The invasion and metastasis of cells were detected by Transwell assay. To detect cell migration, HCC cells in the exponential phase were inoculated in the upper chambers of Transwell plates (Chemicon, USA) at a density of 3 × 104 in 200 μl medium. Then 700 μl normal medium was added to the lower chambers and cultured for 24 h. To detect cell invasion, diluted Matrigel (BD, USA) was placed in the upper chambers of the Transwell plates and incubated at 37°C for 1 h. After hydration of the basement membrane with DMEM, the cells were evenly inoculated in the upper chambers of the Transwell plates at a density of 3 × 104 in 200 μl, and 700 μl normal medium was added to the lower chambers. After 24 h of routine culture, the cells in the lower chambers were fixed and stained with 0.5% methanol crystal purple solution for 30 min. After washing with PBS, the chamber was placed on the slide, and five visual fields were randomly selected under a microscope for imaging.
Transwell plate
Manufactured by BD
Sourced in United States
Transwell plates are a type of cell culture insert system used for in vitro studies. The plates consist of a permeable membrane support that separates the upper and lower compartments, allowing for the study of cell migration, invasion, and transport processes.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (43 mentions)
Inverted microscope, by Olympus (5 mentions)
Matrigel, by Corning (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Inverted light microscope, by Olympus (3 mentions)
Transwell plate, by Corning (3 mentions)
Rpmi 1640 medium, by BD (2 mentions)
Optical microscope, by Olympus (2 mentions)
Matrigel matrix, by BD (2 mentions)
Photomicroscope, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Dynabeads, by BioLegend (2 mentions)
Recombinant mouse il 2, by BioLegend (2 mentions)
Transwell assay, by BD (2 mentions)
Polycarbonate sterile chambers 8 m filters, by BD (2 mentions)
Matrigel, by Leica (1 mentions)
Matrigel, by Thermo Fisher Scientific (1 mentions)
Transwell chamber, by BD (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
119 protocols using transwell plate
1
Transwell Assay for Cell Migration and Invasion
2
Cell Migration Assay using Transwell
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Cells (5 × 104 cells/well) suspended in 200 μl of serum-free medium were added to the upper chambers of transwell plates (8 μm pore size; BD Biosciences, Franklin Lakes, NJ, USA) with or without Matrigel coating. 500 μl of medium containing 10% FBS was added to the lower chamber. After incubation for 48 h, the cells on the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed under a microscope.
3
Cell Migration and Invasion Assay
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Cells were seeded into Boyden chambers containing 24-well Transwell plates with a pore size of 8 μm (BD Bioscience). The upper chamber was either left uncoated for the migration assay or precoated with 50 μl 1:8 diluted Matrigel (BD Bioscience) for the invasion assay. U2OS (5×104), U2OS/MTX300 (5×104) and HOS (3×104) cells were seeded into the upper chamber. The upper chamber was filled with 200 μL serum-free specified medium, whereas the lower chamber was filled with complete medium containing 10% FBS. Following incubation for 12 h (U2OS, HOS) and 24 h (U2OS/MTX300), the cells that had invaded into the lower chamber were fixed with 4% paraformaldehyde and were stained with crystal violet for 1 h at room temperature. The cells in the upper chamber were removed by a cotton swab, and the remaining cells were counted in 5 randomly selected microscopic fields. All experiments were performed in triplicate.
4
Transwell Invasion and Migration Assay
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Briefly, cells were seeded in top chambers of the transwell plates (BD Biosciences, San Jose, CA, USA) in 5% serum media with membrane inserts coated either with or without Matrigel (8%) for invasion and migration tests, respectively. Bottom chambers were filled with DMEM medium with 10% FBS. After 12‐18 hours (for migration) or 36‐48 hours (for invasion) incubation, cells that migrated/invaded to the lower surface of the membrane were fixed and stained, and the cell numbers in six random fields were counted under the light microscope.
