secreting+pancreatic+B-cell.">Influence of membrane potential changes on cytoplasmic Ca2+ concentration in an electrically excitable cell, the insulin-secreting pancreatic B-cell.</a> The Journal of Biological Chemistry. 267 (29), 20713-20720 (1992).</li><li>Valdeolmillos, M., Nadal, A., Soria, B., Garcia-Sancho, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+digital+image+analysis+of+glucose-induced+[Ca2+]i+oscillations+in+mouse+pancreatic+islets+of+Langerhans.">Fluorescence digital image analysis of glucose-induced [Ca2+]i oscillations in mouse pancreatic islets of Langerhans.</a> Diabetes. 42 (8), 1210-1214 (1993).</li><li>Cheng, H., Lederer, W. J., Cannell, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sparks:+elementary+events+underlying+excitation-contraction+coupling+in+heart+muscle.">Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle.</a> Science. 262 (5134), 740-744 (1993).</li><li>Stout, A. L., Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evanescent+field+excitation+of+fluorescence+by+epi-illumination+microscopy.">Evanescent field excitation of fluorescence by epi-illumination microscopy.</a> Applied Optics. 28 (24), 5237-5242 (1989).</li><li>Stozer, A., Dolensek, J., Rupnik, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-stimulated+calcium+dynamics+in+islets+of+Langerhans+in+acute+mouse+pancreas+tissue+slices.">Glucose-stimulated calcium dynamics in islets of Langerhans in acute mouse pancreas tissue slices.</a> PloS One. 8 (1), 54638 (2013).</li><li>Tian, G., Sandler, S., 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-+and+hormone-induced+cAMP+oscillations+in+alpha-+and+beta-cells+within+intact+pancreatic+islets.">Glucose- and hormone-induced cAMP oscillations in alpha- and beta-cells within intact pancreatic islets.</a> Diabetes. 60 (5), 1535-1543 (2011).</li><li>Benninger, R. K., Zhang, M., Head, W. S., Satin, L. S., 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=Gap+junction+coupling+and+calcium+waves+in+the+pancreatic+islet.">Gap junction coupling and calcium waves in the pancreatic islet.</a> Biophysical Journal. 95 (11), 5048-5061 (2008).</li><li>van Gurp, L., 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=Sequential+intravital+imaging+reveals+in+vivo+dynamics+of+pancreatic+tissue+transplanted+under+the+kidney+capsule+in+mice.">Sequential intravital imaging reveals in vivo dynamics of pancreatic tissue transplanted under the kidney capsule in mice.</a> Diabetologia. 59 (11), 2387-2392 (2016).</li></ol>

There are three stages in the protocol that are critical for overall success. Successful injection of the Liberase enzyme into the bile duct determines not just the quantitative success of the isolation procedure but also impacts the quality of the isolated islets. Uninflated pancreata may result in lack of some important metabolic responses in the isolated islets. Secondly, the loading of the dye/the expression of the sensor defines the signal-to-noise ratio of the time-lapse recording. Signals are absent or attenuated ...

