Determination of ATP, ADP, and AMP Levels by Reversed-Phase High-Performance Liquid Chromatography in Cultured Cells
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 wordsReversed-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.
- 2.Szabadkai G, Duchen MR (2008) Mitochondria: the hub of cellular Ca2+ signaling. Physiology (Bethesda) 23:84–94Google Scholar