Measuring Vancomycin-Resistant Enterococcus Oxidative Stress

This assay was adapted from (43 (link)). E. faecalis V583 overnight cultures were diluted 10-fold into 200 μl of complete DMEM in black-walled 96-well plates containing 50 μM DHR123 (Thermo Fisher Scientific), vehicle (DMSO), MTX (0.515 μg/ml), and/or vancomycin (4 μg/ml). Plates were incubated with no shaking at 37°C for 6 hours. At the end, the optical density was measured at 600 nm to determine bacterial growth, and DHR123 fluorescence (excitation = 507 nm, emission = 529 nm) was measured using a Tecan M200 microplate reader to determine cellular ROS levels. ROS levels were normalized to OD₆₀₀ to account for differences in VRE growth.