The purpose of the method is to image real-time changes in cytosolic calcium concentration ([Ca2+]cyt) in minor subpopulations of pancreatic islet cells. This allows uncovering the mechanisms governing hormone secretion in these cells, revealing details about the cross-talk between different cell types and, potentially, introducing a populational dimension into the larger picture of islet signaling.
Islets consist of several cell types. Besides the more well-known insulin-secreting &#946;-cells, there are at least two subpopulations that are also critical in regulating blood glucose3. &#945;-Cells (that make up around 17% of islet cells) secrete glucagon when blood glucose gets too low, which signals for release of glucose into the bloodstream from depots in the liver. Excessive glucagon levels (hyperglucagonemia) and impaired control of glucagon-release accompany (and, technically, can contribute to) the prediabetic condition of impaired insulin sensitivity4. &#948;-Cells (around 2%) secrete somatostatin in response to glucose elevation. This ubiquitous peptide hormone is likely to be present at high concentrations in the vicinity of &#945;- and &#946;-cells within islets, which has a strong Gi receptor-mediated attenuating effect on both glucagon and insulin secretion.
&#945;-Cells and &#948;-cells share a large part of the glucose-sensing machinery with their close lineage relatives, &#946;-cells. All three cells types are equipped with ATP-sensitive K+ channels, elaborate metabolic sensors5 that control the plasma membrane potential of these excitable cells. At the same time, secretion of insulin, somatostatin and glucagon is regulated differently by glucose. Imaging of Ca2+ dynamics in the two minor subpopulations of islet cells can therefore provide an insight into the cross-talk between blood glucose and islet secretory output.
Early attempts of monitoring the excitability of &#945;- and &#948;-cells using patch-clamp electrophysiology were soon followed by imaging of Ca2+ in single &#945;- and &#948;-cells. The identity of cells in these experiments was verified via a posteriori staining with anti-glucagon or anti-somatostatin antibodies. These efforts were frequently hampered by the finding that islet cells behave very differently within the islet and as single cells. Although &#946;-cells may appear to be the main benefactors of the islet arrangement (due to their overwhelming majority that underlies their strong electrical coupling), the main discrepancy was, surprisingly, found in &#945;-cells. Within the intact islet, these cells are constantly and persistently activated at low glucose, which is only true for around 7% of single dispersed &#945;-cells6. Reporting the activity of &#945;- and &#948;-cells within intact islets is therefore believed to represent a closer approximation of in vivo conditions.
In general, there are two ways of reporting Ca2+ dynamics specifically from the &#945;-cell or &#948;-cell subpopulations: (i) expressing a genetically encoded Ca2+ sensor via a tissue-specific promoter or (ii) using marker compounds. The more elegant former approach adds the substantial advantage of true 3D imaging and hence studying of cell distribution within the islet. It cannot however be applied for intact human islet material. Another potential concern is the &#39;leakiness&#39; of the promoter, particularly when the &#946;-/&#945;-cell transdifferentiation or &#945;-cell response to high glucose is in place. The latter approach can be used with freshly isolated tissue including human samples or cultured islets. The data, however, is collected solely from the peripheral layer of islet cells, as delivering the dye/marker molecule in deeper layers without altering the islet architecture is challenging. An unexpected advantage of the latter approach is the compatibility with wide-field imaging mode, which allows scaling up the experiments to simultaneous imaging of tens or hundreds of islets (i.e., thousands to tens of thousands of cells).
Calcium is imaged in vivo using genetically encoded GCaMP7 (or pericam8) family sensors, which are variants of circularly permutated green fluorescent protein (GFP) fused to the calcium-binding protein calmodulin and its target sequence, M13 fragment of myosin light chain kinase7,9. GCaMPs have superb signal-to-noise ratios in the range of nanomolar Ca2+ concentrations and a high 2-photon cross-section, which makes them an ideal choice for in vivo work10,11. The challenging aspect of using recombinant sensors is their delivery into the cells. Heterologous expression requires using a viral vector and multi-hour ex vivo culturing, which frequently raises concerns regarding potential de-differentiation or deterioration of cell functions. Although mouse models pre-engineered to express GCaMP address this problem, they add new challenges by increasing the lead time substantially and limiting the work to a non-human model. Very high sensitivity to changes of intracellular pH is another adverse side of protein-based sensors12, which is, however, less of a problem for sensing oscillatory signals, such as Ca2+.
The advantage of trappable dyes (such as green fluorescent Fluo4) is that they can be loaded into freshly isolated tissue within around an hour. Predictably, trappable dyes have lower signal-to-noise ratios and (much) lower photostability than their recombinant counterparts. We cannot confirm13 the reports of toxicity of the trappable dyes14, however, dye overloading is a frequent problem.
Red recombinant Ca2+ sensors based on circular permutation have been evolving rapidly since 201115, and most recent developments present a strong competition to GCaMPs16 for tissue imaging, given higher depth of penetration of red light. Commercially available red trappable dyes can be used reliably for single-cell imaging but, on the tissue level, cannot compete well with the green analogs.
There is seemingly very little choice of imaging technology for experiments in tissue where out-of-focus light becomes a critical problem. The confocal system provides acceptable single-cell resolution by cancellation of the out-of-focus light with any objective on the NA above 0.3 (for the case of GCaMP6) or 0.8 (trappable dye). In a technical sense, a conventional confocal microscope can be used for simultaneous imaging of [Ca2+]cyt from hundreds (GCaMP) or tens of islets (trappable dye). The only realistic alternative to confocal mode in case of 3D expression of the sensor in tissue is perhaps light-sheet microscopy.
Things are slightly different for the case when the sensor is expressed in the peripheral layer of cells within the islet tissue. For bright recombinant sensors that have a vivid intracellular expression pattern, using a wide-field imaging mode with a low-NA objective may provide sufficient quality and reward the researcher with a substantial increase in the field of view area and hence the throughput. A wide-field system provides poorer spatial resolution, as the out-of-focus light is not cancelled; therefore, imaging tissue with high-NA (low depth of field) objectives is less informative, as the single-cell signal is vastly contaminated by neighboring cells. The contamination is much smaller for low-NA (high depth of field) objectives.
There are tasks, however, for which high throughput and/or sampling rate become a critical advantage. &#945;- and &#948;-cells exhibit substantial heterogeneity, which creates a demand for high sample sizes to reveal the contribution of the subpopulations. Wide-field imaging is fast and more sensitive, with an industrial-scale large field-of-view system imaging hundreds (GCaMP) or tens (Fluo4) of islets at the same signal-to-noise ratio as the confocal experiments on ten or a single islet, respectively. This difference in throughput makes the wide-field system advantageous for populational imaging with a single-cell resolution, which can be especially critical for small subpopulations such as the &#948;-cell one. Likewise, attempts to reconstruct electrical activity from Ca2+ spiking17 would benefit from the higher sampling rate provided by a wide-field imaging mode. At the same time, several &#34;niche&#34; problems like the activity of pancreatic &#945;-cells upon stimulation of the dominating &#946;-cell subpopulation, require the use of a confocal system. A factor that influences the decision towards confocal mode is the presence of substantial contaminating signal from the &#946;-cell subpopulation.
Although using hormone-specific antibody staining to verify the identity of the cells after the imaging experiments is still an option, minor cell subpopulations can be identified using functional marker compounds, such as adrenaline and ghrelin that were shown to selectively stimulate Ca2+ dynamics in &#945;-18 and &#948;-cells19,20, respectively.
The analysis of time-lapse imaging data aims to provide information beyond trivial pharmacology, such as populational heterogeneity, correlation and interaction of different signals. Conventionally, imaging data is analyzed as intensity vs. time and normalized to the initial fluorescence (F/F0). Baseline correction is frequently needed, due to the bleaching of the fluorophore signal or contamination by changes in autofluorescence or pH (typically induced by millimolar levels of glucose12). Ca2+ data can be analyzed in many different ways, but three main trends are to measure changes in the spike frequency, the plateau fraction, or area under the curve, computed vs. time. We found the latter approach advantageous, especially in application to heavily undersampled confocal data. The advantage of the pAUC metric is its sensitivity to both changes in signal frequency and amplitude, whereas computing the frequency requires a substantial number of oscillations21, which is hard to attain using conventional imaging. The limiting factor of pAUC analysis is its high sensitivity to baseline changes.

