Cell invasion ability was determined using Transwell chambers with an 8μm pore size (Corning, USA) and Matrigel (BD Bioscience, China). Cells were incubated in the upper chamber at a density of 2 × 104 cells/chamber with 0.5mg/L Matrigel, and medium with patient serum in a final concentration of 10% was added to the lower chamber. After a 24h incubation, chambers were fixed with paraformaldehyde for 30 min and stained with 0.5% crystal violet for 20 min. Positive cells were visualized using a microscope. Three random fields per chamber were counted using the Image J1.54 software and averages were calculated to reflect invasion activity of the sample. Mean percentage change from post- to preoperative values for each individual patient was calculated and compared between the GA and LA groups. Mean percentage change = [(No. of invaded cells with postoperative serum) − (No. of invaded cells with preoperative serum)]/(No. of invaded cells with preoperative serum) ×100%. Representative fields were photographed with an Olympus fluorescence microscope at 100× magnification.
Transwell chambers with an 8 μm pore size
Manufactured by Corning
Sourced in United States
Transwell chambers with an 8-μm pore size are a type of cell culture insert used for various in vitro studies. The chambers feature a porous membrane with a pore size of 8 micrometers, which allows for the passage of cells, molecules, and other materials between the upper and lower compartments of the chamber.
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4 protocols using transwell chambers with an 8 μm pore size
1
Cell Invasion Assay with Serum
2
Quantifying Cell Invasion and Migration
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Twenty-four-well Transwell chambers with an 8-μm pore size (Corning, New York, NY, United States) were coated with BD Matrigel Basement Membrane Matrix (BD Biosciences, San Diego, CA, United States), maintained overnight at 4 °C, and then polymerized at 37 °C. The transwell chambers used in the migration assay were not coated with Matrigel. Cells were plated in transwell chambers at a density of 1 × 105 cells per well and incubated. After 20 h, the cells that passed through the transwell were stained with 4% paraformaldehyde and then stained with haematoxylin-eosin (HE). By counting the number of stained cells in the four quadrants from each insert and averaging the obtained triplicate values, the number of cells invaded or migrated through Matrigel was quantified.
3
Transwell Invasion Assay for HONE1 and 5-8F Cells
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Transwell chambers with an 8-μm pore size (Corning Incorporated, USA) coated with Matrigel were used to estimate the invasion of HONE1 and 5-8F cells. Briefly, 500 μl of serum-free DMEM containing 2 × 10 5 treated HONE1 and 5-8F cells was added to the upper chamber, while 500 μl of DMEM including serum (10%) was added to the lower chamber. After 24 h of culture, cells remaining on the upper surface were discarded, and those cells on the bottom surface were fixed and stained with 5% crystal violet followed by manual number counting.
4
Transwell Cell Invasion Assay
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Cell invasion capability was measured by Transwell chambers with an 8-μm pore size (Corning, Tewksbury, MA, USA) coated with Matrigel. Brie y, 1×10 5 cells in 100 μL of serum-free RPMI 1640 medium were placed into the upper chamber. 500 μL medium containing 10% FBS was added in the lower chamber. Cells were dyed in 0.5% crystal violet after 48 h culture at 37°C with 5% CO 2 . The cells remaining in the upper chamber were removed. Following washing with PBS and drying, images were taken under a microscope (Olympus, Japan).
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