Fig. 3. Characterization of HKYellow-AM for imaging peroxynitrite in live cells. (a) Validation of HKYellow selectivity with exogenous ROS/RNS donors in SH-SY5Y human neuroblastoma cells. The cells were firstly incubated with HKYellow-AM (10 μM) and then treated with H₂O₂ (100 μM) or the indicated ROS/RNS donors for 1 h, followed by fluorescence imaging. NOC-18 (1 mM), MSB (200 μM), and SIN-1 (100 μM) were used to produce ˙NO, O₂˙^–, and ONOO^–, respectively. FeTMPyP (50 μM) was used as an ONOO^– decomposition catalyst. The scale bar represents 20 μm. (b) Intracellular retention of HKYellow. SH-SY5Y cells were stained with HKYellow-AM (10 μM) and then treated with 500 μM SIN-1 for 30 min. After washing three times, the cells were imaged at indicated time points. The scale bar represents 20 μm. (c) Relative mean fluorescence levels of the cells shown in (b) were quantified. Data are the mean ± S.E.M., n = 211–246 cells. For each experiment, at least three independent biological replicates (n ≥ 3) on different days were performed showing reproducible results.