HT29-MTX-E12 (E12) cells were maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) with 10% (v/v) fetal bovine serum (FBS; HyClone), 1% (v/v) nonessential amino acids, 1% (v/v) L-glutamine, and 1% penicillin and streptomycin (100 IU/mL) at 37 °C in 5% CO2. To study the uptake of NPs quantitatively, E12 cells were seeded into 24-well plates (50,000 cells/well) and cultured in growth medium at 37 °C in a 5% CO2 incubator until they adhered to the plates. To establish cell monolayers for qualitative studies, the E12 cells were seeded at a density of 10×104 cells/mL on a polycarbonate membrane (pore size: 0.4 μm; diameter: 6.5 mm; cell growth area: 0.33 cm2) in Costar Transwell 24-well plates (Corning Costar Corp.). Transepithelial electrical resistance (TEER) was monitored using an electrical resistance meter (Millicell ERS-2, Millipore) to ensure the integrity of the cell monolayers.
Costar transwell 24 well plate
Manufactured by Corning
Sourced in United States
The Costar Transwell 24-well plates are a laboratory equipment product designed for cell culture applications. The plates consist of a 24-well format with a porous membrane insert that separates the upper and lower chambers, allowing for the study of cell migration, permeability, and other cellular processes. The product provides a standardized platform for these types of experiments.
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Lab products found in correlation
Matrigel, by BD (6 mentions)
Human methylcellulose base medium, by R&D Systems (4 mentions)
Image pro plus 6, by Nikon (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Millicell ers 2, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Matrigel matrix, by BD (1 mentions)
Dm4000b microscope, by Leica (1 mentions)
Methanol, by Merck Group (1 mentions)
Optical microscope, by Nikon (1 mentions)
Trypsin, by Solarbio (1 mentions)
Facscalibur, by BD (1 mentions)
Sdf 1b, by Thermo Fisher Scientific (1 mentions)
Sphingosine 1 phosphate (s1p), by Cayman Chemical (1 mentions)
20 protocols using costar transwell 24 well plate
1
Quantitative Nanoparticle Uptake in HT29-MTX-E12 Cells
2
Chemotaxis Assay for CCR4+ Cells
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The chemotaxis assays were performed in Costar Transwell 24-well plates (Costar Corning), each well of which has a lower chamber and an upper chamber separated by a polycarbonate membrane with 5 microM pores. The CCR4-positive cell suspension (100ul, 2×106 cells/ml) was added to the upper chamber of both treatment and control groups, 600ul of chemokine CCL17 (R&D systems) was added to the lower chamber of the treatment group, and an equal volume of the RPMI-1640 medium (Gibco) was added to the lower chamber of the control group. Chemotaxis was performed for 2 h, and the number of cells was counted in the lower wells after staining with CFSE.
3
Transwell Assay for Cell Migration and Invasion
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Transfected cells were harvested, centrifuged, and then resuspended in DMEM without HI-FBS. In total, 200 µL of a cell suspension containing 105 cells were added into an upper compartment of Corning Costar Transwell 24-well plates (Corning Incorporated, Corning, NY, USA), while the lower compartments were covered with 600 µL of DMEM that was supplemented with 10% of HI-FBS. Following incubation for 24 hrs, nonmigratory cells were carefully removed with a cotton swab, whereas the migratory cells were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet (Sigma-Aldrich). The capacity for migration was assessed by counting the migratory cells in five randomly selected visual fields per plate in images captured by means of an Olympus light microscope (Olympus IX83; Olympus Corporation, Tokyo, Japan). The experimental procedures of the Transwell invasion assay were similar to those of the migration assay, except that the plates were precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA).
4
Cell Migration Assay Using Matrigel-Coated Transwell
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Corning costar transwell 24-well plates (8 μm pores; Corning, NY, USA) were coated with matrigel (Biolead, Beijing, China) and incubated at 37 °C for 30 min until the matrigel solidified. Pre-warmed medium with 10% serum was added to the lower chambers. Then cell suspension (2 × 104 cells/well) was seeded to upper chamber in a 1640 medium. After incubation 48 h, the cells on the bottom surface of the membrane were fixed with cold methanol for 15 min and stained with crystal violet for 10 min.
