Optimized Methods for Measuring Extracellular ATP from Human Airway Epithelial Cells and Bronchoalveolar Lavage Fluid

Extracellular adenosine triphosphate (eATP) is a mediator of purinergic signalling in the airways, implicated in mucociliary function, inflammation, and cough via activation of P2X3 receptors. Elevated airway eATP has been associated with multiple respiratory diseases, yet reliable measurement of eATP remains challenging due to its rapid enzymatic degradation and confounding contributions from intracellular ATP. Here, we describe an optimized, microwell plate-based luminescence method for quantifying eATP from human airway epithelial cell cultures and bronchoalveolar lavage (BAL) fluid with enhanced signal stability. Using a commercially available ATP detection assay with a prolonged luminescence half-life, we introduced a simple 0.45 {micro}m syringe filtration step to remove cells and thereby isolate extracellular ATP. This approach demonstrated ATP specificity via apyrase degradation, and provided a linear detection range from 5 nM to 5 {micro}M. Addition of ATP stabilization buffer preserved ATP levels in cell culture media for at least 4 hours at 4 {degrees}C and in human BAL samples for at least 6 weeks at -80{degrees}C. Applying this method to primary human bronchial epithelial cells revealed detectable eATP release, with preferential secretion at the apical surface under air-liquid interface conditions. Collectively, this optimized assay enables robust, high-throughput, and time-flexible quantification of eATP in both experimental and clinical airway samples. These methods support improved investigation of purinergic signalling in airway health and disease and may facilitate biomarker development relevant to eATP in the airways.