Article 21

Reactive oxygen species, 2’,7’-Dichlorodihydrofluorescein acetate, Microvolumetric fluorospectrometer.

Detecting reactive oxygen species (ROS) in living cells is a difficult task because of their short lifetime, presence of cellular antioxidants, risk of cell death and unwanted transformations of the detector substance. Fluorescence-based techniques are currently most widely used, with high sensitivity and easy processing of samples but the classical instruments need a high amount of samples and consumables. We optimized and validated a microvolumetric fluorescence-based method for ROS detection in living cells, using 2’,7’-dichlorodihydrofluorescein acetate (H2DCFDA) and a microvolumetric fluorospectrometer. In our experimental conditions we determined that a measurement volume of 3 µL containing between 125 and 500 cells was sufficient for a reliable measurement of oxidized 2’,7’-dichlorofluorescein fluorescence. The method was applied on H2O2 and catalase treated MEF k41 cells. The catalase preloaded cells presented a lower fluorescent signal under H2O2 stress. Our results show that this microvolumetric method is suitable for oxidative stress specific detection even when compounds able to discriminate the various molecular categories of ROS are added. The measurements could be performed with high accuracy, demonstrating that the microvolumetric method provides a sensitive, fast and cost-effective means for ROS detection in living cells.