Looking for a good protocol to measure OxLDL induced ROS in HUVECs using DCFDA dye. Litreature is all over the place 1 to 24 hrs treatment and self made OxLDL. I would like to know if anybody used Kalen biomedical highly OxLDL to induce ROS in HUVECs.
thanks for your time
Deen
Do Flow cytometry. Higher ROS should lead to greater signal in the FITC channel.
Don't use DCFDA at all. It is probably the worst of all fluorescent probes because it can actually generate ROS on its own. Better probes include Amplex Red, which is fairly specific for hydrogen peroxide or dihydroethidium (DHE) for superoxide. If you use the latter, be careful about the wavelength you employ for detection. There are several references that will help you with assay details.
1. Munzel T, Afanas'ev IB, Kleschyov AL, and Harrison DG. Detection of superoxide in vascular tissue. Arterioscler Thromb Vasc Biol 22: 1761-1768, 2002.
2. Cai H, Dikalov S, Griendling KK, and Harrison DG. Detection of reactive oxygen species and nitric oxide in vascular cells: Comparison of sensitivity and specificity. In: Methods in Molecular Medicine, edited by Sreejayan N, and Ren J. New York, NY: Humana Press Inc., 2005.
3. Nazarewicz RR, Bikineyeva A, and Dikalov SI. Rapid and Specific Measurements of Superoxide Using Fluorescence Spectroscopy. J Biomol Screen 2012.
4. Dikalov SI, and Harrison DG. Methods for detection of mitochondrial and cellular reactive oxygen species. Antioxid Redox Signal 20: 372-382, 2014.
Some of these probes are not as specific as is claimed, but any will be better than DCF. They mainly respond to OH radical although there are some exceptions.
thank you David Harrison and David Kessel for your advice. That is what my opinion was about DCFDA based on my initial experience.