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A Real-Time Extracellular ATP Assay to Identify Modulators of the Innate Immune Response

Niles, A. L. et al., Promega Corporation


AACR 2020 Abstract #1832

Damage-associated molecular patterns (DAMPs) liberated or exposed by dying cells after chemotherapeutic treatment augment innate immune responses and improve clinical outcomes as a result of the immunogenic cell death (ICD) phenotype. Extracellular ATP (eATP) is a potent DAMP that acts as a chemotactic agent for dendritic cells and as an immunomodulator for fine-tuning the inflammatory response. Current in vitro methods for identifying eATP inducers are procedurally difficult and laborious and are hampered by the instability of eATP in cell culture environments.

To overcome these challenges, we have developed an easy-to-use, homogeneous, bioluminescent chemistry that can be introduced with test articles to measure dose-dependent ATP release kinetics and magnitudes of response in real time. We tested the assay using U20S and U937 cells dosed with serial dilutions of doxorubicin, idarubicin, mitoxantrone, and romidepsin. Luminescent data were continuously collected throughout the 24h exposure in 5- or 10- minute increments using a conventional multimode plate reader with CO2-independent medium, or with readers equipped with atmospheric control. When coupled with parallel cell health assays designed to measure either viability or loss of membrane integrity, the data can reveal agents that potentiate ATP release during early or late apoptosis. Our real-time data suggest the potency of eATP release is dependent upon cell model and agent and can be reproducibly and robustly measured in 96 and 384 well environments with kinetic resolution not possible by other methods. This new eATP measurement workflow may help define the capacity of new chemical entities to induce apoptosis and immunogenic cell death.

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