Determination of ATP, ADP, and AMP Levels by Reversed-Phase High-Performance Liquid Chromatography in Cultured Cells

  • Michela MenegolloEmail author
  • Isabella TessariEmail author
  • Luigi Bubacco
  • Gyorgy Szabadkai
Part of the Methods in Molecular Biology book series (MIMB, volume 1925)


Cytoplasmic and mitochondrial Ca2+ signals couple cellular ATP production to activity-related energy demand. In order to accurately determine the bioenergetic effect of Ca2+ signals, cellular energy charge, i.e., the compound ratio of the phosphorylated adenine nucleotides AMP, ADP, and ATP, should be estimated. Reversed-phase high-performance liquid chromatography (RP-HPLC) allows the rapid separation and quantitation of these molecules. Here we describe a protocol applied in our laboratories to quantify ATP, ADP, and AMP nucleotides in cellular extracts.

Key words

Reversed-phase high-performance liquid chromatography (RP-HPLC) ATP/ADP ratio Energy charge Glycolysis Oxidative phosphorylation Ca2+ signal 



Funding was provided by the Biochemical and Biophysical Research Council (BB/L020874/1, BB/P018726/1), the Wellcome Trust (097815/Z/11/Z) to GS in the UK, and the Italian Association for Cancer Research (AIRC, IG13447), Telethon (GEP12066 and GGP16026), and Ricerca Finalizzata, Italian Ministry of Health (RF-2013-02359065) to GS and MM in Italy.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biomedical SciencesUniversity of PaduaPaduaItaly
  2. 2.Department of BiologyUniversity of PaduaPaduaItaly
  3. 3.Department of Cell and Developmental Biology, Consortium for Mitochondrial ResearchUniversity College LondonLondonUK
  4. 4.The Francis Crick InstituteLondonUK

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