The ATPlite 1 Step Luminescence Assay System is used for the quantitative evaluation of proliferation and cytotoxicity of cultured mammalian cells and is an alternative to colorimetric, fluorometric and radioisotopic assays. ATP is a marker for cell viability because it is present in all metabolically active cells and this assay involves a highly sensitive ATP-monitoring method. The assay is based on the production of light caused by the reaction of ATP with added luciferase (firefly) and D-luciferin and the emitted light is proportional to the ATP concentration. The ATPlite 1 Step Luminescence Assay comes with (1) Substrate buffer solution (2) Lyophilized substrate (3 ) Lyophilized ATP standard (4) Instruction booklet and (5) Material Safety Data sheet. The assay can be done in either 96-well or 384-well plates.
I have been using this kit for the last couple of years to determine the effects of potential chemopreventive agents on the proliferation of human bronchial epithelial cells in 96-well plates. The cells were first seeded in a 96-well plate (sterile, tissue culture treated, white or black 96-well plate) for 24 hours and then treated with the agents of our interest for 48 hours in 200 ul of appropriate medium. Before addition of the substrate solution, 100 ul of the culture medium was removed from each well and equilibrated at room temperature for ½ an hour. The substrate buffer solution and the lyophilized substrate were also kept at room temperature for ½ an hour before use. Lyophilized substrate is reconstituted with the substrate buffer solution and 100 ul of this solution is added to each well containing 100 ul of culture media. The plate is then evenly shaken for 3 minutes in the dark and then incubated at room temperature in the dark for 7 more minutes. The emitted luminescence is measured in a microplate luminometer after the incubation. There is a linear correlation between the luminescence signal and the cell number. A very bright signal is obtained within 30 minutes that decreases with time and this decrease is independent of cell number. The final result is normalized with zero hour incubation proliferation data.
Overall, this kit is very easy to use (only one reagent addition step). The results are consistent when I use equal numbers of cells to begin with (2000 cells/well). Although the absolute value varies from experiment to experiment, the relative value is very consistent.
Assistant Research Faculty
Department of Medicine, David Geffen School of Medicine
University of California, Los Angeles