We use a technique of ROS detection developed by Anne-Cecile Ribou at the University of Perpignan.  The technique is based on the change in fluorescence lifetime (tau) of the probe PBA that is directly proportional to the amount of ROS in the sample. An incredible bonus of the method is that simultaneously with the ROS concentration the metabolic rate of a cell (amount of free:protein bound NAD(P)H) can be measured (NADH is autofluorescent).

Principle of time-correlated microfluometry, (c) A. Ribou, with permission.

References:

Oter O, Ribou AC. 2009. Quenching of Long Lifetime Emitting Fluorophores with Paramagnetic Molecules. J Fluoresc 19: 389.

Reinhardt K, Ribou AC 2013. Females become infertile as the stored sperm’s oxygen radicals increase. Sci Rep 3:2888

Rharass T et al. 2008. New method for the detection of reactive oxygen species in anti-tumoral activity of adriamycin: A comparison between hypoxic and normoxic cells. Free Radic Res 42: 124.

Rharass T et al. 2016. Oxidative stress does not play a primary role in the toxicity induced with clinical doses of doxorubicin in myocardial H9c2 cells. Molecular and Cellular Biochemistry. 413: 199-215

Ribou AC, Reinhardt K. 2012. Reduced metabolic rate and oxygen radicals production in stored insect sperm. Proc R Soc B 279: 2196.

Ribou AC, Vigo J, Salmon JM. 2004. Lifetime of fluorescent pyrene butyric acid probe in single living cells for measurement of oxygen fluctuation. Photochem Photobiol 80:274.