Na2cr2o7

RAW 264.7 cells were seeded at a density of 0.25 × 10⁶cells/well in 12-well plates and grown to 50% confluence and incubated with either 0.5 mg/ml or 1 mg/ml of an FSD 8 suspension, or 1 mM Cr (VI) (Na₂Cr₂O_7; Sigma-Aldrich) as a positive control, for 4 and 24 h. Cells were then washed with PBS, scraped, and added to pre-warmed agarose glass slides. Using an alkaline unwinding solution, cells were then lysed and subjected to electrophoresis at 21 V and 50 mA (Trevigen, Inc.; Gaithersburg, MD) for 1 h, causing any fragmented DNA to migrate out of the nuclear region of cells. Cells were then fixed with 70% ethanol overnight and labeled with SYBR green dye (ThermoFisher) to bind double-stranded DNA. Images were acquired using an Olympus AX70 microscope with an Olympus DP73 digital camera (Olympus, Center Valley; PA). The Comet Assay IV software (Perceptive Instruments; Bury Saint Edmonds, UK) was used to count comet tails (n = 50 tails counted per experimental condition).