To verify cell migration, Transwell assay was conducted. Following transfection and 24 h incubation, cells were starved of FBS for 6 hours, then placed into the upper chamber (20,000 cells/chamber) of Transwell assay plates (Corning, USA), and 200 μL of DMEM containing 10% FBS was added. After 24 h, the medium was removed and the cells that migrated to the lower chamber of the plate were fixed in 4% formaldehyde solution, stained with crystal violet for 20 minutes, and counted under light microscope.
Transwell assay plates
Manufactured by Corning
Sourced in United States
Transwell assay plates are a type of cell culture insert used to study cell migration, invasion, and permeability. The plates consist of a permeable membrane that separates the upper and lower chambers, allowing for the exchange of media and solutes between the chambers. The membrane serves as a barrier that cells must migrate through or across to reach the lower chamber.
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5 protocols using transwell assay plates
1
Transwell Assay for Cell Migration
2
Transwell Assay for Cell Migration
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Cell migration and invasion were tested using Transwell assay plates (Corning, New York, U.S.A.). Cells were placed into the upper chamber of the insert with matrigel (Corning, New York, U.S.A.). After 48 h incubation at 37°C, cells adhering to the lower membrane of the insert were stained with 0.1% Crystal Violet and counted using a Leica microscope (Leica, Wetzlar, Germany).
3
Transwell Assay for Cell Migration and Invasion
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EC cell migration and invasion capabilities were evaluated using transwell assay plates (#3422, Corning, Corning, NY, USA). In brief, cells from different groups were suspended in serum-free media and transferred into the upper chambers of transwell plates for migration assays or inoculated into Matrigel (#356234, Corning, USA) coated transwell chambers for invasion assays. The lower chambers were filled with 600 µL of medium containing 20% fetal bovine serum as a chemoattractant. After 24 hr of incubation at 37°C, the migrated and invasive cells in the lower chambers were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 30 min, and subsequently photographed and counted under a microscope.
4
Transwell Assay for Cell Migration and Invasion
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Transfected HepG2 cells were harvested after transfection for 24 h and seeded into the upper chambers (20,000 cells/chamber) of transwell assay plates (Corning, USA) with 200 μl of serum-free DMEM to quantify cell migration. Similarly, transfected HepG2 cells were seeded into the upper chambers (40,000 cells/chamber) of transwell plates with the Matrigel-coated membrane (BD, USA) in 200 μl serum-free DMEM to quantify cell invasion. The lower chambers were filled with DMEM containing 10% FBS. After an incubation period of 24 h, the medium was removed, and cells were fixed with methanol for 20 min. The cells were then stained with crystal violet for 20 min. Air-dried and photographed with a digital microscope. The number of cells was calculated from five random fields for each chamber.
5
Quantifying Cell Migration and Invasion
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Transfected HepG2 cells were harvested after transfection for 24 h and seeded into the upper chambers (20,000 cells/chamber) of Transwell assay plates (Corning Incorporated, Corning, NY, USA) with 200 µL of serum-free DMEM to quantify cell migration. Similarly, transfected HepG2 cells were seeded into the upper chambers (40,000 cells/chamber) of Transwell plates with Matrigel-coated membranes (BD, Franklin Lakes, NJ, USA) in 200 µL serum-free DMEM to quantify cell invasion. The lower chambers were filled with DMEM containing 10% FBS. After an incubation period of 24 h, the medium was removed and cells were fixed with methanol for 20 min. The cells were stained with crystal violet for 20 min, air-dried, and photographed using a digital microscope. The number of cells was calculated from five random fields in each chamber.
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