A transwell insert (Cell Biolabs, Inc. Santiago, CA, USA) without Matrigel (BD Biosciences) was employed to detect cell migration. Following transfection and/or treatment, AC-16 cells suspended in 200 μL serum-free medium were placed into the upper chamber of Transwell, then 600 μL medium fixed with FBS was added into the bottom chamber. After incubation for 24 h, migrated cells on the lower face of the chamber were counted by an inverted light microscope in five random fields (100 ×). Experiments were performed three times.
Transwell insert
Manufactured by Cell Biolabs
Sourced in United States
The Transwell insert is a specialized cell culture device used to study cell migration and permeability. It consists of a porous membrane that separates the upper and lower chambers, allowing for the controlled movement of cells, molecules, and other substances between the two compartments.
Automatically generated - may contain errors
4 protocols using transwell insert
1
Transwell Assay for Cell Migration
2
Transwell Assay for Germ Cell Migration
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Culture mouse germ cells were harvested and depleted of feeder cells by two rounds of preplating. 105 mouse germ cells in single cell suspension were then placed in the upper chamber of a transwell insert containing a polycarbonate membrane with 5 μm pores (Cell Biolabs Inc.). The transwell insert was placed into a well of a 12-well plate containing serum-free medium with or without recombinant CXCL12/SDF-1 (R&D systems; 10 ng/mL) and AMD3100 (Sigma Aldrich, 1.25 μM) [19 (link)]. Cultures were incubated for 24 hrs following which the media containing migrated germ cells were collected and cells stained with cell stain solution (Cell Biolabs Inc.). The absorbance of invading germ cells was quantified at OD at 562 nm. cell stain solution in blank wells containing only medium was used as the reference solution. The higher the number of invading germ cells in the bottom well is, the greater the absorbance quantified is.
3
Transwell Cell Migration and Invasion Assay
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Migration assays were performed using an uncoated chamber (cat. no. 3422; 8-μm pore; Corning, Inc., Corning, NY, USA) and the ability of cells to migrate was measured. Invasion assays were performed by coating the chamber with Matrigel® according to the manufacturer’s protocol. The lower chamber of the Transwell inserts (Cell Biolabs) was filled with 800 μL RPMI 1640 supplemented with 10% FBS. In the upper chamber, 150 μL serum-free medium (Opti-MEM®; cat. no. 31985-070; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 2 × 105 cells was added. The cells were incubated for 24 h at 37 °C in a humidified incubator with 5% CO2. Cells that had migrated/invaded to the bottom of the chamber were stained with crystal violet (cat. no. HT90132-1L; Sigma-Aldrich; Merck KGaA, Burlington, VT, USA) and the cells were counted under a light microscope (400× magnification).
4
Transwell Assay for Cell Migration and Invasion
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Migration assays were performed using an uncoated chamber (Cat. No. 3422; 8 μm pore; Corning, Inc., Corning, USA) and the ability of cells to migrate was measured. Invasion assays were performed by coating the chamber with Matrigel® according to the manufacturer’s protocol. The lower chamber of the Transwell inserts (Cell Biolabs) was filled with 800 µl RPMI 1640 supplemented with 10% FBS. In the upper chamber, 150 µl serum-free medium (Opti-MEM®; Cat. No. 31985-070; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, USA) containing 2 × 105 cells was added. The cells were incubated for 24 h at 37 ℃ in a humidified incubator with 5% CO2. Cells that had migrated/invaded to the bottom of the chamber were stained with crystal violet (Cat. No. HT90132-1 L; Sigma-Aldrich; Merck KGaA, Burlington, USA) and the cells were counted under a light microscope (x400, magnification).
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