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ATP Quantitation in the LMax Microplate Luminometer (MaxLine Application Note #40)

Evelyn McGown, Ph.D. and Michael Su, M.S.
Molecular Devices Corporation, 8/00


INTRODUCTION
Adenosine triphosphate (ATP) is present in all living cells. Because the level isstrictly-controlled, assay of ATP can be used as a indicator of viable cell number.ATP measurements are used to monitor raw materials and manufacturing planthygiene for bacterial contamination in the food, drug, health care and personalhealthcare industries, as well as for waste water analysis.1 In the biotechnologyand pharmaceutical industries, ATP measurements are used to evaluate cellproliferation and cytotoxicity. The bioluminescent assay of ATP has become verypopular because of its high sensitivity and convenience. In this application note,we show the ATP assay results obtained with the Molecular Devices Lmaxmicroplate luminometer.


PRINCIPLE OF
ASSAY
The basis for the assay is the reaction catalyzed by luciferase, obtained from the common American firefly (Photinus pyralis).2 The enzyme catalyzes the ATP-dependent oxidation of luciferin with the concomitant release of light (Figure 1).When ATP is the limiting component in the reaction, the amount of light emittedis proportional to the concentration of ATP. The intensity of the emitted light canbe measured easily with the Lmax microplate luminometer.


EQUIPMENT AND MATERIALS
1. Lmax microplate luminometer with SOFTmax PRO for Lmax (Molecular Devices Corp.)

2. ENLITENTM ATP Assay System, Cat. No. FF2000; Promega Corporation. 608277-2516. The kit contains Luciferase/Luciferin (L/L) reagent, ATP standard 10-7 M in HEPES buffer, pH 7.75) and ATP-free water.

3. Solid white 96-well microplates; e.g. Porvair PS white, E&G Wallac Cat. # 204003; Tel: 1-800-221-9650; or CorningCostar Cat. No.
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