br Ethynyl Deoxyuridine Cells exhibiting logarithmic growth were inoculated
5-Ethynyl-20-Deoxyuridine Cells exhibiting logarithmic growth were inoculated into 96-well plates at a density of 4 103 to 1 105 cells/well and cultured until to normal cell development stage. The cell culture medium was used to dilute 5-ethynyl-20-deoxyuridine (EdU) solution at a ratio of 1000:1, and 50 mM EdU culture medium was prepared accordingly.
The INCB018424 were incubated with 100 mL EdU (50 mM) culture medium for 2 h and then abandoned. The cells were fixed with 50 mL cell fixa-tive solution (PBS containing 4% paraformaldehyde) for 30 min; then the fixative solution was discarded. After 50 mL (2 mg/mL) glycine was added, cells were incubated in the decolorized shaker for 5 min, after which the glycine solution was discarded. The cells were then incubated for 10 min in a decolorized shaker with
100 mL 1 Apollo staining reaction liquid was added to the cells and incubated in a decolorized shaker under conditions void of light for 30 min followed by the removal of the reactive liquid. The cells were then washed with 100 mL infiltrator (PBS containing 0.5% Triton X-100) in decolorized shaker two to three times, each time for 10 min, followed by the removal of infiltrator. Reagent F was diluted by deion-
ized water at a ratio of 100:1 to prepared moderate 1 Hoechst 33342 staining reaction liquid, which was preserved in dark conditions void of sunlight. The cells were added with 100 mL 1 Hoechst 33342 and incubated in a decolorized shaker avoiding exposure to light for
30 min, followed by the removal of reactive liquid.
Detection of cell migration was performed as follows: in the event that cell confluence reached 80%, the third-generation cells were starved in the serum-free DMEM culture medium for 24 h. Serum-free DMEM culture medium was added to the bottom of the Transwell chambers (Corning, NY, USA) and placed at 37 C for 1 h. After digestion, the cells were resuspended with serum-free DMEM, counted, and diluted to 3 105 cells/mL, of which 100 mL was added to the apical chamber. At the same time, 600 mL DMEM with 10% serum (serum was taken as chemokines) was added into the basolat-eral chamber and incubated for 24 h in accordance with the instruc-tions of the Transwell chamber. The cells from the apical chamber were soaked in precooled methanol for 30 min, then fixed and trans-ferred into the basolateral chamber. Subsequently, the cells were stained with 0.1% crystalline violet solution for 10 min. The cells
Table 1. Information of Pancreatic Cancer Chip
36 pancreatic cancer
tumor samples and
16 normal samples
25 pancreatic cancer
tumors and 7 non-
8 normal pancreatic
and 14 pancreatic ductal
18 with pancreatic tumor
pancreatic tissue samples
16 pancreatic cancer
cell lines and 4 normal
were then photographed and counted under an inverted microscope (Olympus, Tokyo, Japan) with six visual fields selected.
Detection of cell invasion was performed as follows: Matrigel pre-served at 20 C was melted at 4 C and diluted using serum-free DMEM with the remaining procedures performed in an identical fashion to the steps used for cell migration.
Annexin V-FITC and/or propidium iodide (PI) double standard staining was employed to detect cell apoptosis. The cells were collected 48 h after transfection with the concentration adjusted to 1 106 cells/mL. Cells were fixed overnight with 70% precooled ethanol solution at 4 C. 100 mL cell suspension (no less than 106 cells/mL) was centrifuged. The cells were subsequently resuspended in 200 mL binding buffer and gently mixed with 10 mL Annexin V-FITC and 5 mL PI under conditions void of light for 15 min, fol-lowed by the addition of 300 mL binding buffer. Cell apoptosis was de-tected using flow cytometry at an excitation wavelength of 488 nm.