Fluo 3 am dye

For the detection of intracellular calcium concentration, BMMSCs were incubated with 5 μM Fluo‐3/AM dye (Invitrogen, Life Technology) for 60 min at 37°C, followed by washing with PBS for three times. After digestion by trypsin, 2 × 10⁵ cells were counted with PBS washing for two times. Then, cells were subjected to flow cytometric analysis (Beckman Coulter). Data are presented as fluorescence intensity.

Calcium imaging was performed using confocal laser microscopy (Zeiss, Oberkochen FV1000, Germany). The intracellular Ca²⁺ level ([Ca²⁺]_i) was determined by Fluo-3 fluorescence intensity, as described previously.^{40 (link)} Briefly, BMSCs were cultured in 12-well plates and were incubated with 5 μmol·L⁻¹ Fluo-3/AM dye (Invitrogen, Life Technology, Carlsbad, CA, USA) for 30 min in α-MEM at 37 °C. BMSCs were again washed three times with calibrated EGTA/Ca²⁺ solutions. KCl (30 mmol·L⁻¹) or TG (20 μmol·L⁻¹, Sigma-Aldrich, St. Louis, MO, USA) was added to test which type of calcium channel was affected. Images were collected every 4 s at 2 Hz with excitation at 488 nm and emission at 530 nm. Data are presented as the Fluo-3 fluorescence intensity increase ratio: R = ΔF/F₀, where ΔF = F – F₀. F is the fluorescence value detected, and F₀ is the minimum fluorescence value.