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
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.