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. 2016 Dec 7;39(1):174–182. doi: 10.3892/ijmm.2016.2822

Figure 4.

Figure 4

Effects of nobiletin on intracellular reactive oxygen species (ROS) formation and protein carbonyl expression in human platelets. (A) Platelet suspensions (3.6×108 cells/ml) were incubated in 24-well microtiter plates with 20 µM of dichlorofluorescein fluorescence (DCF) stock in ethanol. The content of each well was then transferred into a 5 ml tube for centrifugation (335 × g, 10 min, 37°C). After removing the supernatant, suspensions were subsequently re-suspended in Hank's Balanced Salt Solution (HBSS) buffer and transferred to the wells. Following treatment with various concentrations (50, 100 and 500 µM) of nobiletin and 1 µg/ml of collagen, the generation of the fluorescent product DCF was followed in an automated plate fluorescence reader using an excitation wavelength of 485 nm and emission wavelength of 555 nm; (B) the expression of protein carbonyl was monitored in 2,4-dinitrophenylhydrazine (DNPH)-derivatized proteins using an OxyBlot Protein Oxidation Detection kit. (A and B) Profiles are representative of 4 independent experiments.