Cell migration was performed using 24-well Transwell chambers with 8-μm-pore-size polycarbonate membranes (Corning Inc., Corning, NY, USA). After cells grew till the logarithmic phase of growth, they were properly cultured with serum-free DMEM (high glucose) during the 24-h starvation period at 37 °C in a 5% CO2 atmosphere. Then, the starving cells were harvested, resuspended at a concentration of 3 × 105 cells in 100 μL with serum-free DMEM (high glucose), and seeded into the upper chambers of a Transwell plate. Chitinase (0 μg, 2 μg, or 4 μg, Catalog Number C6242, Sigma–Aldrich) was added to the upper chambers of the Transwell plate. The lower chambers were filled with 600 μL of DMEM supplemented with 10% FBS. After incubating the cells for 24 h at 37 °C in a 5% CO2 atmosphere, the noninvasive cells on the top side of the membrane were removed thoroughly but gently using a cotton swab. The invaded cells on the lower membrane surface were fixed in 20% methanol for 15 min and then stained with 0.1% crystal violet for 30 min. The cells were photographed with four randomly visual fields in each well under a light microscope at 100× magnification (Olympus, Tokyo, Japan) and counted using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
Transwell chambers with 8 μm pore size polycarbonate membrane
Manufactured by Corning
Sourced in United States
Transwell chambers with 8-μm pore size polycarbonate membrane are a laboratory tool used for cell culture studies. The chambers consist of a permeable membrane insert that fits into a culture well, allowing for the separation of cell populations or the study of cell migration and invasion.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (9 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Diff quik, by Sysmex (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Matrigel solution, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
13 protocols using transwell chambers with 8 μm pore size polycarbonate membrane
1
Transwell Assay for Cell Migration
2
Cell Migration and Invasion Assay
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Cell migration and invasion assays were performed using 24-well Transwell chambers with 8-μm pore size polycarbonate membranes (Corning, Corning, NY, USA). Cells were plated on the top side of the membrane pre-coated or not with Matrigel (BD, Franklin Lakes, NJ, USA) for invasion or migration assays, respectively. After incubation for 36∼48 hr, cells inside the upper chamber were removed with cottons swabs, whereas those on the lower membrane surface were fixed by methanol and stained with 0.5% crystal violet solution. Five randomly selected fields were analyzed for each well.
3
Transwell Invasion and Migration Assay
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The 24-well Transwell chambers with 8-μm pore size polycarbonate membranes (Corning, USA) were used to test invasion and migration of HTR8/SVneo cells. The 200 ul cell suspension without FBS was added in the upper side of the membrane coated with or without Matrigel (BD, USA), and 600 μl culture medium RPMI 1640 containing 10% FBS was added in the lower chamber and placed in the cell culture incubator. After 24 h (for migration) and 48 h (for invasion), the upper chambers were removed with cottons swabs. The lower chambers were fixed by methanol for 30 min and stained with 0.1% crystal violet solution for 20 min. After the chambers were washed with PBS 3 times, the numbers of cells that have penetrated the filter membrane were observed under optical microscope.
4
Transwell Invasion Assay for Breast Cancer Cells
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Transwell chambers with 8 μm pore size polycarbonate membranes (Corning Incorporated, Corning NY) were coated with diluted Matrigel (BD Biosciences, Sparks, MD) and used for the invasion assay. A total of 5 × 104 BCa cells were collected in serum-free media and placed in the upper chambers. Seven-hundred-fifty microliters of media containing 10% FBS were added to the lower chambers. After 24 h of incubation at 37 °C in a 5% (v/v) CO2 atmosphere, cells that invaded through the transwell chambers were permeabilized with methanol and stained with 0.1% crystal violet. The invading cells were counted from six random microscopic fields. Quantitative results indicate the means ± SD of triplicates.
5
Transwell Assay for Cell Migration and Invasion
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Cell migration and invasion assays were performed using 24-well Transwell chambers with 8-μm pore size polycarbonate membranes (Corning, Corning, NY, USA). Cells were plated on the top side of the membrane pre-coated or not with Matrigel (BD, Franklin Lakes, NJ, USA) for invasion or migration assays, respectively. After incubation for ∼36–48 h, cells inside the upper chamber were removed with cottons swabs, whereas those on the lower membrane surface were fixed by methanol and stained with 0.5% crystal violet solution. Five randomly selected fields were analyzed for each well.
