Wide field of view quantitative imaging of cellular ATP release

Abstract

Although several mechanical stressors promote ATP secretion from eukaryotic cells, few mechanosensitive pathways for ATP release have been precisely characterized and none have been clearly identified. To facilitate progress, we report here a wide field of view (∼20 × 20 mm sample area) imaging technique paired with a quantitative image analysis to accurately map the dynamics of ATP release from a cell population. The approach has been tested on A549 cells stretched at high initial strain rate (2–5 s−1) or swelled by hypotonic shock. The amount of ATP secreted in response to a series of five graded stretch pulses (5–37% linear deformation, 1-s duration at 25°C) changed nonmonotonically with respect to strain amplitude and was inhomogeneous across the cell monolayer. In a typical experiment, extracellular ATP density averaged 250 fmol/mm2, but the area of detectable signal covered only ∼40% of the cells. In some areas, ATP accumulation peaked around 900 fmol/mm2, which corresponded to an estimated concentration of 4.5 µM. The total amount of ATP released from the combined stretch pulses reached 384 ± 224 pmol/million cells ( n = 4). Compared with stretch, hypotonic shock (50%, 30°C) elicited a more homogeneous ATP secretion from the entire cell population but at a lower yield totaling 28 ± 12 pmol/million cells ( n = 4). The quantitative extracellular ATP mapping of several thousand cells at once, with this wide field of view imaging system, will help identify ATP release pathways by providing unique insights on the dynamics and inhomogeneities of the cellular ATP secretion that are otherwise difficult to assess within the smaller field of view of a microscope.