A live-cell assay for the real-time assessment of extracellular ATP levels

• Published in: Analytical biochemistry Vol. 628; p. 114286
• Main Authors: Niles, Andrew L., Cali, James J., Lazar, Dan F.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.09.2021
• Subjects: Apoptosis; Damage associated molecular patterns; DAMP; eATP; Extracellular ATP; ICD; Immunogenic cell death; Necrosis; Non-destructive; Phosphatidylserine PS; Real-time; eATP; Real-time; Non-destructive; Extracellular ATP; Phosphatidylserine PS; ICD; Damage associated molecular patterns; Immunogenic cell death; DAMP; Apoptosis; Necrosis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2021.114286

Extracellular ATP (eATP) is a potent damage associated molecular pattern (DAMP) molecule known to exert profound effects on the innate and adaptive immune responses. As such, it has become an important biomarker for studying means to pro-actively modulate inflammatory processes. Unfortunately, traditional methodologies employed for measuring eATP require cumbersome supernatant sampling, onerous time courses, or unnecessary duplication of effort. Here we describe a new reagent that is tolerable to test cells in extended exposures and enables a fully homogeneous assay method for real-time determinations of extracellular ATP levels. The reagent is introduced into assay plates containing cells at the time of stimulus introduction. The real-time feature of the format allows for sensitive, continuous accounting of eATP levels in the test model over at least 24 h. This work details our efforts to create and characterize this new reagent and to validate utility by demonstrating its use with multiple cell lines and chemically diverse eATP induction stimuli. •The method measures eATP release in real-time throughout at least a 24 h exposure with conventional luminometers.•Is delivered in a fully homogeneous, “add-mix-measure” format at the time of dosing or at time zero of the insult.•The reagent measures release potential and potency from test agents as a function of time.•The method can deliver total ATP viability measure by an endpoint method.•The method was bench marked versus conventional endpoint chemistries.