ROS Detection with DCFH-DA Staining: Measuring Total ROS in Colorectal Cancer Cell Lines

- Reactive oxygen species are produced predominantly from mitochondria when electrons leak out from the electron transport chain. These electrons combine with molecular oxygen giving rise to various reactive oxygen species, or ROS. In cancer cells, there is overproduction of reactive oxygen species. These highly reactive ROS molecules can oxidatively damage membranes, proteins, and DNA inside the cell.

Increased levels of ROS damages DNA leading to activation of an oncogenic signaling pathway causing cancer progression. Therefore, it is vital to detect the levels of reactive oxygen species produced by the cancer cells.

Begin by taking cancer cells from human colorectal cancer in an appropriate culture media. Next, seed these cells to a multi-well plate and add DCFH-DA solution. Incubate the plate for the desired duration. DCFH-DA crosses the cell membrane. Once inside the cell, DCFH-DA converts to DCFH in the presence of a cellular esterase enzyme. Further, the oxidation of DCFH by reactive oxygen species yields green fluorescent molecules.

Observe the cells under a fluorescent microscope. The intensity of green fluorescence indicates the level of reactive oxygen species in the cel