Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have substantial physiological relevance due to their involvement in redox signaling and growth regulation. When abundant, these species cause oxidative stress and can produce detrimental effects on cellular health and survival. The traditional method to measure ROS and RNS is to readout fluorescence from fluorogenic indicator dyes by microscopy or flow cytometry. These methods limit multiplexing since indicators are only available in a few colors. We have developed a method for multiplexed detection of ROS and RNS by capillary electrophoretic separation of up to five green fluorescent species. Specifically, we have optimized a separation of the fluorescent forms of dihydrodichlorofluorescein diacetate (DCFH₂DA), aminophenyl fluorescein (APF), HKSOX-1, and Rhodamine 123, which indicate the levels of general ROS, highly reactive ROS, superoxide, and mitochondrial voltage, respectively. When assessing levels of nitric oxide, dihydrodichlorofluorescein diacetate was substituted with diaminofluorescein-FM diacetate (DAF-FM DA). Additionally, carboxyfluorescein diacetate (CFDA) was included as an internal standard to remove inter-day variation in detector response and to correct for cell loss during sample preparation. We have applied this assay to evaluate the specificity of each indicator in in vitro assays and to answer biological questions about the oxidative species generated by electron transport chain inhibitors in vivo. Using a lysis buffer that maintains the redox state of the indicators, we have performed ensemble analyses of cell lysates from the social amoeba Dictyostelium discoideum. The resulting electropherograms yielded measures of ROS, RNS, and mitochondrial voltage at basal levels and in response to exogenous ROS administration and electron transport chain inhibition.

Michelle Kovarik

Trinity College

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