Assessment of Beta Cell Viability

Sundararajan Jayaraman1

1 University of Illinois at Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.27
DOI:  10.1002/0471142956.cy0627s55
Online Posting Date:  January, 2011
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Abstract

This unit contains detailed protocols for the simultaneous identification of the human pancreatic β cells and determination of their viability by flow cytometry. The enumeration of β cells is based on the ability of the cell‐permeable form of the zinc‐selective dye, FluoZin‐3‐AM, to bind intracellular labile zinc stored at higher levels in these cells than any other types of cells in the body. Although staining of intracellular labile zinc by FluoZin‐3‐AM is dependent on the metabolic activity of β cells, co‐staining with a mitochondrial transmembrane potential indicator allows the accurate determination of viability. Simultaneous measurement of intracellular antioxidant thiols is also compatible with the detection of β cells containing metabolically active mitochondria. The method for assessing the mitochondrial functionality by flow cytometry described herein is simple to perform and sufficient to detect the viability of β cells in human islet preparations. Curr. Protoc. Cytom. 55:6.27.1‐6.27.16. © 2010 by John Wiley & Sons, Inc.

Keywords: beta cells; flow cytometry; glutathione; labile zinc; mitochondrial function; thiols

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Use of the Cell‐Permeable Zinc‐Selective Dye FluoZin‐3‐AM for the Enumeration of β Cells in Isolated Islet Preparations by Flow Cytometry
  • Alternate Protocol 1: Mitochondrial Transmembrane Potential of β Cells
  • Alternate Protocol 2: Simultaneous Determination of β Cells Displaying Polarized Mitochondria and Antioxidant Thiols
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Use of the Cell‐Permeable Zinc‐Selective Dye FluoZin‐3‐AM for the Enumeration of β Cells in Isolated Islet Preparations by Flow Cytometry

  Materials
  • Human islet preparations
  • 0.05% trypsin/EDTA solution (Invitrogen)
  • CMRL 1066 culture medium (Mediatech), pH 7.3, supplemented with 0.25% human serum albumin (Flexbumin 25% human albumin, Baxter)
  • Trypan blue (0.04% in PBS; Sigma‐Aldrich)
  • Flow analysis buffer: 0.2 % bovine serum albumin (Sigma‐Aldrich) dissolved in PBS (Invitrogen) and filter‐sterilized
  • Mouse immunoglobulin (Ig) coupled with fluorescein isothiocyante (MIg‐FITC; BD Pharmingen)
  • 1 mM FluoZin‐3‐AM (Invitrogen; see recipe)
  • Refrigerated benchtop centrifuge
  • 15‐ml and 50‐ml conical polystyrene centrifuge tubes
  • Vortex
  • Cell strainer (40‐µm; Becton Dickinson)
  • Hemacytometer
  • Bright‐field microscope
  • 12 × 75–mm polystyrene Falcon tissue culture tubes with snap caps
  • 37°C CO 2 incubator
  • Flow cytometer equipped with an argon ion (488 nm) laser and filter sets suitable for collection of the emission wavelength at 530 nm (FluoZin‐3)

Alternate Protocol 1: Mitochondrial Transmembrane Potential of β Cells

  • Freshly diluted 10 µM solution of TMRE in CMRL medium supplemented with human serum albumin (see recipe)

Alternate Protocol 2: Simultaneous Determination of β Cells Displaying Polarized Mitochondria and Antioxidant Thiols

  • Monochlorobimane (mBcl, 100 mM; see recipe)
  • Flow cytometer equipped with a UV laser (325 nm) and a 488‐nm laser
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Figures

