Fig. 1. Experimental setup for cell sonication using focused ultrasound. A polystyrene 48-well plate (well dimension, bottom area = 0.75 cm², well volume = 1.4 ml) was placed in a device designed to minimize ultrasound reflection or scattering. Samples were loaded in every two wells to avoid the effect of sonication to the surrounding samples. Focused ultrasound (1.653 MHz center frequency) was delivered vertically to the cells from the bottom through a water tank and each sample was sonicated separately by moving the plate to the target of the focused ultrasound.

Fig. 2. siRNA delivered intracellularly by microbubble-enhanced ultrasound inhibits transient EGFP expression in BJAB cells. Cell viability (A), EGFP-positive populations among viable cells (B), and the mean EGFP fluorescent intensity of EGFP-positive cells (C) are shown. (D) is a representative histogram of our FACScan data. Sonication was at 5.5 W of acoustic power, 10-s continuous-wave exposure in the presence of 2% OPTISON. Cells were recovered 16 h after treatment. EGFP-targeting siRNA (15 μg/ml) did not affect EGFP gene (20 μg/ml) transfection efficiency but suppressed EGFP expression in EGFP-positive cells (C, *p < 0.05). All data are presented as means ± SD of three independent experiments.

Fig. 3. Sonoporation efficiency of C166 cells. The sonoporation efficiency of all (A,C) and viable cells (B,D) is shown. Cells (10⁶ cells/ml) were sonicated with 5% OPTISON and 37.5 μM calcein for 10 s with continuous-wave (A,B) or 100 s with pulse-wave (C,D) at the indicated acoustic power. They were analyzed with FACScan after two PBS washes. Cells were categorized into four groups, sonoporated: viable calcein-positive cells, non-sonoporated: viable calcein-negative cells, dead: non-viable cells (according to the FSC vs. SSC plot), and destroyed: undetectable cells. Data are presented as means ± SD of three independent experiments.

Fig. 4. siRNA delivered intracellularly by microbubble-enhanced ultrasound inhibits the stable expression of EGFP in rat C166-GFP cells. The ratio of cells with suppressed EGFP expression (A,B) and a representative FL1 vs. FSC plot data from continuous-wave sonication (C) are presented. Sonication was at 5.5 W of acoustic power, with 10-s continuous-wave (A) or at 2.75 W acoustic power with 100-s pulse-wave (B) in the presence of 5% OPTISON. Cells were recovered 48 h after treatment. EGFP expression was suppressed in 11% of viable cells with CW sonication and 15% in PW sonication in the presence of 15 μg/ml EGFP-targeting siRNA (egfp-siRNA) but not by siRNA suspension buffer only or by control siRNA (A,B, *p < 0.05). Data are presented as means ± SD of three independent experiments.