TNBC cells after transfection were planted in the upper chamber of Transwell chambers (24-well; Corning Incorporated, Corning, NY, USA). Complete culture medium, meanwhile, was filled in the lower chamber which was cultivated with PBS. Twenty-four hours later, cells migrating to the lower chamber were removed with caution by a cotton swab and then fixed in methanol solution for 15 min. Crystal violet was adopted to stain the membranes for 10 min, and the invaded or migrated cells were observed and counted under a microscope (Olympus) at the magnification of 10 × 10. The experiment was conducted for three times in an independent manner.
Transwell chambers 24 well
Manufactured by Corning
Sourced in United States
Transwell chambers (24-well) are a type of cell culture insert used for various applications, such as cell migration, invasion, and permeability studies. The chambers consist of a membrane insert that fits into a 24-well plate, creating an upper and lower compartment. This design allows for the study of cellular interactions between the two compartments.
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Lab products found in correlation
15 protocols using transwell chambers 24 well
1
Transwell Invasion and Migration Assay
2
Matrigel Transwell Invasion Assay
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To assess cell invasion, cells (5 × 104) suspended in serum‐depleted culture medium were transported to the upper chamber of transwell chambers (24‐well; Corning Incorporated, Corning,) pre‐coated with Matrigel (BD Biosciences). Cell culture medium containing 10% FBS, served as a chemoattractant, was supplemented into the lower chambers. After 24 h‐incubation at 37°C, cells invaded to the low surface were fixed using methanol and stained with 0.1% crystal violet. The images of cell morphology and the number of invaded cells in random 5 fields were recorded using an inverted microscope (×100; Leica).
3
Transwell-based Migration and Invasion Assays
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Transwell chambers (24-well; Corning, Inc.) were used to assess the migratory and invasive ability of the CRC cells (SW-480 and DLD-1). For the migration assay, cells (6×104 cells/well) were re-suspended in serum-free DMEM and seeded into the upper chamber with an 8 µm pore size, and into the lower chamber, DMEM with 10% FBS was added. Following cell culture for 24 h at 37°C, the chambers were washed with PBS. Subsequently, the cells that had migrated to the lower chamber were fixed with 4% paraformaldehyde at room temperature for 30 min, then stained with crystal violet (Beyotime Institute of Biotechnology) at room temperature for 30 min. Finally, images were captured under a microscope (TS100; Nikon Corporation) and the numbers of cells were counted.
For the invasion assays, the experimental procedure applied was similar to that described above for migration, with the difference that the upper chamber was pre-coated with BD Matrigel (BD Biosciences) at 37°C for 8 h according to the manufacture's protocol for the invasion assay, and cells were cultured for 48 h.
For the invasion assays, the experimental procedure applied was similar to that described above for migration, with the difference that the upper chamber was pre-coated with BD Matrigel (BD Biosciences) at 37°C for 8 h according to the manufacture's protocol for the invasion assay, and cells were cultured for 48 h.
4
Transwell Assay for Cell Migration
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A total of 2 × 104 PC cells were collected after transfection, and then seeded in the upper chamber of transwell chambers (24-well; Corning Incorporated, Corning, NY, USA) with serum-free medium for testing cell migration. The lower chamber was filled with the complete culture medium. Twenty-four hour later, the migrated cells were dyed by crystal violet and counted under an optical microscope (DMI1, Leica, Wetzlar, Germany). The assay was independently carried out in triplicate.
5
Transwell Invasion Assay Protocol
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Transwell chambers (24-well; Corning, Inc.) with a 5-µm pore filter were coated with a mixture of 60 ml 1:9 Matrix (BD Biosciences) and serum-free RPMI-1640 medium to form a matrix barrier. Precoating was performed at 37°C for 1 h and the Transwell inserts were refrigerated at 4°C overnight. Before inoculation, RPMI-1640 medium was used to hydrate the basement membrane for 30 min at 37°C. The upper chamber cells were filled with 200 µl serum-free RPMI-1640 medium, and the lower chamber was filled with 500 µl RPMI-1640 medium supplemented with 10% FBS. Following incubation at 37°C for 48 h, the cells were washed twice with PBS (5 min per wash) and fixed with 600 µl polyformaldehyde for 15 min at 4°C. The non-invading cells were wiped with a cotton swab, washed with ddH2O three times and stained with 5% crystal violet solution for 5 min at room temperature (23 (link)). The number of invading cells was counted in four random fields using an inverted phase contrast microscope (Olympus Corporation) at a ×50 magnification.
