Determination of ATP Leakage from Cultured Cells in Toxicity Testing: A Two-Step Bioluminescent Assay

Abstract

The intact plasma membrane of animal cells in culture is considered to be almost impermeable to ATP. Thus, leakage of intracellular ATP to the extracellular medium is indicative of membrane damage. We have developed a rapid and simple method for determination of ATP leakage, using L-1210 and ELD cells incubated with velleral or isovelleral (two terpenes). ATP was determined by a bioluminescent firefly luciferin-luciferase reaction in a two-step assay in the same sample. Extracellular ATP (= leakage) was measured directly in cell suspensions, and total ATP was determined in cell lysate suspensions after addition of a detergent. ATP leakage (% of total ATP) increased after incubation with both terpenes, in a dose-dependent manner.