Plasmid DNA was diluted with distilled water immediately before the transfection. Each experiment was performed on 20 dishes. Cells on each dish were treated with ultrasound (Figure 1(b)). Pulsed wave Doppler, color flow Doppler, and continuous wave Doppler were insonified from PSK-25AT how to order acoustic transducer with Toshiba SSA-380A (Toshiba Medical Systems), and harmonic
Inhibitors,research,lifescience,medical power Doppler was insonified from S3 transducer with Sonos 5500 (Phillips Medical Systems). The experimental results are shown in Figure 2. Continuous-wave Doppler ultrasound was the most efficacious and was used for subsequent experiments. Figure 2 Comparison of four modes of ultrasound for sonoporation. Cells treated with continuous-wave Doppler ultrasound yielded the largest amount of HGF protein indicating this to be the most effective ultrasound mode. CFD: color flow Doppler; PWD: pulsed wave … 2.5. Experiments for Dose-Effect Relations The medium in 35mm Petri dishes containing the cardiomyocytes was changed to fresh defined serum-free medium from DMEM+10% FCS. Rat HGF plasmid DNA was diluted Inhibitors,research,lifescience,medical with distilled water, and a volume corresponding to 60, 120, or 180μg was added to each of the 20 Petri dishes per DNA dose. Cells on each dish were then treated with continuous-wave Doppler ultrasound Inhibitors,research,lifescience,medical (frequency of
2.5MHz and acoustic intensity of 0.5W/cm² from a PSK-25AT acoustic transducer Inhibitors,research,lifescience,medical with Toshiba SSA-380A Ultrasound system) with SHU 508A liposome (1 × 107particles/mL) for acoustic exposure time of 30 or 60 seconds at room temperature (Figure 1(b)).
In a separate series of experiments, we tested four liposome concentrations (0, 1 × 106, 1 × 107 or 1 × 108particles/mL), three insonification repetitions (1 insonification only, 3 or 5 insonifications for 30 seconds), and three DNA incubation times (15, 60 or 120min). After the incubation, the culture medium was changed to normal DMEM+10% FCS and the cells were cultured for 72 hours. In a separate set of experiments, we examined the effect of culture period on the amount of DNA product Inhibitors,research,lifescience,medical that is HGF protein by discontinuing culture at 24, 48, and 72 hours and Enzastaurin measuring the amount of rat HGF protein in the medium. The total amount of protein content in the cultured cells was GSK-3 measured and used to correct the HGF level in each dish. We measured rat HGF protein using an EIA kit (Institute of Immunology Co., Ltd., Tokyo, Japan)  and protein content of cultured cells using a Modified Lowry Protein Assay Kit (Pierce Biotechnology, Rockford). 2.6. Viability of Cultured Cells To determine the safety of sonoporation, in a separate experiment, cultured cells were exposed to 0.1% trypan blue for 5min just after ultrasound insonification. This allowed assessment of sarcolemmal membrane damage and was performed for each concentration of liposome, each insonification time, and each number of repetitions of insonification.