Measurement of Cellular Reactive Oxygen Species

Cellular reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) [45 (link),46 (link)]. Cells were plated into black-walled clear bottom 96-well plates (Corning). For time-course ROS assays, cells grown in 2 mM glutamine were seeded, and the next day, the media was changed to 4 mM glutamine for two, six, twelve, or twenty-four hours. Following treatment, media was removed, and cells were washed once with PBS. Cells were incubated in the dark at 37 °C in 10 μM DCFH-DA in PBS for 20 min. Fluorescence was measured using a Synergy H1 Multi-Mode reader (excitation/emission 485/530 nm). Fluorescence measures were normalized to cell viability, as measured by MTT.