We assayed the invasion and migration activity of cells using a transwell cell culture chamber as described previously [10 (link)]. Briefly, SMMC-7721 and Huh7 cells were seeded into the upper compartment of a Transwell Boyden chamber (BD Biosciences, Franklin Lakes, NJ, USA) at a density of 1 ×104/well, with 100 μl serum-free media added into the upper compartment and 500 μl complete media added into the lower compartment. For the invasion assays, the Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was mixed with serum-free medium at a proportion of 1:5, then applied to the upper compartment of the Transwell Boyden chamber, 100 μl/well, and incubated for 1 h before seeding cells. TGFβ1 with or without indicated concentrations of JR was added into the upper compartment. After incubation at 37°C for 24 h, images of the cells of each group that had migrated to the chamber of the poly carbon membrane were captured and the results quantified.
Transwell boyden chamber
Manufactured by BD
Sourced in United States
The Transwell Boyden Chamber is a laboratory equipment used for cell migration and invasion assays. It consists of an upper and a lower chamber separated by a porous membrane. Cells are placed in the upper chamber and their ability to migrate through the membrane is measured.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (6 mentions)
Crystal violet, by Merck Group (6 mentions)
Recombinant mouse ccl25 protein, by R&D Systems (2 mentions)
Inverted microscope, by Olympus (2 mentions)
Crystal violet, by Beyotime (2 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Light ix71 inverted research microscope, by Olympus (1 mentions)
Fixation buffer, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Ix51 microscope, by Olympus (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Eclipse ts2, by Nikon (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Recombinant murine ccl2, by Thermo Fisher Scientific (1 mentions)
Huvecs, by BD (1 mentions)
Ebm 2, by Lonza (1 mentions)
Huvecs, by Lonza (1 mentions)
28 protocols using transwell boyden chamber
1
Transwell Assay for Cell Invasion and Migration
2
Cell Invasion and Migration Assay
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We performed cells invasion and migration tests with Boyden Transwell chambers (8-mm pore size, BD Biosciences) as illustrated before [10 (link)].
3
Cell Invasion and Migration Assay
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Cell invasion and migration assays were conducted with Boyden Transwell chambers (BD Biosciences) as previously described.13
4
Transwell Invasion Assay for Cell Migration
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Boyden Transwell chambers (BD Biosciences, 353097) coated with Matrigel (BD Biosciences, 356234) were used following the instructions of the manufacturer. Briefly, 2 × 105 cells were seeded into the upper chambers with Matrigel coated and cultured with serum-free medium. The lower chamber of the transwell was filled with 10% foetal bovine serum culture medium as a chemo-attractant. After 18–24 h incubation, cells that successfully invaded were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and counted in 5 random visual fields using a light microscope.
5
Tumor Cell Invasion Assay Using Boyden Chamber
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Cell invasion assay were performed using Boyden transwell chamber (8-μm pore size, Becton Dickinson). Matrigel (Becton Dickinson) diluted with serum-free DMEM was placed in the upper chamber of filter membrane, and incubated at 37 °C for 4 h. A total of 2 × 105 tumor cells were seeded with serum-free DMEM in the upper chamber. Conditioned medium (300 μl) obtained from bone-marrow derived macrophages were placed in the lower chamber. After transwell chambers with cells were maintained at 37 °C for 24 h, cells on the upper membrane surface were completely removed by wiping with cotton swab and the migrant cells on the lower membrane surface were fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet (Sigma-Aldrich). The membranes were examined microscopically and stained migrant cells were counted in at least 5 randomly selected fields.
6
Macrophage-Stimulated Tumor Cell Migration
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Cellular migration assay was performed using Boyden transwell chamber (8-μm pore size, Becton Dickinson, Bedford, MA). Tumor cells (2 × 105 cells/100 μl) were placed in the filter membrane. Conditioned medium (300 μl) obtained from primary mouse macrophages were placed in the lower chamber. After transwell chambers with cells were maintained at 37 °C for 24 h, cells on the upper membrane surface were completely removed by wiping with cotton swab and the migrant cells on the lower membrane surface were fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet (Sigma-Aldrich). The membranes were examined microscopically and stained migrant cells were counted in at least 5 randomly selected fields.
7
Invasion and Migration Assay of MDA-MB-231 Cells
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MDA-MB-231 cells (5×104) were seeded in triplicate onto a 24-well plate with Transwell Boyden chambers (BD Biosciences) coated with Matrigel (37°C for 30 min) or without Matrigel for the invasion and migration assay, respectively. The upper chamber contained serum-free DMEM, and the lower chamber contained DMEM supplemented with 10% FBS as a chemoattractant. Cells were incubated for 36 h at 37°C, fixed with 4% paraformaldehyde at room temperature for 15 min and then stained with 0.5% crystal violet for 5 min at room temperature. Stained cells were visualized in five randomly selected fields using a light IX71 inverted research microscope (magnification, ×200; Olympus Corporation).
8
Chemokine-Dependent Migration of LPLs
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The chemokine-dependent migration of LPLs was measured using an in vitro 2-chamber migration assay with Transwell Boyden chambers (BD Biosciences, USA) followed by flow cytometry analysis [27] (link). The chemotaxis assays were performed in pre-warmed migration buffer (RPMI 1640 containing 1% FCS). Primary LPLs were seeded at a density of 1×105 per well into the upper chamber. A total of 600 µl of migration buffer alone or supplemented with 500 ng/ml recombinant mouse CCL25 protein (Reprokine Research Immunity, USA) was added to the lower chamber. The chambers of cells were incubated for 6 hours at 37°C in an incubator with 5% CO2. The cells in the lower chambers were centrifuged, fixed in 500 µl Fixation Buffer (eBioscience, USA) and counted using a FACS Calibur (BD Biosciences, USA). The results were expressed as a chemotactic index (CI), which is the ratio between the number of migrating cells in the sample and in the medium control.
9
In vitro cell invasion assay
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In vitro cell invasion was evaluated as previously described [10 (link)] using transwell Boyden chambers (8 µm pore size; BD Biosciences, San Jose, CA, USA) coated with basement membrane Matrigel. Two independent assays were performed in triplicates for each condition. Invading cells were counted onto eight fields randomly selected on each membrane.
10
Transwell Invasion and Migration Assay
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Transwell Boyden chambers (BD Biosciences, San Jose, CA, USA) with 8 μm pore size polycarbonate filters were used for in vitro cell migration assays, and chambers coated with Matrigel (BD Biosciences) were used to evaluate the invasive potential of THP-1 and HL-60 cells in vitro. Briefly, ES cells were seeded at a density of 1×105 per well into the upper chamber, and 500 μl complete medium was loaded into the lower chamber. Chambers were then incubated for 24 h. At the time of harvesting, cells remaining inside the upper chambers were removed, while migrated and invaded cells on the lower surface of the membrane were fixed in 1 % paraformaldehyde and stained with crystal violet (Sigma-Aldrich), followed by visualization and counting under an inverted microscope. Cells were imaged using a low power (100×) magnification, and five visual fields per well were randomly selected for cell counting.
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