A colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Chemical Co.) assay was performed to measure the cell viability. Briefly, RAW 264.7 cells were treated with different concentrations of spermidine for 24 h or pretreated with spermidine for 1 h before stimulation with LPS for 24 h. After incubation, the medium was discarded, and MTT solution (5 mg/mL in phosphate-buffered saline, PBS) was added to each well and incubated for another 3 h at 37 °C. The medium was removed, and DMSO was added to dissolve the formazan dye. The optical density was then read at 560 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) to determine the cell viability.
Measurement of NO production …show more content…
The generation of NO and ROS in the zebrafish larvae was analyzed using fluorescent probe dyes, 4-amino-5-methylamino-2’7’ difluorofluorescein diacetate (DAF-FM-DA, Molecular Probes) and DCF-DA, respectively. After 4 dpf, the larvae were transferred into 24-well plates and incubated with DAF-FM-DA (5 μM) and DCF-DA (20 μg/mL) solution for 1 h in the dark at 28.5 °C, and then anaesthetized using 1-phenoxy-2-propanol (1/500 dilution, Acros Organics, Morris Plains, NJ, USA). The images of stained larvae were observed for the NO and ROS generation under a fluorescence microscope, and fluorescence intensity of individual larvae was quantified at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a spectrophotometer and ImageJ 1.46r software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA), respectively. The generation of NO and ROS were calculated by comparing the fluorescence intensity of treatment larvae to the controls [Ko and Jeon,
Measurement of NO production …show more content…
The generation of NO and ROS in the zebrafish larvae was analyzed using fluorescent probe dyes, 4-amino-5-methylamino-2’7’ difluorofluorescein diacetate (DAF-FM-DA, Molecular Probes) and DCF-DA, respectively. After 4 dpf, the larvae were transferred into 24-well plates and incubated with DAF-FM-DA (5 μM) and DCF-DA (20 μg/mL) solution for 1 h in the dark at 28.5 °C, and then anaesthetized using 1-phenoxy-2-propanol (1/500 dilution, Acros Organics, Morris Plains, NJ, USA). The images of stained larvae were observed for the NO and ROS generation under a fluorescence microscope, and fluorescence intensity of individual larvae was quantified at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a spectrophotometer and ImageJ 1.46r software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA), respectively. The generation of NO and ROS were calculated by comparing the fluorescence intensity of treatment larvae to the controls [Ko and Jeon,