5
Transwell Invasion Assay for Metastatic Cells
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Corning Costar Transwell 24-well plates (8-µm pores; Corning Incorporated, Corning, NY, USA) coated with BD Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA) were maintained for 1 h at 37°C, followed by the addition of 1×105 transfected cells suspended in 200 µl medium with 1% serum into the top of each well insert. Normal growth medium was added to the bottom wells. The cells were allowed to migrate for 24 h at 37°C. The migrated cells were fixed with 10% methanol for 15 min. The invading cells on the lower surface of the membrane were stained with 0.5% crystal violet for 5 min at room temperature. The stained cells were counted under a microscope (Nikon Corporation). To minimize the bias, ≤5 randomly selected fields at ×100 magnification were counted, and the average number was calculated.
6
Transwell Migration and Invasion Assay
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Transwell migration assay was conducted on Costar Transwell 24-well plates (Corning Incorporated, Corning, NY, USA) to assess cell migration. In brief, 6×104 virus-transduced cells were seeded into the upper chamber with 0.2 mL serum-free medium. Cultured medium supplemented with 10% FBS were added to the lower chamber. After 24 hours at 37°C with 5% CO2, the cells remaining on the upper surface were completely removed. The cells that migrated through the membranes were fixed, stained with crystal violet, and counted under a microscope.
A Transwell invasion assay was carried out in the same manner, except that the membranes were pre-coated with Matrigel (1 µg/µL; BD Biosciences, San Jose, CA, USA).
A Transwell invasion assay was carried out in the same manner, except that the membranes were pre-coated with Matrigel (1 µg/µL; BD Biosciences, San Jose, CA, USA).
7
Boyden Chamber Cell Invasion Assay
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Boyden chamber invasion assay (24-well plate format) was used to investigate the ability of cell invasion. Corning Costar Transwell 24-well plates (8-μm pores; Corning, USA) were coated with Matrigel (BD) and placed in a cell culture hood for 3 h at 37 °C. Then a total of 20,000 cells/well were seeded in the inserts and cultured in the medium without serum. Cultured medium with 20% serum was placed in the bottom wells. Cells were then allowed to invade for 48 h. Invaded cells were fixed with 1% formaldehyde solution for 15 min and stained with 0.1% of crystal violet for 15 min. Then, a cotton swab was used to erase the noninvasive cells and Matrigel on the top surface of the membrane. Invasive cells on the lower surface of the membrane were stained by crystal violet in purple and the stained cells were captured using a microscope (Nikon) and counted by software Image-Pro Plus 6.0.
8
Invasion Assay for Transfected Cells
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Before TU212 cells were seeded, Corning Costar Transwell 24-well plates (Corning, Corning, NY, U.S.A.) with Matrigel were placed in RPMI 1640 medium for 1 h at 37°C. A total of 2 × 104 transfected cells were seeded in the wells, and were cultured in 500 µl RPMI 1640 medium without FBS. 500 µl RPMI 1640 medium containing 10% FBS was placed in the bottom wells. After incubation for 48 h at 37°C, the cells that did not migration through the pores were carefully removed by cotton swabs. Then, the invasive cells were fixed with methanol (Sigma–Aldrich, MO, U.S.A.) and stained with 0.1% crystal violet. Images were captured and number of invasive cells was counted under a Leica DM4000B microscope.
9
Cell Migration Assay with Transfected Cells
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Following 12 h of transfection, SW480 and SW620 cells were treated with 0.25% trypsin (Beijing Solarbio Science & Technology Co., Ltd.) and suspended in serum-free RPMI-1640 medium at a density of 3×105 cells/ml. A total of 200 µl cell suspension was placed in the top chamber of a 2-chamber Costar Transwell 24-well plates (8-µm pores; Corning Inc.) and 600 µl RPMI-1640 medium containing 15% FBS was added in the lower chamber. The cells were cultured at 37°C for 48 h. The cells on the surface of the upper chamber were swabbed and those under the surface of the lower chamber were stained with crystal violet (0.1%) at room temperature for 20 min. Cell migration was evaluated by counting the cells that had migrated into the filters using an optical microscope (magnification, ×100; Nikon Corporation).
10
Boyden Chamber Cell Invasion Assay
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Cell invasion abilities were determined using Boyden chamber invasion assay (24-well plate format). In brief, the transfected cells (5 × 104 cells each well) were plated in upper well of Corning Costar Transwell 24-well plates (8-μm pores; Corning, U.S.A.) pre-coated with Matrigel (BD, Biosciences) and cultured in serum-free medium. DMEM with 20% FBS was placed in the bottom wells as a chemoattractant. After 48 h of incubation at 37°C, a cotton swab was used to remove the noninvasive cells remaining on upper well, while invaded cells were fixed and stained in 0.1% Crystal Violet for 5 min. The stained cells were captured using a light microscope (Nikon) and counted in five randomly selected fields by software Image Pro Plus 6.0.
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