Fura 2 acetoxymethyl am ester

To measure [Ca²⁺]_c, C2C12 cells were grown to confluence, washed twice with sterile PBS 1× (Euroclone), and incubated with 5 μM fura-2/acetoxymethyl (AM) ester (Sigma, Milan, Italy) in DMEM (Euroclone) containing 10% FBS and without phenol red for 30 min in the dark. After additional washings with DMEM (Sigma), the coverslips were mounted in a thermostatted quartz cuvette and placed in an agitation system at 37 °C. The measurement was performed using a Hitachi F-4500 Fluorescence Spectrometer (Hitachi High-Technologies Corporation) for a continuous duration of 300 s at an excitation wavelength of 340 nm and an emission wavelength of 510 nm. Fura-2/AM-loaded C2C12 cells were stimulated with EVs as described for HUVECs, in the presence or absence of Ca²⁺ in the incubation medium with 50 mM ethylene glycol tetraacetic acid (EGTA) (Sigma). The quantification of [Ca²⁺]_c was obtained using the following equation, as previously reported [74 (link),75 (link),76 (link)]: (Ca²⁺) = K_d ((R − R_min)/(R_max − R)). The K_d of fura-2/AM for Ca²⁺ was considered as 224. R_min and R_max were the minimum and maximum values of fluorescence ratio obtained under Ca²⁺-free (EGTA 0.1 M) or Ca²⁺-saturated conditions, respectively. The fluorescence intensities obtained were corrected for cell autofluorescence at the respective wavelengths employed [75 (link)].

To measure [Ca²⁺]_c, C2C12 cells were grown to confluence, washed twice with sterile PBS 1× (Euroclone), and incubated with 5 μM fura-2/acetoxymethyl (AM) ester (Sigma, Milan, Italy) in DMEM (Euroclone) containing 10% FBS and without phenol red for 30 min in the dark. After additional washings with DMEM (Sigma), the coverslips were mounted in a thermostatted quartz cuvette and placed in an agitation system at 37 °C. The measurement was performed using a Hitachi F-4500 Fluorescence Spectrometer (Hitachi High-Technologies Corporation) for a continuous duration of 300 s at an excitation wavelength of 340 nm and an emission wavelength of 510 nm. Fura-2/AM-loaded C2C12 cells were stimulated with EVs as described for HUVECs, in the presence or absence of Ca²⁺ in the incubation medium with 50 mM ethylene glycol tetraacetic acid (EGTA) (Sigma). The quantification of [Ca²⁺]_c was obtained using the following equation, as previously reported [74 (link),75 (link),76 (link)]: (Ca²⁺) = K_d ((R − R_min)/(R_max − R)). The K_d of fura-2/AM for Ca²⁺ was considered as 224. R_min and R_max were the minimum and maximum values of fluorescence ratio obtained under Ca²⁺-free (EGTA 0.1 M) or Ca²⁺-saturated conditions, respectively. The fluorescence intensities obtained were corrected for cell autofluorescence at the respective wavelengths employed [75 (link)].