5
Matrigel Invasion Assay for Cell Migration
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Matrigel invasion assay was performed using Transwell plates (BD, Biosciences, CA, USA). Transwell membrane inserts were precoated with Matrigel (BD), and the analyzed cells were appropriately seeded onto the upper compartment of the chamber. The bottom chamber contained complete RPMI-1640 medium, and the upper chamber contained serum-free medium. After 24 h of incubation at 37°C, the cells on the Matrigel-coated upper surface were wiped off with a cotton swab, and the invaded cells throughout the filter were fixed and stained with Giemsa at 37°C for 3 h. The images of invaded cells were photographed with a microscope (Leica) at × 100 magnification. For each membrane isolated from triplicate chambers, the number of invaded cells was counted in three randomly selected fields. The results were presented as bar graphs.
6
Transwell-based GC Cell Invasion Assay
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Transwell plates (BD, Biosciences, United States) were used for GC cell invasion assay. The bottom chamber contained complete medium and the upper chamber has serum-free medium. Matrigel (BD) was added to the RPMI 1640 medium for detecting invading cells. After transfection with NC, miR-NC, or miR-596 mimics, GC cells were appropriately seeded into the cell culture. After incubation at 37 °C for 24 h, the invaded cells were fixed and stained using Giemsa. The images of cells were photographed with a microscope (Leica) at × 100 magnification and the cell number counted in three random fields of view. The results are presented as a column graph with statistics.
7
Transwell Assay for Cell Migration and Invasion
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Cells were seeded into Boyden chambers containing 24-well Transwell plates with a pore size of 8 µm (BD Bioscience). The upper chamber was either left uncoated for the migration assay or precoated with 50 µL of 1:8 diluted Matrigel (BD Bioscience) for the invasion assay. For the migration assay, the Hs578T (4×104) and MDA-MB231 (5×104) cells were seeded into the upper chamber; for the invasion assay, 8×104 Hs578T and 1×105 MDA-MB231 cells were seeded into the upper chamber. The upper chamber was filled with 200 µL serum-free specified medium whereas the lower chamber was filled with specified medium containing 10% FBS. Following incubation for 16 h or 24 h at 37 °C, cells that had invaded into the lower chamber were fixed with 4% paraformaldehyde and stained with crystal violet for 1 h at room temperature, and counted in five randomly-selected microscopic fields. All these experiments were performed in triplicate.
8
IL-6 Induced Cell Migration and Invasion Assay
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These assays were performed in 24-well Transwell plates (BD Biosciences, San Jose, CA, USA) with 8-μm pore inserts coated with or without Matrigel (Invitrogen). Cells (1 × 105) were seeded in a culture insert in serum-free media and treated with 20 ng/ml IL-6 into the upper chamber. Complete medium was applied to the lower chamber. Cells were allowed to migrate or invade the layer for 12 or 24 h, respectively. Migrating/invading cells were stained with 0.4% SRB and visualized under a microscope. The experiment was repeated three times.
9
Establishing Nasal Epithelial Cultures
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HNECs were grown until 80% confluent then harvested for seeding onto collagen coated 6.5 mm permeable Transwell plates (BD Biosciences, San Jose, California, USA) at a density of 5 × 104 cells per well. Cells were maintained with B-ALI™ growth medium for 2–3 days in a cell incubator at 37°C with 5% CO2. On day 3 after seeding, the apical media was removed and the basal media replaced with B-ALI™ differentiation media, exposing the apical cell surface to the atmosphere. Human nasal epithelial cultures at air liquid interface (HNEC-ALI) were maintained for a minimum of 21 days prior to experimentation for development of tight junctions (Ramezanpour et al., 2019b (link)).
10
Transwell Assay for Cell Invasion
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Transwell plates (BD Biosciences) were used for transwell experiments. According to the manufacturer's instructions, 5×104 cells and 1% fetal bovine serum were added to each upper compartment, and 20% fetal bovine serum was added to each lower compartment for transmembrane induction. Six hours later, TGF-β1 solution (5ng/ml) was added to the lower chambers. After 24 hours incubation at 37°C, taken out the upper compartment and the cells were fixed with methanol for 30 minutes, then the upper surface cells of the upper compartment were removed with cotton swabs. Cells invading to the lower side of the upper compartment were stained with 1% crystal violet. Cells in three different regions were photographed and counted under a microscope. All the experimental data were repeated three times.
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