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Analysis of Regulated Secretion Using PC12 Cells

Laurent Taupenot1,2

1University of California at San Diego, La Jolla, California
2Veterans Affairs San Diego Healthcare System, San Diego, California


Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
UNIT 15.12
DOI: 
10.1002/0471143030.cb1512s36
Print Publication Date: 
September, 2007
Online Posting Date: 
September, 2007
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Abstract

The catecholamine-secreting PC12 cell line derived from the rat adrenal medulla has long been considered a model system for neurosecretion and neuronal differentiation. PC12 cells contain a large number of secretory granules (otherwise known as large dense-core vesicles) for storage of small molecules, processing enzymes, neuropeptides, and peptide hormones. Secretory granule exocytosis in PC12 cells is tightly regulated by calcium and occurs in response to a secretagogue. This unit provides protocols for maintenance and transfection of PC12 cells. Several secretion assays are described to measure the release of secretory granule cargo molecules by detection of radioactive catecholamine, or by immunochemical or chemiluminescence detection of transfected regulated secretory proteins. Curr. Protoc. Cell Biol. 36:15.12.1-15.12.13. © 2007 by John Wiley & Sons, Inc.

Keywords: granule; PC12; exocytosis; chromogranin; catecholamine

     
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Radiolabled Catecholamine Release
  • Support Protocol 1: Routine Growth and Maintenance of PC12 Cells
  • Basic Protocol 2: Measuring Exocytosis After Transient Transfection of the Regulated Secretory Reporter Protein CgA-EAP
  • Alternate Protocol 1: Measurement of Human Growth Hormone Release
  • Support Protocol 2: Transfection of PC12 Cells Using Cationic Lipids
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
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Materials

Basic Protocol 1: Measurement of Radiolabled Catecholamine Release

 Materials
  • Early-passage PC12 cells or ATCC-PC12 cells (#CRL-1721)
  • Poly-l-lysine (Sigma, cat. no. P6282)
  • Rat tail collagen Type I (Upstate, cat. no. 08-115)
  • DMEM or F12K basal and complete culture media (see recipe)
  • Levo-[ring-2,5,6-3H]-norepinephrine (specific activity 40 to 80 Ci/mmol; PerkinElmer Life and Analytical Sciences)
  • Calcium saline buffer (CaSB; see recipe)
  • Stimulation buffer (see recipe)
  • Scintillation fluid, water soluble
  • Triton X-100
  • 6-well tissue-culture treated polystyrene plates (e.g., Falcon, Costar)
  • 37°C, CO2 incubator
  • Motorized pipetting aid
  • Scintillation vials
  • Orbital shaker
  • Liquid scintillation beta-counter

CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and surroundings. Carry out the experiment and dispose of wastes in appropriately designated areas, following guidelines provided by the local radiation safety officer (also see appendix 1D).

Support Protocol 1: Routine Growth and Maintenance of PC12 Cells

 Materials
  • Early-passage PC12 cells or ATCC-PC12 cells (#CRL-1721)
  • DMEM or F12K complete culture media (see recipe)
  • 100 µg/ml poly-l-lysine hydrobromide solution (Sigma, no. P6282)
  • 100 µg/ml rat tail collagen Type I (Upstate, no. 08-115) in PBS (appendix 2A)
  • 100-mm cell culture dishes, tissue-culture treated polystyrene (e.g., Becton Dickinson-Falcon, Corning-Costar)
  • 37°C, 6% CO2, humidified incubator

Basic Protocol 2: Measuring Exocytosis After Transient Transfection of the Regulated Secretory Reporter Protein CgA-EAP

 Materials
  • Early-passage PC12 cells or ATCC-PC12 cells (see Support Protocol 1)
  • pCMV-CgA-EAP(N2) [available from the authors]
  • Expression plasmid containing transgene of interest
  • Calcium saline buffer (CaSB; see recipe)
  • Stimulation buffer (see recipe)
  • 0.1% (v/v) Triton X-100 in CaSB
  • Secreted Alkaline Phosphatase Chemiluminescence assay kit (e.g., Phospha-Light, Applied Biosystem, no. T1017) containing:
    • Assay Buffer
    • Reaction Buffer Diluent
  • 3-(4-methoxyspiro[1,2-dioxetane-3,2¢-(5¢chloro)tricyclo[3.3.1.1(3,7)]decan]-4-yl) phenyl phosphate (CSPD)
  • 37°C incubator
  • Cell scraper
  • Ethanol/dry ice or liquid N2 baths
  • Luminometer (tube or plate)
  • Absorbance microplate reader
  • Additional reagents and equipment for co-transfecting PC12 cells (Support Protocol 2)

