Migration assays were performed using a 24-well Transwell chamber system (Corning, USA). 5 × 104 cells were seeded in the upper chamber of an insert with 0.4 ml serum-free culture media in 24-well plates. 0.6 ml culture media with 20% FBS were added to the lower chamber. After incubation for 24 h, cells were fixed in methanol for 15 min and then stained with 0.1% crystal violet (Sigma-Aldrich, USA) for 30 min. After rinsing with water, the membranes of the chambers were mounted, covered on slides. For invasion assays, the 24-well Transwell chamber system (Corning, USA) and the upper chamber of an insert was coated with Matrigel (Sigma-Aldrich, USA) before plating cells. Migrated or invaded cells were imaged and counted under a 20× microscope.
24 well transwell chamber system
Manufactured by Corning
Sourced in United States
The 24-well Transwell chamber system is a laboratory equipment designed for cell culture and cell-based assays. It consists of a 24-well plate with permeable membrane inserts that allow the passage of cells, molecules, or other substances between the upper and lower chambers. This system facilitates the study of cell migration, invasion, and permeability, as well as the interaction between different cell types or the effects of various treatments on cell behavior.
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12 protocols using 24 well transwell chamber system
1
Transwell Cell Migration and Invasion Assay
2
Transwell Migration Assay Protocol
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Migration capability was measured using 24-well transwell chamber systems (Corning, USA) with 8.0 µm pore size. Cells were seeded in the upper chamber insert and cultured in serum-free medium. The bottom chambers were filled with 10% fetal bovine serum medium. After 24 hours, the migrated cells were fixed with 4% paraformaldehyde for 20 min at room temperature and then stained with 1% crystal violet. The migrated cells were counted and photographed in three randomly selected views.
3
Transwell Migration Assay for Cancer Cells
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The modified Boyden chamber assay was performed in 24-well transwell chamber systems (Corning, USA) with 8.0 µm pore size. The breast and pancreatic cancer cells were cultured with or without ADSCs for 3 days and then trypsinized. Cancer cells (5×10 4 cells/well) were seeded in the upper chamber insert and cultured in serumfree media. The lower chamber was filled with complete medium (600 μL, 10% FBS). After incubated at 37°C for 24 hours, the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. The membranes were placed under an inverted phase contrast microscope and imaged to count the migrated cells. Three independent experiments were conducted.
4
Cell Migration Capability Assay
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Migration capability was measured using 24-well transwell chamber systems (Corning, USA) with 8.0 µm pore size. Cells were seeded in upper chamber insert and cultured in serum-free medium. The bottom chambers were lled with 10% fetal bovine serum medium. After incubated at 37°C for 24 hours, the cells on the lower surface of the membrane were xed with 4% paraformaldehyde and stained with crystal violet. The membranes were placed under an inverted phase contrast microscope and imaged to count the migrated cells. Three independent experiments were conducted.
5
Cell Migration Assay Using Transwell
6
Cell Migration Assay with Iron Modulation
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A 6.5 mm (0.8 μm pore size) 24-well transwell chamber system (Corning) was used in Migration assay. 3×105 Cells were suspended in 100 µl serum-free DMEM, and added into the upper chamber. 600 µl DMEM with 10% FBS was added into the bottom of 24-wells. After culture at 37 °C for 24 h, cells were co-incubated with DMT1 and TfR1 neutralizing antibody and DFO for 24 h. The upper chamber was washed with PBS for 10 s 3 times, and the cells on the on the upper layer of the filter were scrubbed. Then the upper chamber was fixed in 4% paraformaldehyde for 10 min, and treated with crystal violet for another 15 min. Images were captured in 5 random fields by an invert Microscope with a 5× objective. 33% acetic acid was used to dissolve the migrated cells which were stained by crystal violet. Migrated cells were measured at OD 595 nm of the eluted crystal violet. All experiments were performed in triplicate.
7
Transwell Assay for Cell Migration and Invasion
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Transwell assays include migration and invasion assays, which were conducted in a 24‐well transwell chamber system (Corning, USA). For the migration assay, 5 × 104 cells with 0.2 ml FBS‐free DMEM were seeded into the upper chamber of the well. The wells were inserted into the lower chambers with 0.6 ml DMEM and 20% FBS. In terms of invasion, the upper chamber was first coated with Matrigel, and other steps were similar to migration assay. The membrane of the well was fixed with methanol and subsequently stained with crystal violet after 16 to 24 h of incubation. After staining, wells were washed with water and the cells on the membrane were taken with photos under a microscope.
8
Cell Migration and Invasion Assay
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A 24-well Transwell chamber system (Corning, USA) was used to conduct migration and invasion assay. For migration, in the upper chamber of the 24-well Transwell, 200 μl of suspension cells (4×104) in serum-free were seeded. In the lower chamber, 600 μl of culture media containing 20% FBS was added. For the invasion, 50 μl of Matrigel diluted with serum-free media was put into the upper chamber and incubated until solidification. Then 200 μl of suspension cells (4×104) in serum-free culture media were seeded in the upper chamber of the 24-well Transwell, and 600 μl culture media with 20% FBS were added to the lower chamber. Cells were fixed with 4% formaldehyde for 30 minutes after 24 hours of incubation and then stained for 2 hours with 0.1% crystal violet (Sigma-Aldrich, USA). The chamber membranes were mounted and covered on slides after being washed with PBS. Under a 200× microscope, migrated or invaded cells were visualized and counted.
9
Matrigel-coated Invasion Assay Protocol
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Matrigel-coated invasion assays were performed using a 24-well Transwell chamber system (Corning, NY, USA) as previously described 37 (link). Briefly, 5×104 cells in 400 μL serum-free culture medium were placed into the upper chamber, which was coated with Matrigel (BD, New Jersey, USA). A total of 600 μL medium supplemented with 20% FBS was added to the lower chamber. After incubation for 24 h, cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The stained cells were analyzed.
10
Cell Migration and Invasion Assay
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Cell migration and invasion assays were carried out using a 24-well transwell chamber system (Corning, 3422). A transwell apparatus was separated into upper and lower compartments by polycarbonate filters (8-μm pores). For migration assays, the NIH3T3 cells stably expressed empty vector or wild-type SS18-SSX1, and its mutants (4.0×104) were suspended in 200 μL of growth medium in the upper chamber. For invasion assays, the polycarbonate filters were coated with 300 μg/mL matrigel (Corning, 356234) before the cell seeding. The lower chamber contained a 750 μL growth medium supplemented with 10% FBS. After 24 h incubation at 37 °C in a 5% CO2 incubator, cells on the upper filter surface were removed by wiping with a cotton swab. Filters were then fixed in 4% paraformaldehyde (Dingguo Biotechnology, AR-0211) and stained with 0.1% crystal violet (Sigma-Aldrich, V5265). All cells that migrated to the lower filter surface were counted under a microscope at ×200 magnification. Each assay was performed in triplicates.
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