Cell experiment:

HeLa cells and RAW264.7 macrophages are cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplied with 10% fetal bovine serum (FBS) and 1% antibotics (penicillin and streptomycin) at 37℃ in humidified incubator containing 5% CO2. The cells are seeded into glass-bottomed dishes and cultured for 24 h. Subsequently, the cells are incubated with FHZ for 30 min at 37℃ and then washed with PBS buffer three times. Each treatment of cells with H2O2, EDTA-Fe2+, HClO or scavengers kept 30 min[1].

Animal experiment:

Wild type zebrafish is used in this study. Seven-day old fertilized zebrafish embryos are cultured in 50 μM FHZ for 30 min, and then the zebrafish embryos are transferred to fresh water. A FHZ-loaded zebrafish is fixed under confocal microscope using 2% agarose gel to keep its living state for fluorescent imaging. In order to observe the release of oOH in fresh wound, the ventral fin of the FHZ-loaded zebrafish is carefully cut a small wound using a blade. After raised for 20 min in water, the wound of injured zebrafish is imaged using confocal microscope[1].

FHZ is a fluorescent probe.

After loaded with probe FHZ and treated with HClO and H2O2/EDTA-Fe2+ in order, HeLa cells display the bright fluorescences from both cyan and green channels. FHZ can give out two different fluorescent signals in the presence of both •OH and HClO, suggesting the synchronous discrimination of •OH and HClO by a dual-channel detection model with two exciting wavelengths. Probe FHZ shows very high specificity to the detections of •OH and HClO with the excitations at 410 and 490 nm, respectively. The probe FHZ can efficiently enter the cellular mitochondria and exhibit the differentiable/visual capabilities to the endogenous •OH and HClO by the dual fluorescent responses[1].

Probe FHZ can rapidly be absorbed into the blood circulation system from the zebrafish intestine, and spread out whole zebrafish tissues, and keep its stability in the blood, organs and tissues in the absence of ROS. The probe can keep its stability in biological environments and only selectively react with •OH and HClO species[1].

[1]. Zhang R, et al. Real-Time Discrimination and Versatile Profiling of Spontaneous Reactive Oxygen Species in Living Organisms with a Single Fluorescent Probe. J Am Chem Soc. 2016 Mar 23;138(11):3769-78.