Dose-dependent inhibition of ultrasound-induced cell killing and free radical production by carbon dioxide

Previous studies have shown that if a solution for cell suspension is saturated with CO₂, ultrasound-induced in vitro cell killing and free-radical production are inhibited. However, the dose dependency of this observation has not been explored. Here, we used NaHCO₃ and HCl to produce a predictable concentration of CO₂ within the culture medium. Using 1 MHz continuous wave 4 W/cm² ultrasound, we sonicated U937 cells suspension for 1 min at 37 °C with CO₂ at different concentrations. At 2 mM, reduced cell killing was observed that further decreased with increasing CO₂ concentration until 100% protection was attained at 20 mM. Ultrasound-induced free-radical production was significantly decreased at 1 mM and became undetectable at 2 mM CO₂. This finding shows that CO₂-mediated inhibition is concentration dependent and that the threshold for free-radical production is one order of magnitude higher than the threshold for cell killing induced by ultrasound. In addition, it also cautions researchers when adding acids and acid-based agents to a culture medium, which almost always contains NaHCO₃, in experiments related to the bioeffects of ultrasound.