DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit

Protocol tips

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	- Cells were treated with 25 μM DCFDA in essential medium with 10% FBS in 37°C incubator for 45 min. - Before analysing cells, they are treated with hyperosmotic media by adding NaCl for different time periods (30–180 min).

Protocol tips
- Cells were treated with 25 μM DCFDA in essential medium with 10% FBS in 37°C incubator for 45 min. - Before analysing cells, they are treated with hyperosmotic media by adding NaCl for different time periods (30–180 min).

Publication protocol

Cellular ROS production was measured using a DCFDA assay kit according to manufactory’s protocol. DCFDA (2’,7’-dichlorofluorescein diacetate), a cell-permeable fluorogenic dye, is deacetylated by cellular esterases to a non-fluorescent compound, and later oxidized by ROS into highly fluorescent 2’,7’-dichlorofluorescein (DCF), which measures hydroxyl, peroxyl and other ROS activity within the cell. HCECs were grown on 96-well plates or 8-chamber slides. When reached confluence, the cells were washed twice with phosphate-buffered saline (PBS) and then incubated with 25 μM DCFDA in essential medium with 10% FBS in 37°C incubator for 45 min. After PBS washed twice, the cells were exposed to hyperosmotic media (350, 400 or 450 mOsM) by adding NaCl (19, 44 or 69 mM, respectively) for different time periods (30–180 min). Cells images were taken under fluorescent microscope. Cell fluorescence in 96-well plates was measured at 488 nm excitation and 525 nm emission using Tecan Infinite M200 Multimode Microplate Reader (Tecan US, Inc. Morrisville, NC) after adding NaCl for 15–120 mins. Relative changes of DCF fluorescence were expressed as fold increase over untreated cells.

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Manufacturer protocol

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