Background:

H2DCFDA is a cell-permeable probe used to detect intracellular reactive oxygen species (ROS).

Analysis of H2DCFDA probe oxidation by means of flow cytometry technique showed that the basal level of ROS in difESCs is about 6-7 times larger than that in embryonic stem cells (ESCs). Both H2DCFDA and DCFDA probes are non-fluorescent in their initial form but they undergo multistep conversion inside the cell that results in the formation of fluorescent product dichlorofluorescein (DCF). The only difference between these two probes is that the conversion of H2DCFDA involves oxidation. Therefore, fluorescence of the H2DCFDA-treated cells depends on the intracellular ROS level, in contrast to fluorescence associated with the DCFDA probe. Surprisingly, flow cytometry analysis shows that the difference between fluorescence levels of ESCs and difESCs loaded with H2DCFDA is close to the difference between the signals from DCFDA-treated cells. The latter indicates a ROS-independent reason for low fluorescent signal of oxidized H2DCFDA in ESCs[1].

[1]. Lyublinskaya OG, et al. Redox environment in stem and differentiated cells: A quantitative approach. Redox Biol. 2017 Aug;12:758-769.