Fura-2 Imaging of Calcium Dynamics in MuSCs

Fura-2 imaging was performed as previously described with minor modifications (Tsuchiya et al, 2018 (link)). For indicator loading, MuSCs were plated on glass-bottomed dishes (Matsunami) coated with Matrigel and incubated with 5 μM Fura-2 AM (Dojindo) at 37°C for 60 min. Time-lapse images were obtained every 2 s. The base composition of HBS was (in mM) 107 NaCl, 6 KCl, 1.2 MgCl₂, 11.5 glucose, and 20 HEPES (pH = 7.4 adjusted with NaOH). HBS with 2 mM Ca²⁺ (2Ca) in addition contained 2 mM CaCl₂, whereas that without Ca²⁺ (0Ca) in addition contained 0.5 mM EGTA instead of CaCl₂. Ratiometric images (F₃₄₀/F₃₈₀) were analysed using Physiology software (Zeiss). Yoda1-induced Ca²⁺ influx was measured as the difference in the Fura-2 ratio between its maximum value and that at 1 min from the initiation of imaging. These experiments were performed using a heat chamber (Zeiss) to maintain the temperature at 37°C throughout the imaging process. The amplitude was calculated using the following formula: $\text{Amplitude}=\left(\left[{\text{maximum} \text{value} \text{of} \text{the} \mathrm{F}}_{340}{/\mathrm{F}}_{380} \text{ratio}\right]-\left[{\text{minimum} \text{value} \text{of} \text{the} \mathrm{F}}_{340}{/\mathrm{F}}_{380} \text{ratio}\right]\right)/2.$