ATP Measurement in Cerebrospinal Fluid Using a Microplate Reader

Part of the Methods in Molecular Biology book series (MIMB, volume 2041)


Imbalance in extracellular ATP levels in brain tissue has been suggested as a triggering factor for several neurological disorders. Here, we describe the most sensitive and reliable technique for monitoring the ATP levels in mice cerebrospinal samples collected by cisterna magna puncture technique and quantified using a microplate reader.

Key words

ATP Luciferin Luciferase Cerebrospinal fluid In vivo Neurological disorders Microplate reader 



This work was supported by funding from Spanish Ministry of Science and Education BFU2012-31195 to M.D.-H. European Union project H2020-MSCA-ITN-2017 number 766124 to M.D.-H. and from Universidad Complutense of Madrid (UCM)-Santander Central Hispano Bank PR41/17-21014 to M.D.-H. A.S.-S. was supported by BFU2012-31195 grant, and L.d.D.-G. held a UCM predoctoral fellowship supervised by M.D.-H. C.d.L. and C.B. were hired for an H2020-MSCA-ITN action supervised by M.D.-H.


  1. 1.
    Robson SC, Sevigny J, Zimmermann H (2006) The E-NTPDase family of ectonucleotidases: structure function relationships and pathophysiological significance. Purinergic Signal 2(2):409–430. Scholar
  2. 2.
    Zimmermann H, Zebisch M, Strater N (2012) Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal 8(3):437–502. Scholar
  3. 3.
    Burnstock G (2008) Purinergic signalling and disorders of the central nervous system. Nat Rev Drug Discov 7(7):575–590. Scholar
  4. 4.
    Diaz-Hernandez M, Diez-Zaera M, Sanchez-Nogueiro J, Gomez-Villafuertes R, Canals JM, Alberch J, Miras-Portugal MT, Lucas JJ (2009) Altered P2X7-receptor level and function in mouse models of Huntington’s disease and therapeutic efficacy of antagonist administration. FASEB J 23(6):1893–1906. Scholar
  5. 5.
    Wang XH, Xie X, Luo XG, Shang H, He ZY (2017) Inhibiting purinergic P2X7 receptors with the antagonist brilliant blue G is neuroprotective in an intranigral lipopolysaccharide animal model of Parkinson’s disease. Mol Med Rep 15(2):768–776. Scholar
  6. 6.
    Diaz-Hernandez JI, Gomez-Villafuertes R, Leon-Otegui M, Hontecillas-Prieto L, Del Puerto A, Trejo JL, Lucas JJ, Garrido JJ, Gualix J, Miras-Portugal MT, Diaz-Hernandez M (2012) In vivo P2X7 inhibition reduces amyloid plaques in Alzheimer’s disease through GSK3beta and secretases. Neurobiol Aging 33(8):1816–1828. Scholar
  7. 7.
    Martin E, Amar M, Dalle C, Youssef I, Boucher C, Le Duigou C, Bruckner M, Prigent A, Sazdovitch V, Halle A, Kanellopoulos JM, Fontaine B, Delatour B, Delarasse C (2018) New role of P2X7 receptor in an Alzheimer’s disease mouse model. Mol Psychiatry.
  8. 8.
    Engel T, Gomez-Villafuertes R, Tanaka K, Mesuret G, Sanz-Rodriguez A, Garcia-Huerta P, Miras-Portugal MT, Henshall DC, Diaz-Hernandez M (2012) Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice. FASEB J 26(4):1616–1628. Scholar
  9. 9.
    Sebastian-Serrano A, Engel T, de Diego-Garcia L, Olivos-Ore LA, Arribas-Blazquez M, Martinez-Frailes C, Perez-Diaz C, Millan JL, Artalejo AR, Miras-Portugal MT, Henshall DC, Diaz-Hernandez M (2016) Neurodevelopmental alterations and seizures developed by mouse model of infantile hypophosphatasia are associated with purinergic signalling deregulation. Hum Mol Genet 25(19):4143–4156. Scholar
  10. 10.
    Llaudet E, Hatz S, Droniou M, Dale N (2005) Microelectrode biosensor for real-time measurement of ATP in biological tissue. Anal Chem 77(10):3267–3273. Scholar
  11. 11.
    Corriden R, Insel PA, Junger WG (2007) A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells. Am J Physiol Cell Physiol 293(4):C1420–C1425. Scholar
  12. 12.
    Hayashi S, Hazama A, Dutta AK, Sabirov RZ, Okada Y (2004) Detecting ATP release by a biosensor method. Sci STKE 2004(258):pl14. Scholar
  13. 13.
    Liu L, Duff K (2008) A technique for serial collection of cerebrospinal fluid from the cisterna magna in mouse. J Vis Exp (21).
  14. 14.
    Vanderstichele H, Demeyer L, Janelidze S, Coart E, Stoops E, Mauroo K, Herbst V, Francois C, Hansson O (2017) Recommendations for cerebrospinal fluid collection for the analysis by ELISA of neurogranin trunc P75, alpha-synuclein, and total tau in combination with Abeta(1-42)/Abeta(1-40). Alzheimers Res Ther 9(1):40. Scholar
  15. 15.
    Czarnecka J, Cieslak M, Michal K (2005) Application of solid phase extraction and high-performance liquid chromatography to qualitative and quantitative analysis of nucleotides and nucleosides in human cerebrospinal fluid. J Chromatogr B Analyt Technol Biomed Life Sci 822(1–2):85–90. Scholar
  16. 16.
    Xu P, Xu Y, Hu B, Wang J, Pan R, Murugan M, Wu LJ, Tang Y (2015) Extracellular ATP enhances radiation-induced brain injury through microglial activation and paracrine signaling via P2X7 receptor. Brain Behav Immun 50:87–100. Scholar
  17. 17.
    Zierhut M, Dyckhoff S, Masouris I, Klein M, Hammerschmidt S, Pfister HW, Ayata K, Idzko M, Koedel U (2017) Role of purinergic signaling in experimental pneumococcal meningitis. Sci Rep 7:44625. Scholar
  18. 18.
    Sebastian-Serrano A, de Diego-Garcia L, Henshall DC, Engel T, Diaz-Hernandez M (2018) Haploinsufficient TNAP mice display decreased extracellular ATP levels and expression of Pannexin-1 channels. Front Pharmacol 9:170. Scholar
  19. 19.
    Morciano G, Sarti AC, Marchi S, Missiroli S, Falzoni S, Raffaghello L, Pistoia V, Giorgi C, Di Virgilio F, Pinton P (2017) Use of luciferase probes to measure ATP in living cells and animals. Nat Protoc 12(8):1542–1562. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Biology, Veterinary SchoolComplutense University of MadridMadridSpain
  2. 2.Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC)MadridSpain
  3. 3.Department of Physiology and Medical PhysicsRoyal College of Surgeons in IrelandDublinIreland
  4. 4.FutureNeuro Research CenterDublinIreland
  5. 5.Instituto de Investigaciones Biomédicas “Alberto Sols”Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM)MadridSpain

Personalised recommendations