Videos

Literature Cited

Literature Cited
   Ashcroft, P.M. and Ashcroft, S.J.H. 1992. Insulin: Molecular Biology to Pathology Oxford University Press, New York.
   Atkinson, M.A. and Gianani, R. 2009. The pancreas in human type 1 diabetes: Providing new answers to age‐old questions. Curr. Opin. Endocrinol. Diabetes Obes. 16:279‐285.
   Baertschiger, R.M., Berney, T., and Morel, P. 2008. Organ preservation in pancreas and islet transplantation. Curr. Opin. Organ Transplant. 13:59‐66.
   Baetens, D., Malaisse‐Lagae, F., Perrelet, A., and Orci, L. 1979. Endocrine pancreas: Three‐dimensional reconstruction shows two types of islets of langerhans. Science 206:1323‐1325.
   Barnett, M.J., McGhee‐Wilson, D., Shapiro, A.M., and Lakey, J.R. 2004. Variation in human islet viability based on different membrane integrity stains. Cell Transplant. 13:481‐488.
   Berg, J.M. and Shi, Y. 1996. The galvanization of biology: A growing appreciation for the roles of zinc. Science 271:1081‐1085.
   Canzoniero, L.M., Turetsky, D.M., and Choi, D.W. 1999. Measurement of intracellular free zinc concentrations accompanying zinc‐induced neuronal death. J. Neurosci. 19:RC31.
   Chen, L.B. 1989. Fluorescent labeling of mitochondria. Methods Cell Biol. 29:103‐123.
   CITR Research Group Collaborators (196). 2009. 2007 update on allogeneic islet transplantation from the Collaborative Islet Transplant Registry (CITR). Cell Transplant. 18:753‐767.
   Dunn, M.F. 2005. Zinc‐ligand interactions modulate assembly and stability of the insulin hexamer—a review. Biometals 18:295‐303.
   Ehrenberg, B., Montana, V., Wei, M.D., Wuskell, J.P., and Loew, L.M. 1988. Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophys. J. 53:785‐794.
   Eisenbarth, G.S. 2007. Update in type 1 diabetes. J. Clin. Endocrinol. Metab. 92:2403‐2407.
   Eizirik, D.L., Colli, M.L., and Ortis, F. 2009. The role of inflammation in insulitis and beta‐cell loss in type 1 diabetes. Nat. Rev. Endocrinol. 5:219‐226.
   Emamaullee, J.A. and Shapiro, A.M. 2007. Factors influencing the loss of beta‐cell mass in islet transplantation. Cell Transplant. 16:1‐8.
   Emdin, S.O., Dodson, G.G., Cutfield, J.M., and Cutfield, S.M. 1980. Role of zinc in insulin biosynthesis. Some possible zinc‐insulin interactions in the pancreatic B‐cell. Diabetologia 19:174‐182.
   Fernández‐Checa, J.C. and Kaplowitz, N. 1990. The use of monochlorobimane to determine hepatic GSH levels and synthesis. Anal. Biochem. 190:212‐219.
   Figlewicz, D.P., Formby, B., Hodgson, A.T., Schmid, F.G., and Grodsky, G.M. 1980. Kinetics of 65zinc uptake and distribution in fractions from cultured rat islets of langerhans. Diabetes 29:767‐773.
   Foster, M.C., Leapman, R.D., Li, M.X., and Atwater, I. 1993. Elemental composition of secretory granules in pancreatic islets of Langerhans. Biophys. J. 64:525‐532.
   Frederickson, C. 2003. Imaging zinc: Old and new tools. Sci STKE 182:pe18.
   Galluzzi, L., Zamzami, N., de La Motte Rouge, T., Lemaire, C., Brenner, C., and Kroemer, G. 2007. Methods for the assessment of mitochondrial membrane permeabilization in apoptosis. Apoptosis 12:803‐813.
   Gee, K.R., Zhou, Z.L., Qian, W.J., and Kennedy, R. 2002. Detection and imaging of zinc secretion from pancreatic beta‐cells using a new fluorescent zinc indicator. J. Am. Chem. Soc. 124:776‐778.
   Haist, R.E. 1971. Functions of the islets of Langerhans. Can. Med. Assoc. J. 105:956‐968.
   Hall, A.G. 1999. Review: The role of glutathione in the regulation of apoptosis. Eur. J. Clin. Invest. 29:238‐245.
   Harlan, D.M., Kenyon, N.S., Korsgren, O., and Roep, B.O; Immunology of Diabetes Society. 2009. Current advances and travails in islet transplantation. Diabetes 58:2175‐2184.
   Haugland, R.P. 2001. Handbook of Fluorescent Probes and Research Products, 8th Ed. Molecular Probes, Eugene, Oreg.
   Hedley, D.W. and Chow, S. 1994. Evaluation of methods for measuring cellular glutathione content using flow cytometry. Cytometry 15:349‐358.
   Hellman, B., Idahl, L.A., Lernmark, A., Sehlin, J., and Täljedal, I.B. 1975. Stimulation of insulin release by thiols. Biochim. Biophys. Acta. 392:101‐109.