Alternate Protocol 1: Measurement of Human Growth Hormone Release

 Additional materials (also see Basic Protocol 2)
  • Human growth hormone ELISA kit (e.g., Roche Applied Science, no. 11585878001)

Support Protocol 2: Transfection of PC12 Cells Using Cationic Lipids

 Materials
  • Early-passage PC12 cells or ATCC-PC12 cells (#CRL-1721)
  • Poly-l-lysine (Sigma, no. P6282)
  • DMEM or F12K complete culture medium (see recipe)
  • Rat tail collagen type I (Upstate, cat no. 08-115)
  • Cationic lipid reagent GenePORTER 2 (Genlantis)
  • Plasmid DNA, purified by anion-exchange chromatography (e.g., Qiagen)
  • DNA diluent (Genlantis)
  • 6-well cell culture plates, tissue-culture treated polystyrene (e.g., Becton Dickinson-Falcon, Corning-Costar)
  • 37°C CO2 incubator
  • Clear polystyrene plastic snap tubes (e.g., Becton Dickinson Falcon)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 15.12.1
    Electron micrograph of early passage PC12 cells. Dense-core secretory granules (arrows) are seen in the vicinity of or docked at the plasma membrane (A). Scale bar = 1 µm. High magnification electron micrograph in (B) shows the presence of electron-dense, membrane-delimited vesicles of ~100 to 130 nm in diameter (arrows). Scale bar = 100 nm. Cells were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde and post-fixed in 1% OsO4.

    View Image
  • Figure 15.12.2
    Detection of secretagogue stimulated exocytosis of secretory granules in PC12 cells. Cells labeled with [3H]-norepinephrine, or transfected for 48 hr with CgA-EAP or hGH, were incubated for 15 min in secretion medium alone (CaSB, mock) or with 2 mM BaCl2.

    View Image

Literature Cited

Literature Cited
    Berger, J., Hauber, J., Hauber, R., Geiger, R., and Cullen, B.R. 1988. Secreted placental alkaline phosphatase: A powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66:1-10.
    Courel, M., Rodemer, C., Nguyen, S.T., Pance, A., Jackson, A.P., O'Connor, D.T., and Taupenot, L. 2006. Secretory granule biogenesis in sympathoadrenal cells: Identification of a granulogenic determinant in the secretory prohormone chromogranin A. J. Biol. Chem. 281:38038-38051.
    Greene, L.A. and Tischler, A.S. 1976. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. U.S.A. 73:2424-2428.
    Kelly, R.B. 1985. Pathways of protein secretion in eukaryotes. Science 230:25-32.
    Mahata, S.K., O'Connor, D.T., Mahata, M., Yoo, S.H., Taupenot, L., Wu, H., Gill, B.M., and Parmer, R.J. 1997. Novel autocrine feedback control of catecholamine release. A discrete chromogranin a fragment is a noncompetitive nicotinic cholinergic antagonist. J. Clin. Invest. 100:1623-1633.
    Martin, T.F. and Grishanin, R.N. 2003. PC12 cells as a model for studies of regulated secretion in neuronal and endocrine cells. Methods Cell Biol. 71:267-286.
    Sugita, S. 2004. Human growth hormone co-transfection assay to study molecular mechanisms of neurosecretion in PC12 cells. Methods 33:267-272.
    Taupenot, L., Mahata, M., Mahata, S.K., and O'Connor, D.T. 1999. Time-dependent effects of the neuropeptide PACAP on catecholamine secretion: Stimulation and desensitization. Hypertension 34:1152-1162.
    Taupenot, L., Harper, K.L., and O'Connor, D.T. 2003. The chromogranin-secretogranin family. N. Engl. J. Med. 348:1134-1149.
    Taupenot, L., Harper, K.L., and O'Connor, D.T. 2005. Role of H+-ATPase-mediated acidification in sorting and release of the regulated secretory protein chromogranin A: Evidence for a vesiculogenic function. J. Biol. Chem. 280:3885-3897.
    Wick, P.F., Senter, R.A., Parsels, L.A., Uhler, M.D., and Holz, R.W. 1993. Transient transfection studies of secretion in bovine chromaffin cells and PC12 cells. Generation of kainate-sensitive chromaffin cells. J. Biol. Chem. 268:10983-10989.
 Internet Resources
    http://medicine.ucsd.edu/hypertension

UCSD chromaffin and PC12 cell biology online protocols.

     
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