Intracellular reactive oxygen species (ROS) was determined by using the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) [54 (link)]. DCFH-DA readily diffuses through the cell membrane and is enzymatically hydrolyzed by intracellular esterases to form non-fluorescent 2′,7′-dichlorofluorescein (DCFH), which is then rapidly oxidized to form highly fluorescent 2′,7′-dichlorofluorescin (DCF) in the presence of ROS. The DCF fluorescence intensity is indicative of the amount of ROS formed intracellularly. Here, after treating the cells with IC50 of the complex for 6, 12 and 24 h, the medium was poured out, the cells were washed with PBS and then incubated with 10 µM DCFH-DA in the loading medium. After DCFH-DA was removed, the cells were washed with PBS and lysed by sonication for 1 min at room temperature. Immediately, the fluorescence intensity (relative fluorescence units) was measured at 485 nm excitation and 530 nm emission in a Jasco (Easton, MD, USA) F 6500 spectrofluorometer.
F 6500 spectrofluorometer
Manufactured by Jasco
Sourced in United States
The F 6500 spectrofluorometer is a laboratory instrument designed for fluorescence measurements. It provides accurate and reliable data acquisition for various fluorescence-based applications. The core function of the F 6500 is to excite samples with a light source and measure the resulting fluorescence emission.
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2 protocols using f 6500 spectrofluorometer
1
Intracellular ROS Measurement Protocol
2
Comprehensive Spectroscopic Analysis of Compounds
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NMR spectra were recorded on a JEOL JNM-AL 400 Fourier-transform NMR spectrometers (400 MHz). The chemical shifts of the 1H, 13C{1H}, 11B{1H} and 19F{1H} NMR spectra determined in CDCl3 were given in ppm relative to tetramethylsilane (0.00 ppm for 1H and 13C{1H}) as an internal standard, or boron trifluoride diethyl-ether complex (0.00 ppm for 11B{1H}) and hexafluorobenzene (−164.9 ppm vs. CFCl3 for 19F{1H}) as external standards. High-resolution fast-atom bombardment mass spectroscopies (HR-FAB-MS) were carried out on a JEOL JMS-700N spectrometer. Elemental analyses (C, H, N) were performed by a Perkin Elmer 2400II elemental analyzer. UV-vis absorption spectra were recorded on a Jasco V-560 spectrophotometer. The corrected emission spectra were obtained by using a Jasco F-6500 spectrofluorometer (excitation wavelength = 365 nm). Fluorescence decay measurements were conducted by using a Hamamatsu Photonics picosecond fluorescence lifetime measurement system C11200 equipped with picosecond light pulser PLP-10 as a 405 nm excitation light source. Emission quantum yields were determined by using a Hamamatsu Photonic Absolute PL Quantum Yield Measurement System C9920-02 (excitation wavelength = 365 nm). For the photophysical measurements, spectrophotometric-grade toluene was used as supplied.
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