FHZ

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°C 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°C 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 •OH 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].

References:

[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.