Whole Cell Detection of Reactive Oxygen Species

7′-dichlorofluorescin diacetate (DCFDA) was used to detect whole cell ROS. Cells were seeded in 6-well plates at 275,000 cells/well if suspension or 100,000 cells/well if adherent and allowed to incubate overnight. DCFDA was pretreated for 30 minutes at a final concentration of 20 μM at 37 °C then cells were treated with the negative control (DMSO vehicle) or the compounds being studied (dissolved in DMSO or water) for 3 or 6 hours protected from light. For the 24-hour time point, cells were treated first with studied compound or DMSO then treated with DCFDA at the 18-hour time point for 6 hours. Cells were then collected in 2 mL eppendorfs followed by centrifugation at 2000 RPM for 5 minutes. Media was aspirated and resuspended in 1xPBS. Results were quantified using the Tali Image-Based Cytometer (LifeTechnologies Inc, Cat. No. {"type":"entrez-nucleotide","attrs":{"text":"T10796","term_id":"390950","term_text":"T10796"}}T10796, Burlington, ON, Canada) and were measured as the percent of cells positive for the green fluorescent product, DCF relative to the DMSO control.