Determination of ROS by DCFH-DA fluorescence

Reactive oxygen species (ROS) production during ER stress-induced apoptosis was detected using 7′-dichlorodihydrofluorescein diacetate (DCFH-DA), a redox-sensitive fluorescent probe from Cell Signaling (Danvers, MA). First, MC3T3 cells were plated at a cell density of 1.5 × 10⁴ cells per well in a black 96-well fluorescence assay plate with clear bottom (Costar, Catalog no. 07-300-562). Triplicate wells were treated with vehicle alone, etoposide (100 μM), brefeldin A (1 μM), and thapsigargin (1 μM) for 24 h. At the end of incubation, cells were incubated with 10 μM DCFH-DA for an additional period of 30 min at 37 °C. Cells were washed to remove any unbound excess fluorescence probe. Fluorescence intensity was determined in Synergy-H4, fluorescence microplate reader (BioTek, Winooski, VT) at 485/535 nm. Relative fluorescence intensity for each treatment condition was calculated after subtracting the background fluorescence. Experiment was repeated twice.