Reactive oxygen species (ROS) are a by product of a number of natural cellular processes such as oxygen metabolism and inflammation. They can additionally result from exposure of cells to UV light and ionizing radiation (IR). In fact, a large proportion of the cellular damage induced by IR is caused through ROS generation, especially the hydroxyl radical. ROS are generally ‘kept in check’ by antioxidant proteins and systems within cells, but overproduction of ROS can lead to cell death. ROS are highly reactive and can damage DNA, protein and lipids.
DCF-DA has been used as a detector of ROS for a number of years in many applications such as fluorescence microscopy and flow cytometry. This dye is non fluorescent when chemically reduced, but after cellular oxidation and removal of acetate groups by cellular esterases it becomes fluorescent. CM-H2DCFDA ( 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester) is a derivative of DCF-DA, but with an additional thiol reactive chloromethyl group, which enhances the ability of the compound to bind to intracellular components, thereby prolonging the dye’s cellular retention.
The protocol I have used for ROS detection by this dye is straightforward:
1) Stable cells derived from MDA-MB-231 mammary carcinoma cells were plated in 6 well plates at 3 x 105 in full serum media
2) Approx. 24 hours later the cells were treated with 10 uM Hydrogen Peroxide (H2O2) for 2 hours (as a positive control)
3) Untreated and H2O2 treated cells were harvested in full serum media, spun down for 5 minutes at 1000 rpm and resuspended in PBS containing 10 uM CM-H2DCFDA
4) Cells were maintained at 37°C in the dark for 30 minutes to ‘load’ the dye (N.B. Untreated cells not loaded with dye were used as a negative control to examine cellular autofluorescence.)
5) Cells pelleted and resuspended in PBS
6) Analysis by flow cytometry
Other negative controls that can be included, but are not imperative, are an assessment of the fluorescence of cell free media/buffer (whatever you have resuspended your cells in) and determination if the forward and side scatter of the cells changes after dye loading.
Additionally, positive controls other than H2O2 can be used. Examples include PMA (4beta-phorbol, 12-myristate, 13-acetate) and tert-butyl hydroperoxide (TBHP). Initially I used H2O2 at 3 different concentrations (10 uM, 50 uM and 100 uM) in order to ascertain which concentration was required to elicit a ROS response in MDA-MB-231 cells; 10 uM was found to be adequate.
Another parameter that can be altered is what cells are resuspended in for analysis; I have tested serum free media, full serum media and the physiological buffer PBS. I found that for analysis of untreated cells, full serum media was acceptable, but it interfered with the cellular response to H2O2, quenching the effect. Serum free media and PBS do not have this effect. The best responses I have seen have been in cells resuspended in PBS, but cells must be analyzed immediately as a healthy cell population cannot be maintained for extended periods in PBS.
In summary, once a cell specific protocol has been optimized, this reagent is easy to use and reproducible.
PhD Student
The Liggins Institute
University of Auckland