Assessment of Extracellular ATP Concentrations

  • Lucia Seminario-Vidal
  • Eduardo R. Lazarowski
  • Seiko F. Okada
Part of the Methods in Molecular Biology™ book series (MIMB, volume 574)


Most cells release ATP to the extracellular milieu. Extracellular ATP plays important signaling roles by activating a score of broadly distributed cell surface purinergic receptors (purinoceptors). Biological responses regulated by purinergic receptors include neurotransmission, smooth muscle relaxation and contraction, epithelial cell ion transport, inflammation, platelet activation, immune responses, cardiac function, endocrine and exocrine secretion, glucose transport, and cell proliferation. ATP concentrations at the cell surface, and consequently the magnitude of purinergic receptor stimulation, reflect a well-controlled balance between rates of ATP release and extracellular metabolism. Given the broad spectrum of responses triggered by extracellular ATP, there is a growing interest in accurately assessing the concentrations of this nucleotide at the cell surface. In this chapter, we discuss the use of the luciferin/luciferase-based reaction to measure extracellular ATP concentrations with high sensitivity. Protocols are adapted to assess ATP levels either in sampled extracellular fluids or in situ at the cell surface. Although our focus is on studies of ATP release from epithelial cells, protocols described here are applicable to practically all cell types.

Key words

ATP release extracellular ATP ecto-ATPase luciferase protein A-luciferase luciferin 


6 × His



arbitrary light unit




β,γ-methyleneadenosine 5′-triphosphate


bovine serum albumin


Dulbecco’s modified eagle’s medium




fetal bovine serum


Hank’s balanced salt solution


4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid


minimum essential medium


phosphate-buffered saline


room temperature


Staphylococcus protein A-fused luciferase.


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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Lucia Seminario-Vidal
    • 1
  • Eduardo R. Lazarowski
    • 1
  • Seiko F. Okada
    • 1
  1. 1.Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel HillChapel HillUSA

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