The membrane was removed and fixed with methanol and stained with HE. Five vision fields were selected randomly under the BX50 microscope, and the number of cells that kinase inhibitor SB203580 pen etrated the membrane was counted. Each group consisted of duplicates. The invasion inhibition rate was calculated using the following equation Invasion inhibition rate number of cells in control group that penetrated the membrane 100%. MTT assay A549 cells were inoculated in 96 well plates at 1 105 cells well in phenol red free RPMI 1640 medium. After drug treatment, 12 L MTT was added into each well and the cells were incubated at C for 4 h. After incubation, 100 L isopropanol was added into each well and the samples were mixed well. The purplish blue Inhibitors,Modulators,Libraries crys tals were dissolved at room temperature.
The absorbance at 540 nm was read on the spectra Max Plus 384 FACS apoptosis assay Cells Inhibitors,Modulators,Libraries were collected and digested with 0. 25% trypsin and 0. 02% EDTA at 37 C for 3 4 min. Cells were Inhibitors,Modulators,Libraries pipet ted gently and then collected by centrifugation at 1000 rpm for 5 min. They were then washed twice with cold PBS and resuspended in the residual PBS. After adding 1 mL prechilled 80% ethanol, the cells were stored at 20 C over night. After washing twice with PBS, 60 80 L RNAase was added and cells incubated at 37 C for 30 min. After chilling on ice for 2 min, PI solution was added and the sample was incubated in the dark for 30 min. The cell cycle distribution and apoptosis were analyzed by flow cytometry. Data acquired by CEL LQuest was analyzed using the software ModFit LT that came with the machine.
Transmission electron microscope Inhibitors,Modulators,Libraries A549 cells cultured for 24 h, were collected from the cul ture flask wall with a cell shovel and prepared as a cell sus pension of 1 106 cells ml. The samples were centrifuged at 1000 rpm min for 10 min, and the supernatant was dis carded. Samples were rinsed with 0. 01 M PBS twice. Phos phate buffer containing 2. 5% glutaraldehyde was added and the samples were fixed for 30 min. The samples were then fixed with 1% osmium tetroxide for 30 min, dehy drated using a concentration series of ethanol and embed ded using epoxy resin Epon812. Thin sections were made using a LKB 5 type ultra thin slicing instrument. The slices were stained with uranyl acetate lead citrate and observed under the H 600 transmission electron microscope.
Western blot analyses Cytoplasmic and cell membrane proteins were Inhibitors,Modulators,Libraries prepared as described. 5 mL protein extracting liquid A, 0. 125 mol L sucrose, 2 mmol L EDTA, 015 mmol L EGTA, 10 mg L leupeptin, selleck chemicals and 50 mmol L mercaptoethanol containing 1 mmol L freshly added protease inhibitor mix was added to the cell samples. The samples were dissolved on an ice bath for 10 min and centrifuged at 4 C, 100,000 g for 1 h. The col lected supernatant contained cytoplasmic proteins.