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A Novel Cell-Based ATP Assay with Enhanced Operational Stability and Ease of Use

Part # PS201


James J. Cali1, Michael P. Valley1, James Unch2, Poncho L. Meisenheimer2, and Dan F. Lazar1
1Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711; 2Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401
Abstract #282

Bioluminescent ATP assays use the ATP dependence of firefly luciferase to correlate luminescence with cell viability. The assay reagents minimally provide a luciferase enzyme, luciferin and Mg2+ in a lytic buffer that lacks ATP. This is combined with a sample to produce an amount of light that depends on ATP released from cells and that correlates to cell number. The luciferase is typically a dried component that is reconstituted with a liquid formulation at the time of use. Stability of the reconstituted reagents is relatively poor at room temperature or 4°C so they are stored frozen as best practice for maintaining optimal performance over an extended time period. To improve on this format we focused on making a one component liquid reagent with sustained stability at room temperature and 4°C. We applied substrate and buffer optimization to reaction formulations for the thermostable variant of Photuris pennsylvanica luciferase (Ultra-Glo™ Recombinant Luciferase). The enzyme was durable under a wide range of conditions tested for providing cell lysis, ATPase inhibition and formulation stability. These efforts produced an all liquid formulation that retains >85% of its activity when stored at room temperature for one week. At 4°C it retains >90% activity for one month with no detectable change in the first week. This represents a significant improvement to stability over existing formulations and eliminates operational inconveniences associated with freezing and thawing. The all liquid formulation eliminates a need to reconstitute a reagent and delivers sensitive cellular ATP detection (detection limit at low 10s of cells) in a one addition protocol that provides signal half-lives greater than 3 hours. By providing a high level of operational stability, sensitivity and ease of use this new all liquid formulation represents the next generation in cell-based ATP detection assay chemistry.

Printed in USA.