Fura 2 am

Cortical cultures were incubated with 2.5 mm Fura2-AM (Molecular Devices) in calcium-free HBSS (ThermoFisher) supplemented with 20 mm HEPES and 0.02% pluronic F-127 (ThermoFisher) for 1 h and imaged on a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices). Light excitation at 340 nm and 380 nm and measurement of detection at 510 nm allowed the ratio of unbound:bound calcium signal to be calculated (F340/F380). After establishing a baseline signal for 30 s, calcium compounds were injected into wells and the ratio measured for 60/120 s. Δ (F₁—F₀) was calculated to assess calcium store content.

The differentiated SKPs (up to > 15 days with maturation medium) were seeded on X-well (Sarstedt) loaded with 4 µM Fura 2-AM (Molecular Devices, Sunnyvale, CA, USA) and 2 µM pluronic acid (Thermo Scientific, Waltham, MA, USA) in the dark for 45 min at 37 °C and 5% CO₂. The cells were washed to remove the excess dye, and intracellular Ca²⁺ measurements were performed according to the protocol described by Sakka et al. [28 (link)]. For the data analysis, the F340/F380 ratio of emitted fluorescence signals intensity recorded at 340 and 380 nM excitation wavelengths was calculated for each measurement time point. The amplitude of the elicited Ca²⁺ responses was measured by calculating the difference between the basal and maximal F340/F380 ratio values. Cells were stimulated with capsaicin at 10 µM (Sigma, M2028), SLIGKV at 50 µM (PAR2-AP, Sigma, S9192), polygodial at 3 µM (Santacruz, sc-201489), and histamine at 1 mM (Sigma, H7125) to evaluate the functionality of the receptor and the capacity of differentiated cells to induced a Ca²⁺ signal following stimulation. For each agonist, a random field was recorded. Each activation by an agonist was recorded in a compartment that was distinct from other activations.