Optimized Methods for Measuring Extracellular ATP from In Vitro Airway Epithelial Cell Cultures
Bibliographic record
Abstract
Abstract Rationale: Extracellular adenosine triphosphate (eATP) is a neurotransmitter detectable in airway surface lining fluid. Patients with refractory chronic cough (RCC) demonstrate an exaggerated cough response to ATP inhalation, suggesting eATP plays a role in evoking cough. Abnormally high eATP concentrations in human airways have been found to correlate with COPD severity, asthma, and CF-associated neutrophilic inflammation, suggesting dysfunctional ATP release and/or metabolism in disease states. In vitro measurement of eATP in diverse lung cell types is typically performed in real-time via transient chemiluminescence reactions, limiting the potential for higher throughput or time-delayed experimental designs. This work presents methods for measuring eATP from in vitro cell cultures with high signal stability and resolution.Methods: Calu-3 bronchial epithelial cells and primary human bronchial epithelial cells (HBECs) were cultured under submerged monolayer conditions on tissue culture plastic or under air-liquid interface (ALI) conditions in Transwell™ inserts. The ATPlite™ Luminescence ATP Detection Assay System (Revvity) was used to measure eATP from cell culture media and apical wash samples. This kit is intended for measuring intracellular ATP using a lysis buffer containing ATP-stabilizing agents that irreversibly inhibit ATPases, maintaining a luminescence signal with a half-life over 5 hours. To measure only eATP while retaining the ATP-stabilizing benefits of the lysis buffer, we explored using centrifugation or syringe filters to remove cells and isolate eATP in collected samples. Characterizations were performed for luminescence assay sensitivity, stability of frozen samples, and effectiveness of cell removal via centrifugation or syringe filters. Concentrations of eATP in apical and basal compartments of ALI-differentiated primary HBECs were compared. Results: Luminescence signals were directly proportional to the ATP standard concentration with a minimum sensitivity of 0.61 nM ATP. Removing cells from samples via centrifugation yielded lower ATP signals than syringe filtered samples. Syringe filters with 0.22 and 0.45 µm pore sizes removed all cells from cell suspensions without inducing detectable ATP release. ATP release was 8.5× higher in the apical compartment of ALI-differentiated primary HBECs compared to the basolateral compartment. Apyrase, an ATP-degrading enzyme, demonstrated specificity of the eATP signal in these experiments. Conclusions: Our data describe a method that enables in vitro measurement of eATP with higher signal stability than typical transient luminescence-based assays and an apical preference for ATP secretion in human airway epithelial cells. Funding: This study was funded by a research grant to I.S. (McMaster University) from the Investigator Initiated Studies Program of Merck Inc.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.003 | 0.002 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.003 | 0.005 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".