The invasion assay was performed using 8 µm transwell chambers (Corning Life Science) placed in 24-well plates. Initially, the upper chamber was coated with a 1:20 dilution of Matrigel (BD Biosciences) in a volume of 100 µl per well. The plates were then incubated at 37 °C in a humidified incubator for 2-4 hours to allow for gel formation. Subsequently, a cell suspension containing 2-5×104 cells in serum-free medium was added to the upper chamber of a Transwell insert, while 750 μl of medium containing 10% FBS was added to the lower chamber. After incubating for 24 hours, the non-invaded cells in the upper chamber were removed by washing. The invaded cells that passed through the insert and adhered to the lower surface were fixed with methanol and stained with a 0.1% (w/v) solution of crystal violet. To ensure accuracy and consistency, three wells were used to measure the invaded cells of each group, and the experiment was conducted at least three times.
8 m transwell chamber
Manufactured by Corning
Sourced in United States
The 8 µm transwell chamber is a cell culture insert designed for use in transwell assays. It features a microporous membrane with 8 micron pore size, allowing for the study of cell migration, invasion, and co-culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (6 mentions)
Inverted microscope, by Olympus (1 mentions)
Extracellular matrix gel, by Corning (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Crystal violet, by Solarbio (1 mentions)
Inverted microscope, by Leica (1 mentions)
Cck 8 solution, by Dojindo Laboratories (1 mentions)
Matrigel, by Corning (1 mentions)
Matrigel coating, by BD (1 mentions)
Eclipse ti s inverted microscope, by Nikon (1 mentions)
Matrigel coated membrane, by BD (1 mentions)
18 protocols using 8 m transwell chamber
1
Transwell Invasion Assay Protocol
2
Transwell Invasion Assay Protocol
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Transwell assays were performed using 8-µm transwell chambers (Corning, Tewksbury, MA, USA) coated with Matrigel (BD Bioscience). The upper chamber with stromal cells (5 × 10 4 /well) and a lower chamber containing DMEM/F12 supplemented with 10% FBS were prepared. After 24 h, the cells were fixed and stained. Finally, invading cells were measured using an inverted microscope (Olympus Corporation, Tokyo, Japan). The relative cell invasion level was calculated as a percentage of untreated control cells.
3
Transwell Migration and Invasion Assay
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The transwell assay was performed using 8µm-transwell chambers (Corning Life Science). For the invasion assay, the filters were precoated with 50 µL matrigel at 1:4 dilution in serum-free RPMI-1640 to form a genuine reconstituted basement, then 500 µL RPMI-1640 medium containing 10% FBS was added to the lower chamber. For the migration and invasion assays, BGC823 and SNU216 were placed into the upper chamber at a density of 2×104 cells/200 µL and 5×104 cells/200 µL, respectively. After incubation for 24 hours, cells remaining on the upper membrane were removed with a cotton swab, while cells that had invaded through the membrane were fixed in methanol, stained with Wright–Giemsa. Five representative fields of each insert were randomly counted under a microscope (Olympus, Tokyo, Japan) and analyzed statistically.
4
Cell Migration and Invasion Assay
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For the migration assay, 4 × 10 4 Bel7402 cells in serum-free medium were seed in the upper compartment of an 8 µm transwell chamber (Corning, NY, USA). For the invasion assay, the upper chambers were previously coated with 50 µg extracellular matrix gel (Corning, NY, USA). After incubation for 24-48 hours, the migrated or invaded cells on the lower membrane were stained with 4% paraformaldehyde, then stained with 0.1% crystal violet. The upper cells were moved gently by the soft medical cotton ball. Each group random took 10 pictures under a microscope with 400× magni cation (Olympus, Japan). Cell numbers per eld were calculated by ImageJ.
5
Migratory Effect of SDF-1 and EX-4 on PDLSCs
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The migratory effect of SDF‐1 and EX‐4 on PDLSCs was evaluated by an 8 µm transwell chamber (Corning). PDLSCs were seeded onto the upper chamber at a density of 5 × 104 cells/well and cultured in maintenance media. The lower plates were supplemented in 500 µL maintenance media with SDF‐1, EX‐4, or SDF‐1+EX‐4. 500 µL maintenance media served as a negative control (NC) and 500 µL basic media served as a positive control (PC). After 20 hours, cells that had migrated through the membrane were fixed with 4% paraformaldehyde (Sigma Aldrich) and stained with 0.1% crystal violet (Solarbio). Then cells were observed under a microscope and six randomly selected high‐power microscopic fields (×200) per filter were counted. Experiments were performed in triplicate (N = 3).
6
Transwell Invasion Assay for Evaluating Cancer Cell Metastasis
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For Transwell analysis, an 8-µm Transwell chamber (Corning, Inc.) was coated with 250 mg/ml Matrigel™ (BD Biosciences) at room temperature for 24 h (18 (link)–20 (link)), then placed into a 24-well plate. The upper chambers were seeded with control or transfected MCF-7 cells suspended in 200 µl serum-free DMEM at a density of 1×105 following Anlotinib treatment. RPMI-1640 medium containing 10% FBS was added to the lower chamber for 24 h of incubation at 37°C. Finally, the invading cells into the bottom of chamber were fixed with 4% methanol at 37°C for 10 min, then stained with 0.1% crystal violet solution at 37°C for 15 min. The number of invasive cells in five random fields were counted using a light microscope (magnification, ×100).
7
Transwell Invasion Assay Protocol
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Cells were suspended in serum-free DMEM medium. 100 µl of the cell suspension was seeded on the upper part of a 24-well culture plate with an 8 µm transwell chamber (Corning, USA) for 48 h, and 500 µl of DMEM medium supplemented with 10% FBS was added to the lower chamber. The chambers uniformly covered with Matrigel (BD Biosciences) are used to invasion assay. The transwell chambers were fixed in 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 30 min at room temperature. Then, cells above the chambers were wiped off by a cotton swab. And stained cells were washed with PBS and observed with a microscope. The capacities of cell migration and invasion were assessed with average stained cells in 5 areas.
8
Transwell Migration Assay Protocol
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Transwell migration assays were performed as previously described (Yang et al., 2020 (link)) with 4 × 104 cells seeded per 8-µm Transwell chamber (Corning, Corning, NY, USA).
9
Analyzing Cell Migration and Invasion
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The ability of cell migration was analyzed by a wound scratch test. According to the experimental requirements, cells were seeded into 6-well plates, after the cells grew to complete fusion, the layer of cells was scratched to form wounds using a sterile 20 µl pipette tip and cultured for 48 hours. The image of the scratched area was captured under the phase contrast microscope and the cell healing area was analyzed by ImageJ software (NIH, USA). Transwell invasion assay was used to evaluate cell invasion. 8 µm transwell chamber (Corning, cat:354578) was applied to this experiment. Apply 5 µl Matrige (Becton Dickinson, Franklin Lakes, NJ, USA) to the chamber and adjust the cell concentration, then continuing to culture normally for 4 hours, the excess cells in the inner side of the chamber were washed with PBS twice, xed with polyformaldehyde. Finally, the images were observed by the inverted microscope (Leica).
10
Transwell Invasion Assay for Cell Migration
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Transfected cells were seeded into the upper part of the 8-µm Transwell chamber (Corning, NY, USA) coated with Matrigel. The medium (500 µL) containing 15% FBS was added to the lower part. Through incubation under the conventional conditions, the non-invasive cells in the upper part were removed while the invasive cells in the lower part were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet solution, followed by photography under an optical microscope [31 (link)].
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