6
Cell Migration and Invasion Assay
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Cell migration and invasion assays were carried out using 24-well Transwell chambers with 8-μm pore size polycarbonate membrane (Corning, NY, USA). A total of 6 × 104 or 8 × 104 cells in 200 μl serum-free medium were seeded in the upper chambers coated without or with Matrigel (BD Biosciences, NY, USA) for migration or invasion assays, respectively, after which 600 μl medium with 10% fetal bovine serum (FBS) (20% FBS for invasion assays) was added to the lower chambers. The cells on the upper surfaces of the membranes were removed 36–48 h later. Cells on the bottom surfaces of the membranes were fixed, stained with 0.1% crystal violet, and counted in five fields using a Zeiss microscope (Melville, NY, USA).
7
Transwell Migration and Invasion Assay
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Transwell chambers with 8-μm pore-size polycarbonate membrane (Corning Life Sciences, Tewksbury, MA, USA) were used. For migration assay, FTC-133 cells (0.8 × 104 cells/well) or 8505C cells (1.5 × 104 cells/well) resuspended in serum-free medium with different concentrations of GB1107 or TD139 were seeded onto the upper chamber, while the complete medium containing 10% fetal bovine serum as the chemoattractant was added to the lower chamber [13 (link)]. After 24 h, the migrated cells on the lower surface of the porous membrane were fixed and stained with Diff-Quik (Sysmex, Kobe, Japan). For invasion assay, FTC-133 cells (1 × 104 cells/well), 8505C cells (1 × 104 cells/well), or Nthy cells (2 × 104 cells/well) were allowed to invade through BioCoat cell culture inserts precoated with Matrigel. The invaded cells were fixed and stained after 24 h. Migrated or invaded cells were photographed under the microscope, and five random fields were counted for each assay.
8
Transwell Assay for Cell Migration and Invasion
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The abilities of cell migration and invasion were determined using 24-well Transwell chambers with 8-μm pore size polycarbonate membrane (Corning, Corning, NY, USA). For the cell migration assay, the harvested cells were resuspended in 200 μl of serum-free medium and seeded on the upper culture chamber. Six hundred microliters of complete medium was added into the lower compartment. For invasion assay, the upper chambers were precoated with Matrigel solution (BD, Franklin Lakes, NJ, USA) before the invasion started. After incubation at 37°C for 48 h, the migrated and invaded cells in the lower chamber were fixed with 100% methanol and stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 20 min. Nontraversed cells were removed by cotton swabs from the upper surface of the filter. Five random fields were counted in each well using an inverted microscope (Olympus, Tokyo, Japan).
9
Invasion and Migration Assay of Glioma Cells
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After glioma cell lines were harvested, the migration and invasion abilities were tested by the 24-well Transwell chambers with 8-μm pore size polycarbonate membrane (Corning, Corning, NY, USA) according to the manufacturer’s instructions. Cells were resuspended in serum-free medium into the upper Transwell chamber (preapplied with 50 μl of Matrigel) with 1 × 105 cells/well, and 600 μl of medium containing 20% FBS (Gibco) was added into the lower chamber.
Cells were incubated at 37°C for 4 h before the assay was started. After 96 h, cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min. The upper surface of the membrane was wiped with a cotton swab and washed by PBS. Five areas of the membrane were randomly selected, photographed, and counted under an inverted microscope.
Cells were incubated at 37°C for 4 h before the assay was started. After 96 h, cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min. The upper surface of the membrane was wiped with a cotton swab and washed by PBS. Five areas of the membrane were randomly selected, photographed, and counted under an inverted microscope.
10
Transwell Assay for Cell Migration and Invasion
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For migration and invasion assays, 24-well Transwell chambers with 8-μm-pore size polycarbonate membrane was used (Corning Incorporated, Corning, NY, USA). For invasion assays, 1 × 105 cells in serum-free RPMI-1640 medium were seeded on the top side of the membrane pre-coated with Matrigel (BD, Franklin Lakes, NJ, USA; without Matrigel for migration assays). RPMI-1640 containing with 10% fetal bovine serum was added to the lower chamber. After incubation for 24 h, cells inside the upper chamber were removed with cottons swabs, while cells on the lower membrane surface were fixed and then stained with 0.5% Crystal violet solution. Five fields were counted randomly in each well.
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