Wound-healing and transwell assays were used to evaluate migration. Cells were seeded into 6-well plates, and a 200 µL pipette tip was applied to wound the cell monolayer when grown to 100% confluency. The wound was photographed, which served as the 0 hour time point. Cells were incubated in medium without serum for 24 or 48 hours, and wound closure was photographed. For the transwell assay, cells (2×104 cells/200 µL) were seeded into the top chamber without serum in a 24-well polycarbonate transwell filter (8 µm pore size, Corning Incorporated, USA), which was precoated with (for invasion assay) or without (for migration assay) 30μL matrigel, and 20% foetal bovine serum (700 µL) was placed into the lower chamber. After incubation for 24 or 48 hours, cells grown in the polycarbonate transwell filter were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. A cotton swab was used to wipe cells from the top chamber, and migrated cells were photographed with an inverted microscope.
24 well polycarbonate transwell filter
Manufactured by Corning
Sourced in United States
The 24-well polycarbonate transwell filter is a laboratory equipment used for cell culture applications. It features a polycarbonate membrane that allows for the separation of cells and media between the upper and lower compartments of the well. The transwell system facilitates the study of cell-cell interactions, diffusion, and transport processes.
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4 protocols using 24 well polycarbonate transwell filter
1
Evaluating Cell Migration and Invasion
2
Transwell and Wound Healing Assays for Cell Migration
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The migration ability of the cells was determined by transwell assay and wound healing assay, which were carried out as previously described16 (link). For transwell assay, a total of 1 × 106 transfected cells were seeded into the top chamber of a 24-well polycarbonate transwell filter (8 µm pore size, Corning Incorporated, USA). Media containing 20% FBS was placed in the lower chamber. After incubation for 12 h, the migration cells on the lower surface were fixed with 4% paraformaldehyde and stained with 5% crystal violet. The numbers of crystal violet stained cells in five random fields were counted using an inverted microscope (Olympus, Tokyo, Japan). For wound healing assay, cells were seeded in 6-well plates, and grown to 100% confluency. A 10-µl pipette tip was applied to wound the cell monolayer. Subsequently, the cells were incubated in free-serum medium and cultured for 48 h. Wound closure was photographed using an inverted microscope.
3
Matrigel-Coated Transwell Invasion Assay
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The upper transwell chambers (8-μm pore) were coated with 100 μl of Matrigel at a dilution of 1:6 (BD Biosciences, San Jose CA, USA). A total of 1 × 105-transfected cells were seeded into the top chamber of a 24-well polycarbonate transwell filter (Corning Incorporated, Glendale, AZ, USA). Then, cells were treated with RANKL (1 μg/ml) for 48 h. The numbers of crystal violet-stained cells in five random fields were counted using an inverted microscope.
4
Evaluating Cell Migration Mechanisms
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Wound-healing and transwell assays were used to evaluate migration. LX2 cells were inoculated into 6-well plate, and when they grew to 100% confluence, the cell monolayer was wrapped with a 200 μL pipette tip. The wound was photographed, which served as the 0-h time point. Cells were incubated in serum-free culture medium for 48 h, and wound healing was photographed. For the transwell assay, LX2 cells (2 × 104 cells/200 µL) were seeded in the top chamber with 5% serum in a 24-well polycarbonate transwell filter (8 µm pore size, Corning Incorporated, USA), which was filled with 20% fetal bovine serum (700 µL) and placed in the lower chamber. After 48 h of incubation, cells grown in the polycarbonate transwell filter were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cells were wiped from the apical chamber with a cotton swab, and migrated cells were photographed with an inverted microscope [17 (link)]. The grouping and processing of cells are the same as CCK8 assay.
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