   Ichii, H., Inverardi, L., Pileggi, A., Molano, R.D., Cabrera, O., Caicedo, A., Messinger, S., Kuroda, Y., Berggren, P‐O., and Ricordi, C. 2005. A novel method for the assessment of cellular composition and beta‐cell viability in human islet preparations. Am. J. Transplant. 5:1635‐1645.
   Iglesias, I., Bentsi‐Barnes, K., Umeadi, C., Brown, L., Kandeel, F., and Al‐Abdullah, I.H. 2008. Comprehensive analysis of human islets using flow and laser scanning cytometry. Transplant. Proc. 40:351‐354.
   Jayaraman, S. 2005. Flow cytometric determination of mitochondrial membrane potential changes during apoptosis of T lymphocytic and pancreatic beta cell lines: Comparison of tetramethylrhodamineethylester (TMRE), chlorometyl‐X‐rosamine (H2‐CMX‐Ros) and MitoTracker Red 580 (MTR580). J. Immunol. Methods 306:68‐79.
   Jayaraman, S. 2008. A novel method for the detection of viable human pancreatic beta cells by flow cytometry using fluorophores that selectively detect labile zinc, mitochondrial membrane potential and protein thiols. Cytometry A 73:615‐625.
   Jayaraman, A.K. and Jayaraman, S. 2010. Increased level of exogenous zinc induces cytotoxicity and up‐regulates the expression of the ZnT‐1 zinc transporter gene in pancreatic cancer cells. J. Nutr. Biochem. PMID:20392624.
   Khan, M.H. and Harlan, D.M. 2009. Counterpoint: Clinical islet transplantation: Not ready for prime time. Diabetes Care 32:1570‐1574.
   Kin, T., Johnson, P.R., Shapiro, A.M., and Lakey, J.R. 2007. Factors influencing the collagenase digestion phase of human islet isolation. Transplantation 83:7‐12.
   Kroemer, G. and Reed, J.C. 2000. Mitochondrial control of cell death. Nat. Med. 6:513‐519.
   Lacy, P.E. and Scharp, D.W. 1986. Islet transplantation in treating diabetes. Annu. Rev. Med. 37:33‐40.
   Lakey, J.R., Mirbolooki, M., and Shapiro, A.M. 2006. Current status of clinical islet cell transplantation. Methods Mol. Biol. 333:47‐104.
   Latif, Z.A., Noel, J., and Alejandro, R. 1988. A simple method of staining fresh and cultured islets. Transplantation 45:827‐830.
   Leninger, A.L. 1965. The mitochondrion. W.A. Benjamin, New York.
   London, N.J., Swift, S.M., and Clayton, H.A. 1998. Isolation, culture and functional evaluation of islets of Langerhans. Diabetes Metab. 24:200‐207.
   Lukowiak, B., Vandewalle, B., Riachy, R., Kerr‐Conte, J., Gmyr, V., Belaich, S., Lefebvre, J., and Pattou, F. 2001. Identification and purification of functional human beta‐cells by a new specific zinc‐fluorescent probe. J. Histochem. Cytochem. 49:519‐528.
   Marí, M., Morales, A., Colell, A., García‐Ruiz, C., and Fernández‐Checa, J.C. 2009. Mitochondrial glutathione, a key survival antioxidant. Antioxid. Redox Signal. 11:2685‐2700.
   Mårtensson, J., Lai, J.C., and Meister, A. 1990. High‐affinity transport of glutathione is part of a multicomponent system essential for mitochondrial function. Proc. Natl. Acad. Sci. U.S.A. 87:7185‐7189.
   McNary, W.F. Jr. 1954. Zinc‐dithizone reaction of pancreatic islets. J. Histochem. Cytochem. 2:185‐194.
   Métivier, D., Dallaporta, B., Zamzami, N., Larochette, N., Susin, S.A., Marzo, I., and Kroemer, G. 1988. Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO‐1‐triggered apoptosis of Jurkat T lymphoma cells. Comparison of seven mitochondrion‐specific fluorochromes. Immunol. Lett. 61:157‐163.
   Mineo, D., Pileggi, A., Alejandro, R., and Ricordi, C. 2009. Point: Steady progress and current challenges in clinical islet transplantation. Diabetes Care 32:1563‐1569.
   Modica‐Napolitano, J.S. and Aprille, J.R. 1987. Basis for the selective cytotoxicity of rhodamine 123. Cancer Res. 47:4361‐4365.
   Moriarty‐Craige, S.E. and Jones, D.P. 2004. Extracellular thiols and thiol/disulfide redox in metabolism. Annu. Rev. Nutr. 24:481‐509.
   Nath, D.S. and Hering, B.J. 2005. Islet cells replacement therapy. Clin. Lab. Med. 25:541‐556.
   Noguchi, H. and Matsumoto, S. 2008. Islet transplantation at the Diabetes Research Institute Japan. J. Hepatobiliary Pancreat. Surg. 15:278‐283.