6
Transwell Cell Migration and Invasion Assay
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Cell migration assay was performed using Transwell chambers (24-well; Corning, Corning, NY, USA). The lower chamber was added with complete culture medium. Cells were suspended in serum-free medium and plated into the upper chamber. Then the chambers were cultivated with 5% CO2 at 37 °C for 24 h. Cells in the upper layer were removed with caution by a cotton swab and then the cells in the lower chamber were fixed in methanol solution for 15 min. After that, migrated cells were counted using 0.1% crystal violet dye and observed via a light microscope (DMi1, Leica, Wetzlar, Germany). Transwell chambers were pre-coated with Matrigel (Clontech, Madison, WI, USA) for invasion assay. Cell migration and invasion were determined by counting 5 random fields under a microscope (DMi1, Leica, Wetzlar, Germany).
7
Evaluating Cell Invasion under Normoxia/Hypoxia
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Cell invasion under normoxic or hypoxic conditions was evaluated in Transwell chambers (24-well; Costar, Cambridge, MA, USA) as described previously.22 (link) Initially, fibronectin (2 µg/filter) was dissolved in 100 µL of minimal essential medium and poured into the upper section of the polyethylene filter (pore size, 8 µm). The wells were coated overnight in a laminar flow hood, after which 5×105 cells (in 100 µL growth medium) were added to the top of the filter in the upper well. The chamber was incubated for 12 h under normoxic or hypoxic conditions with 5% CO2 at 37℃. Non-migrant cells located in the upper section of the filter were removed using a cotton swab. Finally, attached cells in the lower section were stained with crystal violet and counted using a light microscope.
8
Transwell Invasion Assay for Cell Lines
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Transwell chambers (24-well, Costar, Cambridge, MA, USA) were used to assess cellular invasion as described previously7 (link). Briefly, Matrigel (Coring, MA, USA) was dissolved in 100 μL MEM and applied to a polyethylene membrane filter with a pore size of 8 μm. Wells were coated overnight in a laminar flow hood. Next, cDNA or siRNA-transfected cells (1 × 104 in 100 μL growth medium) were added to the top of the filter in the upper well. The chamber was incubated for 48 h for SNU1041 cells and for 72 h for SCC15 cells in 5% CO2 at 37 °C. Finally, attached cells in the lower section (invading cells) were stained with crystal violet and counted in four representative fields by light microscopy (200x magnification).
9
Cell Migration Assay with Transwell
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Transwell chambers (24-well; Costar, Boston, MA, U.S.A.) were applied for cell migration assay. Cells in serum-free media were put into upper chambers afterwards, while medium with 10% FBS was put into the bottom chambers. After incubation for 24 h, migratory cells were treated with 4% PFA (Sigma-Aldrich, Burlington, Massachusett, U.S.A.) and 0.5% Crystal Violet. The cell numbers of three replications were counted in five different fields by a microscope.
10
Transwell-Based Cell Invasion Assay
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The invasion assay was carried out using Transwell chambers (24 wells; Corning, Tewksbury, MA, USA) and a previously described protocol.8 , 9 Cells (2 × 104) were seeded on the top side of the Transwell filter coated with Matrigel in the chamber. Cells were suspended in the serum‐free medium. The medium supplemented with 10% FBS was added into the lower chamber. After culturing for 24 hours, cells that had invaded the membrane were fixed with 75% methanol and stained with 0.1% crystal violet. The number of invaded cells was observed using an inverted microscope and calculated by counting 5 random views. All experiments were carried out in triplicate.
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