These selected clones were taken for identification and frozen for future use. Analysis of transfectants RT-PCR and Western blotting analysis were respectively performed to detect the mRNA and protein of FBG2, and immunocytochemical analysis was used to detect the expression of FBG2 protein in situ. Cell growth curve assay All of 12 MKN-FBG2 cell clones and 9 HFE-FBG2 which stable expressed

FBG2 were used. 12 clones which were transfected by PCDNA3.1 empty vector and untreated cell strains were used as control groups. The cells of each clone were inoculated into 24-well culture plate at the concentration of 5 × 104/ml. After learn more the cells completely adhered to the wall, they were washed once with PBS and then trypsinized in 0.5 ml of Trypsin/EDTA and counted in triplicates at 1 to 7 day using a cell counter (Beckman Coulter, Inc., Fullerton, CA). The mean values of all 12 MKN-FBG2 cell clones and 9 HFE-FBG2 on different time were calculated, and growth curves were plotted. In addition, MKN-PC cell clones, HFE-PC cell clones and untreated cell clones were used as control groups. Analysis of cell cycle and apoptosis FBG2 gene stable expression cell groups(MKN-FBG2, HFE-FBG2), PCDNA3.1 empty vector transfection groups(MKN-PC, HFE-PC) and untreated cell control groups were detected by flow cytometry. When the cells covered 70% of the area of cell culture plates in each group, serum-free culture medium was used

for synchronization. After 24 hours’ ICG-001 research buy continuous culture, the cells were harvested and fixed by 100% ethanol, then prepared for single cell suspensions. After DNA staining, the cell cycles of the Teicoplanin samples were measured on a FACS Calibur cytometer. The analysis software was CellQuest. After synchronization and 24 hours’ continuous culture, the cells were harvested and fixed, PI and AnexinV-FITC double staining was performed, and flow cytometry was used to detect the apoptosis of cells. 3 replicate tests on every clone were performed in each group, the average values of three groups were calculated respectively, and comparison

between three groups was conducted. Colony RG-7388 nmr formation assay MKN-FBG2, HFE-FBG2, MKN-PC, HFE-PC and untreated cell control groups were detected. 1000 cells of each clone were respectively seeded in a 9 cm cell culture dish. After 18 days’ culture in DMEM containing fetal calf serum, the number of cell clones with more than 50 cells was counted under microscope in each dash (clone formation rate = number of clones in each dish/1000). Three reduplicate dishes were used from each clone. Cell colonies were then fixed and stained with 0.5% methylene blue (Sigma, Poole, Dorset, U.K.) in ethanol. All colonies visible by eye were counted separately for each sample and evaluated their clone formation rates. Cell migration assay Cell migration assays were performed using FCS-coated polycarbonate filters (8 μm pore size; Transwell)[10].