Figure 2.

ROS was one of the lethal factors for antibacterial toxicity of silver and antibiotics in combinations. E. coli DHB4 cells grown to OD_{600 nm} of 0.4 were incubated with 80 µM antibiotics and 5 µM Ag⁺ in combinations, and 80 µM ebselen and 5 µM Ag⁺ in combination was used as the positive control. (A) The producion level of ROS was detected by flow cytometry (CyAnadp, Beckman coulter), and mean fluorescent intensity (MFI) ± .s. d. of H₂DCF-DA-stained E. coli was detected. (B) The production level of H₂O₂ was detected by the Amplex® Red Hydrogen Peroxide/Peroxidase method (Invitrogen). Reaction buffer contains 50 μM Amplex® Red reagent, 0.1 U/mL HRP, and the indicated amount of H₂O₂ in 50 mM sodium phosphate buffer (pH 7.4), which was incubated for 30 minutes at 25 °C and further verified with absorbance at 560 nm. The background obsorbance was detected by a non-H₂O₂ control reaction, which has been subtracted from each value. Data that are presented here as means ± s. d. of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).