<table border="0" cellpadding="0" cellspacing="0" style="width:779px;" width="779">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">40x/1.3 objective</td>
			<td style="width:277px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Axiovert 200 microscope</td>
			<td style="width:277px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">emission</td>
			<td style="width:277px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">Excitation</td>
			<td style="width:277px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum</td>
			<td style="width:277px;">Sigma-&#1040;ldrich</td>
			<td style="width:119px;">F7524-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo4</td>
			<td style="width:277px;">ThermoFisher (Life Technologies)</td>
			<td style="width:119px;">&#160; F14201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GCaMP6f, in (human type 5) adenoviral vector</td>
			<td style="width:277px;">Vector Biolabs</td>
			<td style="width:119px;">1910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hanks&#39; solution&#160;</td>
			<td style="width:277px;">ThermoFisher (GibCo, Life Technologies)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liberase</td>
			<td style="width:277px;">Sigma-&#1040;ldrich</td>
			<td style="width:119px;">5401020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin</td>
			<td style="width:277px;">ThermoFisher (GibCo, Life Technologies)</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI medium</td>
			<td style="width:277px;">ThermoFisher (GibCo, Life Technologies)</td>
			<td style="width:119px;">61870044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss LSM510-META confocal system</td>
			<td style="width:277px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Islets load fairly well with the trappable dyes (Figure 1A), unless the lipid composition of the membrane has been affected (e.g., by chronic exposure to fatty acids). The human adenovirus type 5 (Ad5) vector also targets all islet cells (Figure 1B). Problems may arise when more than one recombinant sensor is being expressed in the same cell. Furthermore, islets are typically very well immobilized using the technology described ...

Watch this Scientific Journal Video about Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells at JoVE.com

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic 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 ...

Research

JoVE Journal

Biology

Murine Pancreatic Islet Isolation

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

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

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...