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About 4 to 6 different parts of the slide were randomly selected for analysis. Fluorescence-activated cell sorting analysis Cells were washed in PBS and fixed in methanol overnight. Subsequently, cells were washed and resuspended in PBS containing 50 ��g/ml propidium iodide, 100 ��g/ml RNase, and 0.1% Nonidet P-40 for 30 minutes at 37��C. The distribution of cells in different phases of the cell cycle was determined by measuring the nuclear DNA content using a FACS Calibur cell flow cytometer (Becton, Dickinson and Company). Cell proliferation assay MTS assay was applied to assess cell proliferation as instructed by the manufacturer (Promega). Cells were seeded at [http://www.selleck.co.jp/products/atezolizumab.html http://www.selleck.co.jp/products/atezolizumab.html Atezolizumab anti-PD-1 antibody inhibitor click here] 3��103 cells/well in 100 ?l/well using 96-well culture plates. The absorbance of the samples was measured at 490 nm on a scanning multi-well spectrophotometer. The experiment was repeated 3 times. Cell proliferation was compared at four time point (6 h, 24 h, 48 h, and 72 h) after the cells were seeded. Cell migration and invasion assay In vitro cell migration and invasion assays were performed as described previously [18]. Cells growing in the log phase were trypsinized, re-suspended in serum-free medium, and seeded into Boyden chambers (8 ��m pore size with polycarbonate membrane). The chambers see more were then inserted into transwell apparatus (Costar, Cambridge, MA, USA). The chambers were coated with Matrigel (BD Biosciences, San Jose, USA) when cell invasion assay was done. selleck screening library Medium with 10% FBS (600 ��l) was added to the lower chamber. After incubation of 48 hours, cells on the top surface of the insert were removed by wiping with cotton swab. Cells that migrated to the bottom surface of the insert were stained in 0.3% crystal violet for 30 min, rinsed in PBS and then subjected to microscopic inspection. Images of four random fields (10��) were captured from each membrane, and the number of migratory or invasive cells was counted. The migration and invasion results were normalized by cell proliferation under the same treatment conditions. Triplicate assays were used or each experiment. Statistical analysis Statistical analyses were done using the SPSS 19.0 software version (SPSS, Inc., Chicago, IL). Mann-Whitney U nonparametric test was used for comparing two different groups such as tumor size, LN metastasis and Lymphovascular invasion, but if more than two groups were compared, Kruskal-Wallis nonparametric test was used. A value of p