Bovine Chromaffin Cells: Culture and Fluorescence Assay for Secretion

  • Tamou Thahouly
  • Emeline Tanguy
  • Juliette Raherindratsara
  • Marie-France Bader
  • Sylvette Chasserot-Golaz
  • Stéphane Gasman
  • Nicolas VitaleEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2233)


Over the last four decades, chromaffin cells originating from the adrenal medulla have been probably one of the most popular cell models to study neurosecretion at the molecular level. Accordingly, numerous seminal discoveries in the field, including the characterization of role of the cytoskeleton, fusogenic lipids, and soluble N-ethylmaleimide-sensitivefactor attachment protein receptor (SNARE) proteins, have been made using this model. In this chapter, we describe a standard method currently used to isolate and culture bovine chromaffin cells, and we illustrate a catecholamine secretion assay based on the successive transformation of adrenaline into adrenochrome and adrenolutine for fluorescence measurements. We also provide some guidelines for efficient cell recovery and for the use of this assay in the laboratory.

Key words

Chromaffin cells Fluorescence assay Catecholamine secretion 



We thank Dr. F.A. Meunier (University of Queensland, Brisbane, Australia) and Dr. Robert D. Burgoyne (University of Liverpool) for sharing an original fluorescence assay for catecholamine secretion. We are indebted to the Abattoir Municipal of Haguenau (France) for providing us with the fresh bovine adrenal glands. This work was supported by Fondation pour la Recherche Médicale and the ANR grant (ANR-19-CE44-0019) to NV.


  1. 1.
    Bader MF, Holtz RW, Kumakura K, Vitale N (2002) Exocytosis: the chromaffin cell as a model system. Ann N Y Acad Sci 971:178–183CrossRefGoogle Scholar
  2. 2.
    Vitale N, Gensse M, Chasserot-Golaz S, Aunis D, Bader MF (1996) Trimeric G proteins control regulated exocytosis in bovine chromaffin cells: sequential involvement of go associated with secretory granules and Gi3 bound to the plasma membrane. Eur J Neurosci 8:1275–1285CrossRefGoogle Scholar
  3. 3.
    Zeniou-Meyer M, Liu Y, Béglé A, Olanich ME, Hanauer A, Becherer U, Rettig J, Bader MF, Vitale N (2008) The coffin-Lowry syndrome-associated protein RSK2 is implicated in calcium-regulated exocytosis through the regulation of PLD1. Proc Natl Acad Sci U S A 105:8434–8439CrossRefGoogle Scholar
  4. 4.
    Béglé A, Tryoen-Tóth P, de Barry J, Bader MF, Vitale N (2009) ARF6 regulates the synthesis of fusogenic lipids for calcium-regulated exocytosis in neuroendocrine cells. J Biol Chem 284:4836–4845CrossRefGoogle Scholar
  5. 5.
    Domínguez N, Rodríguez M, Machado JD, Borges R (2012) Preparation and culture of adrenal chromaffin cells. Methods Mol Biol 846:223–234CrossRefGoogle Scholar
  6. 6.
    Kloppenborg PW, Island DP, Liddle GW, Michelakis AM, Nicholson WE (1968) A method of preparing adrenal cell suspensions and its applicability to the in vitro study of adrenal metabolism. Endocrinology 82:1053–1058CrossRefGoogle Scholar
  7. 7.
    Hochman J, Perlman RL (1976) Catecholamine secretion by isolated adrenal cells. Biochim Biophys Acta 421:168–175CrossRefGoogle Scholar
  8. 8.
    O’Connor DT, Mahata SK, Mahata M, Jiang Q, Hook VY, Taupenot L (2007) Primary culture of bovine chromaffi n cells. Nat Protoc 2:1248–1253CrossRefGoogle Scholar
  9. 9.
    Krause W, Michael N, Lubke C, Livett BG, Oehme P (1996) Catecholamine release from fractionated chromaffin cells. Eur J Pharmacol 302:223–228CrossRefGoogle Scholar
  10. 10.
    Livett BG, Boksa P, Dean DM, Mizobe F, Lindenbaum MH (1983) Use of isolated chromaffin cells to study basic release mechanisms. J Auton Nerv Syst 7:59–86CrossRefGoogle Scholar
  11. 11.
    Bader MF, Ciesielski-Treska J, Thierse D, Hesketh JE, Aunis D (1981) Immunocytochemical study of microtubules in chromaffi n cells in culture and evidence that tubulin is not an integral protein of the chromaffin granule membrane. J Neurochem 37:917–933CrossRefGoogle Scholar
  12. 12.
    Baker PF, Knight DE (1981) Calcium control of exocytosis and endocytosis in bovine adrenal medullary cells. Philos Trans R Soc Lond Ser B Biol Sci 296:83–103Google Scholar
  13. 13.
    Bader MF, Trifaró JM, Langley OK, Thiersé D, Aunis D (1986) Secretory cell actin-binding proteins: identification of a gelsolin-like protein in chromaffin cells. J Cell Biol 102:636–646CrossRefGoogle Scholar
  14. 14.
    Bader MF, Thiersé D, Aunis D, Ahnert-Hilger G, Gratzl M (1986) Characterization of hormone and protein release from alpha-toxin-permeabilized chromaffin cells in primary culture. J Biol Chem 261:5777–5783PubMedGoogle Scholar
  15. 15.
    Strober W (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol. Appendix 3, Appendix 3BGoogle Scholar
  16. 16.
    Meunier FA, Feng ZP, Molgo J, Zamponi GW, Schiavo G (2002) Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity. EMBO J 21:6733–6743CrossRefGoogle Scholar
  17. 17.
    Heacock RA, Laidlaw BD (1958) Reduction of adrenochrome with ascorbic acid. Nature 182:526–527CrossRefGoogle Scholar
  18. 18.
    Vitale N, Mukai H, Rouot B, Thiersé D, Aunis D, Bader MF (1993) Exocytotis in chromaffin cells. Possible involvement of the heterotrimeric GTP-binding protein Go. J Biol Chem 268:14715–14723PubMedGoogle Scholar
  19. 19.
    Chasserot-Golaz S, Vitale N, Umbrecht-Jenck E, Knight D, Gerke V, Bader MF (2005) Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles. Mol Biol Cell 16:1108–1119CrossRefGoogle Scholar
  20. 20.
    Tryoen-Tóth P, Chasserot-Golaz S, Tu A, Gherib P, Bader MF, Beaumelle B, Vitale N (2013) HIV-1 Tat protein inhibits neurosecretion by binding to phosphatidylinositol 4,5-bisphosphate. J Cell Sci 126:454–463CrossRefGoogle Scholar
  21. 21.
    Gabel M, Delavoie F, Royer C, Thahouly T, Gasman S, Bader MF, Vitale N, Chasserot-Golaz S (2019) Phosphorylation cycling of Annexin A2 Tyr23 is critical for calcium-regulated exocytosis in neuroendocrine cells. Biochim Biophys Acta, Mol Cell Res 1866:1207–1217CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Tamou Thahouly
    • 1
  • Emeline Tanguy
    • 1
  • Juliette Raherindratsara
    • 1
  • Marie-France Bader
    • 1
  • Sylvette Chasserot-Golaz
    • 1
  • Stéphane Gasman
    • 1
  • Nicolas Vitale
    • 1
    Email author
  1. 1.Centre National de la Recherche ScientifiqueUniversité de Strasbourg, Institut des Neurosciences Cellulaires et IntégrativesStrasbourgFrance

Personalised recommendations