These experiments confirmed the former obser vation that MD

Proteins were then quantitatively de termined through densitometry by using FUJIFILM Multi Gauge V3. 0 software. Measuring of p53, p21, and p27 levels Intracellular levels of the levels of p53, p21, and p27 were オーダー Ivacaftor de termined by enzyme linked immunosorbent assay. In brief, T 24 cells were seeded in 5 cm dishes and grown to 85 90% confluence, and were then treated with 0, 20, and 30 ug ml of apigenin for 6, 12, 24, and 48 h. Cell lysates were placed in 96 well microtiter plates coated with monoclonal detective antibodies, and then incubated for 2 h at room temperature. After the unbound antibodies were removed through washing with a washing buffer, the detection antibody, which is bound by horseradish peroxidase conjugated streptavidin, was added to bind to the antibodies.<br><br> Horseradish peroxidase catalysed the con version of a chromogenic substrate to a coloured solution with colour intensity propor tional to the protein level present in the sample. The absorbance of each well was measured at 450 nm, and p53, purchase LBH589 p21, and p27 levels were determined by interpol ating from standard curves obtained with known levels of standard proteins. Data are expressed as pg p53, p21, or p27 levels. Measurement of intracellular levels of ROS Intracellular ROS was estimated using a fluorescent probe, 2,7 dichlorofluorescein diacetate. DCFH DA readily diffuses through the cell membrane and is enzymatically hydrolysed by intracellular esterases to non fluorescent dichlorofluorescin, which is then rapidly oxidised to highly fluorescent DCF in the presence of ROS.<br><br> Briefly, the cells were treated with 30 ug ml of apigenin for various periods. T 24 cells were then incu LY2109761 製造者 bated in a culture medium containing 20 uM DCFH DA for 30 min at 37 C, and were washed with PBS. The cell suspensions were centrifuged at 412 × g for 10 min and the medium was removed. Cells were dissolved with 1% Triton X 100, and DCF fluorescence intensity was de tected at various time intervals with an excitation wave length of 503 nm and an emission wavelength of 529 nm by using the FLUOstar OPTIMA spectrofluorophot ometer. DCF fluorescence intensity is proportional to the intracellular ROS levels. Measurement of intracellular levels of GSH The intracellular levels of GSH were measured using the method of Hissin and Hilf. After treatment, cells were washed with PBS and scrapped into 6. 5% trichloroacetic acid.<br><br> Phosphate EDTA buffer at pH 8. 0 was added to 0. 5 ml of the supernatant obtained after cen trifugation at 12000 × g. The final assay mixture contained 100 ul of diluted supernatant, 1. 8 ml of phosphate EDTA buffer, and 100 ul of 0. 1% o phthalaldehyde solution. After thorough mixing and incubation at room temperature for 15 min, fluores cence was read at wavelengths of 350 nm and 420 nm using the FLUOstar OPTIMA spectrofluorophotometer. The reduced form of GSH was used as a standard. Data are expressed as nano mole GSH per 106 cells. Statistical analysis Data are expressed as means standard deviation of 3 in dependent experiments and analysed using Students t test. P values of 0. 05 were considered statistically significant. Results Apigenin inhibited T 24 cells proliferation The chemical structure of apigenin is illustrated in Figure 1A.