It is possible to measure ATP in living cells using Luciferase as a reporter system, because the Luciferase needs the ATP as a substrate for the light-emitting reaction. In our setup ATP is the rate-limiting step, i.e. the signal is proportional to ATP. We stable transfected the HEK293 cell line with a vector carrying the Luciferase under con-trol of an constitutive promoter. The reaction of D-luciferin to oxyluciferin is ATP dependent. D-luciferin, dissolved in PBS (w/o Ca, Mg) 60mg/100ml, was added to the medium of the cells at a ratio of 1:1. Luminescence was measured immediately thereafter (Hamamatsu C2400-40) and integrated for 10 min. To test our system we applied adenosine as a precursor of ATP. Adenosine was added to a final concentration of 2.5*10^-5M to the test wells. The control wells did not receive this in-tervention. Luminescence was again integrated for 10 min. Integrated light density (ILD) was quantified with a software (GelPro-Analyzer 4.5, Media Cybernetics Inc.). The ILD ratio of test wells and control wells was calculated before and after adding adenosine. After adding adenosine the integrated light density (ILD) ratio increases significantly.

In conclusion, we established a working system for intracellular ATP measurement with no need of cell lysis.