Figure 2D shows that the luciferase activ ities of u PA were significantly suppressed, as

DMSO at 0. 33% is non toxic to the cell line mentioned above. Cytotoxicity of EADs Cytotoxicity of EADs on MCF 7 cells was determined by the MTT 2,5 diphenyltetrazo lium bromide assay. Briefly, 1x105 of cells were seeded in each well of a 96 well plate. After 24 hours incubation, cells were treated with EADs. Untreated control cells オーダー KU-0063794 were also included. After incubation with EADs for 24, 48 and 72 hours, 20 uL of 5 mg mL of MTT was added into each well and incubated for 3 hours. Active mitochondria in live cells reduced MTT to crystalline purple blue formazan. The number of living cells was proportionate to the amount of crystalline purple blue formazan produced. After incubation, media in each well was discarded and 100 uL of DMSO was added to solubilize the purple blue formazan.<br><br> The absorbance was measured with an ELISA plate reader at wavelength of 570 nm, and 630 nm as background. A graph of percentage of cell viability versus concentration of EADs was plotted and the IC50 was determined. Cell morphology study of apoptosis by inverted light microscope Briefly, 3 × 105 of MCF 7 cells were seeded in each well of a 6 well plate. After 24 hours of incubation, cells オーダー Lenalidomide were treated with EADs at concentration of 25 and 50 ug mL. Control untreated cells were also included. Morphological changes of cells untreated and treated with EADs were examined under an inverted light microscope after 24, 48 and 72 hours. The cells were captured at the same spot at different time interval. Cell cycle analysis Briefly, 3 × 105 of MCF 7 cells were seeded in each well of a 6 well plate and treated with EADs at 25 and 50 ug mL.<br><br> Control untreated cells were also included. After incubation for 24, 48 and 72 hours, cells were trypsinized and washed with PBS. After centrifugation, cell suspension was resuspended repeatedly into single cells prior fixation with 70% ethanol. Fixed cells were kept at 20 C for at least 2 hours. Later, fixed cells LY294002 154447-36-6 were washed with PBS twice and the supernatant was discarded. Cell pellets were resuspended with 425 uL of PBS in a round bottom tube. Next, 50 uL of RNAse and 25 uL of propidium iodide were added into the cell suspension and incubated for 15 minutes on ice in the dark. FACS Calibur and Cell Quest Pro software was used to determine the cell cycle distribution. A total of 10,000 of cells were acquired each time using FACS Calibur flowcytometer.<br><br> Flowcytometric data were analyzed using Modfit software and displayed in histogram cell count against DNA content. Annexin V PI apoptosis assay Briefly, 3 × 105 of MCF 7 cells were seeded in each well of a 6 well plate and treated with EADs at 25 and 50 ug mL. Control untreated cells were also included. After incubation for 24, 48 and 72 hours, cells were trypsinized, washed twice with PBS and the supernatant was discarded. The cell pellets were mixed with 185 uL of 1X binding buffer. Next, 5 uL of Annexin V FITC and 10 uL of propidium iodide were added into the suspension and incubated at room temperature for 10 minutes in the dark. Subsequently, 300 uL of 1X binding buffer was added prior to measurement using FACS calibur flowcytometer and Cell Quest Pro software. Samples were kept on ice.