Application Note

It is time for a new measure of cell function: quantifying cellular ATP production rate using Agilent Seahorse XF technology


denosine triphosphate (ATP) is the universal high energy intermediate of living organisms. The free ener-

gy released from ATP hydrolysis can be used to sustain various cellular functions, including cell growth, movement and response to the environment. In mam- malian cells, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) pathways provide the majority of cellular ATP. Most cells can readily switch between these two pathways, thereby adapting to changes in their environment. The Agilent Seahorse XF Real-Time ATP

Rate Assay provides a new measure to quantify ATP Production Rates in live cells in real time, allowing for simultaneous measurement of the two-main bioenergetic pathways – to calculate the total rate of cel- lular ATP production as well as the frac- tional contribution from each pathway (Figure 1). Because ATP generation is high- ly regulated to meet cellular energy demands, quantifying ATP production rates provides valuable insight into cellular function, disease liabilities and potential strategies for intervention. In particular, cancer cells are known to

exhibit high glycolytic activity during rapid proliferation, even in the presence of nor- mal oxygen concentrations in culture. However, the metabolic switch to glycoly- sis may not be necessary as a major con- tributor of ATP, but rather to allow nutri- ent assimilation into biosynthetic precur- sors. To investigate the relative contribu- tions of glycolysis and OXPHOS on malig- nant cancer cell energy requirements and cell growth, the XF Real-Time ATP Rate Assay was used to measure ATP produc- tion rates and fractional contributions of ATP from OXPHOS and glycolysis for a representative panel of 18 frequently used cancer cell lines (Figure 2). Results showed wide variations in the

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in response to gene modification, com- pound exposure and/or other types of interventions. The need for improved tools to study cel-

Figure 1: Representative scheme of Agilent Seahorse XF Real-Time ATP Rate Assay. Kinetic profile of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurement. OCR and ECAR rates are first measured. Injection of oligomycin results in an inhibition of mitochondrial ATP synthesis that results in a decrease in OCR, allowing mitochondrial ATP production rates to be quantified. Complete inhibition of mitochondrial respiration with rotenone plus antimycin A enables determination of mitochondrial-associated acidification, allowing calculation of glycolytic ATP production rate

metabolic phenotypes across individual cell lines. Interestingly, even in cells typi- cally considered highly glycolytic, the ATP production from glycolysis ranged only between 30-60% of total ATP production. Furthermore, results showed that mitoATP and total ATP production rates, but not glycoATP production rates, inversely cor- relate with cell doubling times in the 16 adherent cells of the cancer panel analysed (data not shown), suggesting significance of mitochondrial OXPHOS as energy sup- plier

driving cell proliferation.

Collectively, these results highlight distinct functionality between the two-main energy metabolic pathways and their roles in can- cer cell proliferation. Future studies with the XF Real-Time ATP Production Rate Assay may provide greater insight into energy metabolism and cellular functions

Figure 2: (A) Glycolytic and mitochondrial ATP production rate distribution in a representative panel of cancer cells lines. (B) Relative contribution of ATP production from glycolysis and oxidative phosphorylation to total ATP production


lular function is clear, and is reflected in the increasing number of cell-based assays techniques developed over the last decade. Cell energy metabolism is a fundamental driver of cell phenotype and function. However, very few tools are available to study changes in cell bioenergetics in real time. This application brief describes a new quantitative method to measure the rate of ATP production from glycolysis and mito- chondria simultaneously, using label-free technology in live cells, in real time.

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