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  • 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.
    Transwell Assay
    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
    Gene or
    Accession No.
    36 pancreatic cancer
    tumor samples and
    16 normal samples
    25 pancreatic cancer
    tumors and 7 non-
    malignant pancreas
    8 normal pancreatic
    and 14 pancreatic ductal
    adenocarcinoma tissues
    18 with pancreatic tumor
    pancreatic tissue samples
    16 pancreatic cancer
    cell lines and 4 normal
    pancreatic samples
    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.
    Flow Cytometry
    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.