LIPUS-Induced Calcium Response in Cells

The cells were incubated with 2.5 μmol/L Fluo-4 AM (Beyotime) at 37 °C for 10 min. After washing with PBS, Opti-α-MEM was added and cells were observed under a laser scanning confocal microscope. The total observation and baseline times were 12 min and 30 s, respectively, whereas the LIPUS exposure period was 0.5–2.5 min; the treatment parameters were the same as those described in the “LIPUS treatment” subsection, and the interval between image captures was 4 s. The calcium response of each frame of image was quantified as ΔF/F₀, where F₀ is the average baseline fluorescence intensity before LIPUS processing, ΔF = F_P − F₀, and F_P is the real-time fluorescence intensity [34 (link)]. We compared the highest F_P values of each group during and after LIPUS treatment. The analysis was carried out using the NIS-Elements AR 4.20 (Nikon), GraphPad Prism 7.0, and Origin 2019 software packages.