br ized water at a ratio of to
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 GW501516 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.
Isolation and Identification of BMSCs
Under sterile conditions, 10 mL bone marrow was extracted from the femoral shaft fracture end with a 20-mL syringe (containing 2,000 IU heparin) and mixed with heparin quickly. The bone marrow was centrifuged in a centrifuge tube at 258 g for 10 min in order to re-move the upper adipose tissue, followed by three washes with DMEM, and resuspended using 15 mL medium. Bone marrow was centrifuged in a centrifuge tube containing the same volume of Ficoll-Paque PLUS lymphocyte separation fluid at 716 g for 20 min. Nucleated cells were noted to be located predominately in the boundary and up-per liquids, while most of the erythrocytes had precipitated to the bot-tom. The nuclear cells were withdrawn from the interface with a straw, centrifuged at 179 g for 8 min, after which the supernatant was discarded. Next, 5 mL cell culture medium was added to make Molecular Therapy: Nucleic Acids
nuclear cells evenly spread. The cell suspension (10 mL) was evenly mixed with 490 mL PBS. After that, 10 mL of mixture was obtained and counted under the microscope. The cells were inoculated in a cul-ture bottle (1 105 cells/bottle) and incubated with 5 mL low-glucose DMEM culture medium at 37 C with 5% CO2 and saturated humid-ity. After 24 h, BMSCs began to adhere to the wall, and half of the me-dium was replaced to remove non-adherent cells. The medium was replaced every 2–3 days, during which a small amount of hematopoi-etic stem cells, as well as the red blood cell suspension that failed to be removed by means of centrifugation, along with the other non-adherent mixed cells, was removed in a progressive manner. Cell adhesion and growth were observed using an inverted phase-contrast microscope. When the monolayer adherent cells grew to 80%–90% confluence at days in vitro (DIV) 10–14, the cells were treated with 0.25% trypsin and sub-cultured at ratio of 1:2–1:3. Flow cytometer was used to detect surface markers CD29, CD34, CD44, CD45, CD71, and HLA-DR of BMSCs. The adipogenic and osteogenic dif-ferentiation of BMSCs was identified according to the ability of inducing differentiation in vitro, and the formation of lipid droplets was observed by oil red O staining under an inverted microscope. The calcium deposits of osteoblast differentiation were observed through the application of alizarin red staining 4 weeks after osteo-blast induction and differentiation.