Stretch-Induced Cytosolic Calcium Signaling

To measure cytosolic calcium increases in response to stretch, cells were cultured in a silicone chamber (STB-CH-04, STRETX Inc.) to a 50% confluence and loaded with 5 μM fura- 2 AM (Thermo Fisher Scientific) as previously described (17 (link)). Isotonic bath solutions contained 140 mM NaCl, 2.5 mM KCl, 1.2 mM CaCl₂, 0.5 mM MgCl₂, 5 mM glucose, 300 mOsm (adjusted with d-mannitol), and 10 mM Hepes (adjusted to pH 7.3 with tris base). For mechanical stimulation of Piezo1, two steps of 0.5-s uniaxial stretching (40 and 80% of the initial chamber length) were applied with a delay of 8 min, which is the time necessary to recover basal calcium levels after the first stimulus. The fluorescence ratio (F₃₄₀/F₃₈₀) was acquired with an imaging processing software (HCImage, Hamamatsu Photonics). Signals were normalized to the F₃₄₀/F₃₈₀ measured before cell stimulation. Intracellular [Ca²⁺] was also measured in cells loaded with the single-wavelength Ca²⁺ dye Calbryte (23 ). Signals were normalized to the fluorescence intensity measured before the application of different stimuli.