   Okamoto, K., Aoyama, Z., Ibaraki, T., Narumi, H., Shibata, M., Kawasaki, Y., Shibata, D., and Komatsu, K. 1951. Production of experimental diabetes mellitus and zinc reaction of islets of Langerhans. Hyogo J. Med. Sci. I:77‐88.
   Paget, M., Murray, H., Bailey, C.J., and Downing, R. 2007. Human islet isolation: semi‐automated and manual methods. Diab. Vasc. Dis. Res. 4:7‐12.
   Papas, K.K., Suszynski, T.M., and Colton, C.K. 2009. Islet assessment for transplantation. Curr. Opin. Organ Transplant. 14:674‐682.
   Petit, P.X., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B., and Gougeon, M.L. 1995. Alterations in mitochondrial structure and function are early events of dexamethasone‐induced thymocyte apoptosis. J. Cell. Biol. 130:157‐167.
   Poot, M., Zhang, Y.Z., Krämer, J.A., Wells, K.S., Jones, L.J., Hanzel, D.K., Lugade, A.G., Singer, V.L., and Haugland, R.P. 1996. Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J. Histochem. Cytochem. 44:1363‐1372.
   Qian, W‐J., Gee, K.R., and Kennedy, R.T. 2003. Imaging of Zn2+ released from pancreatic beta‐cells at the level of single exocytotic events. Anal. Chem. 75:3468‐3475.
   Rahman, I., Biswas, S.K, Jimenez, L.A, Torres, J.M., and Forman, H.J. 2005. Glutathione, stress responses, and redox signaling in lung inflammation. Antioxid. Redox. Signal 7:42‐59.
   Salvioli, S., Dobrucki, J., Moretti, L., Troiano, L., Fernandez, M.G., Pinti, M., Pedrazzi, J., Franceschi, C., and Cossarizza. A. 2000. Mitochondrial heterogeneity during staurosporine‐induced apoptosis in HL60 cells: Analysis at the single cell and single organelle level. Cytometry 40:189‐197.
   Sensi, S.L., Canzoniero, L.M., Yu, S.P., Ying, H.S., Koh, J.Y., Kerchner, G.A., and Choi, D.W. 1997. Measurement of intracellular free zinc in living cortical neurons: routes of entry. J. Neurosci. 17:9554‐9564.
   Shapiro, H.M. 1988. Practical flow cytometry, 2nd Ed. Alan R. Liss, New York.
   Sweet, I.R., Gilbert, M., Scott, S., Todorov, I., Jensen, R., Nair, I., Al‐Abdullah, I., Rawson, J., Kandeel, F., and Ferreri, K. 2008. Glucose‐stimulated increment in oxygen consumption rate as a standardized test of human islet quality. Am. J. Transplant. 8:183‐192.
   Szot, G.L., Lee, M.R., Tavakol, M.M., Lang, J., Dekovic, F., Kerlan, R.K., Stock, P.G., and Posselt, A.M. 2009. Successful clinical islet isolation using a GMP‐manufactured collagenase and neutral protease. Transplantation 88:753‐756.
   Vaithilingam, V., Sundaram, G., and Tuch, B.E. 2008. Islet cell transplantation. Curr. Opin. Organ Transplant. 13:633‐638.
   Vallee, B.L. and Falchuk, K.H. 1993. The chemical basis of zinc physiology. Physiol. Rev. 73:79‐118.
   Vesce, S., Jekabsons, M.B., Johnson‐Cadwell, L.I., and Nicholls, D.G. 2005. Acute glutathione depletion restricts mitochondrial ATP export in cerebellar granule neurons. J. Biol. Chem. 280:38720‐38728.
   Wiederkehr, A. and Wollheim, C.B. 2006. Minireview: Implication of mitochondria in insulin secretion and action. Endocrinology 147:2643‐2649.
   Yamamoto, T., Horiguchi, A., Ito, M., Nagata, H., Ichii, H., Ricordi, C., and Miyakawa, S. 2009. Quality control for clinical islet transplantation: Organ procurement and preservation, the islet processing facility, isolation, and potency tests. J. Hepatobiliary Pancreat. Surg. 16:131‐136.
   Zalewski, P.D., Millard, S.H., Forbes, I.J., Kapaniris, O., Slavotinek, A., Betts, W.H., Ward, A.D., Lincoln, S.F., and Mahadevan, I. 1994. Video image analysis of labile zinc in viable pancreatic islet cells using a specific fluorescent probe for zinc. J. Histochem. Cytochem. 42:877‐884.
   Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssière, J.L., Petit, P.X., and Kroemer, G. 1995. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181:1661‐1672.
   Zhang, Y., Wang, H., Li, J., Jimenez, D.A., Levitan, E.S., Aizenman, E., and Rosenberg, P.A. 2004. Peroxynitrite‐induced neuronal apoptosis is mediated by intracellular zinc release and 12‐lipoxygenase activation. J. Neurosci. 24:10616‐10627.
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