For transwell migration assays or invasion assays, transfected SW1116 or SW1463 cells with serum-free medium (2×105 cells/100 μl) were seeded on uncoated or Matrigel coated upper chambers (Costar, Boston, MA, USA) respectively, while total of 500 μl medium containing 20% FBS was added in the bottom chambers. After incubation for 24 h, the migrated or invaded cells were fixed by 4% paraformaldehyde for 30 min and then stained for 30 min using 0.1% crystal violet. Then, the migrated or invaded cells were visualized and counted in four random fields via the microscope.
Matrigel coated upper chamber
Manufactured by Corning
Sourced in United States
Matrigel-coated upper chamber is a laboratory equipment component designed for cell culture applications. It provides a coated surface for cell attachment and growth within an experimental setup.
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8 protocols using matrigel coated upper chamber
1
Transwell Migration and Invasion Assay
2
Transwell Assay for Investigating GC Cell Invasion and Migration
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Transwell assays were used to detect whether ELMO3 knockdown affects GC cell invasion and migration and were performed as described previously, with slight modifications.
For migration assays, GC cell suspensions (0.4 ml, 1 × 105) diluted in serum-free culture medium were seeded in the upper chambers (Corning Costar, Acton, MA, USA) without a coating of Matrigel. For the invasion assays, GC cells (0.4 ml, 1 × 105) diluted in serum-free culture medium, were seeded on Matrigel-coated upper chambers (Corning Costar, Acton, MA, USA). In both assays, 0.6 ml RPMI Medium 1640 containing 10% FBS was added to the lower chamber [30 (link)]. After incubation for 12 h, 24 h, and 48 h, the Matrigel and the non-migrating cells in the upper chambers were carefully removed or cleaned. Cells moving to the bottom chamber were fixed with 4% paraformaldehyde for 20 min and then stained with 0.1% crystal violet solution at room temperature for 20–30 min. The cells were subsequently washed twice with PBS, and the stained cells were observed under a microscope. Three fields on each membrane were randomly selected, and the average number of penetrating cells was counted under a 100 × inverted microscope.
For migration assays, GC cell suspensions (0.4 ml, 1 × 105) diluted in serum-free culture medium were seeded in the upper chambers (Corning Costar, Acton, MA, USA) without a coating of Matrigel. For the invasion assays, GC cells (0.4 ml, 1 × 105) diluted in serum-free culture medium, were seeded on Matrigel-coated upper chambers (Corning Costar, Acton, MA, USA). In both assays, 0.6 ml RPMI Medium 1640 containing 10% FBS was added to the lower chamber [30 (link)]. After incubation for 12 h, 24 h, and 48 h, the Matrigel and the non-migrating cells in the upper chambers were carefully removed or cleaned. Cells moving to the bottom chamber were fixed with 4% paraformaldehyde for 20 min and then stained with 0.1% crystal violet solution at room temperature for 20–30 min. The cells were subsequently washed twice with PBS, and the stained cells were observed under a microscope. Three fields on each membrane were randomly selected, and the average number of penetrating cells was counted under a 100 × inverted microscope.
3
Matrigel-based Cell Invasion Assay
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The treated HCT‐116 and Caco‐2 cells were resuspended in serum‐free medium and seeded into the Matrigel‐coated upper chamber (Corning, USA) at a final concentration of 1 × 105 per well. The complete medium containing 10% foetal bovine serum was added to the lower chamber. After 24 h, the unpassed cells were wiped off with a cotton swab, and the passed ones were fixed with 4% paraformaldehyde for 30 min. After staining with 0.1% crystal violet for 10 min and washing with running water for 2 min, the invaded cells were photographed under a microscope (Nikon, Japan).
4
Transwell Invasion Assay Protocol
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The transfected cells (1 × 104) were resuspended in 200 μL serum-free medium and plated in the Matrigel-coated upper chamber (Corning, USA). Meanwhile, 600 μL medium with 10% FBS was added to the lower chamber. After incubation for 48 h, the non-invaded cells were removed with cotton swab, and the invasive cells were fixed with 4% polymethanol and then stained with 0.1% crystal violet for 20 min. Finally, the cells that passed through the Matrigel were photographed and calculated in 5 randomly selected fields.
5
Transwell Invasion Assay Protocol
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The cells were collected by trypsinization 48 h after transfection and the cell density was adjusted to 5×105/ml using serum-free medium. A total of 60 µl Matrigel (diluted 7 times with serum-free medium) was added into the upper chambers and incubated at 37°C in 5% CO2 atmosphere for 1 h. Then, 200 µl of cell suspension (5×105 cells/ml) was inoculated into the Matrigel-coated upper chamber (8 µm pore size; Costar; Corning, Inc.). Subsequently, 600 µl culture medium containing 20% FBS was added to the lower chamber. After incubation at 37°C in 5% CO2 atmosphere for 24 h, cells were fixed and stained with 0.2% crystal violet for 1 h at room temperature. The excess stain was then washed away slowly with water and the images were obtained using a light microscope at ×40 and ×100 magnification. The cell number was counted, and the result was compared among all groups.
6
Evaluating Cell Migration and Invasion
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Cell migration and invasion were measured with the wound healing assay and Transwell chamber assay, respectively. For the wound healing assay, BEAS-2B cells transfected with LV-FAM13A, shFAM13A, or corresponding negative control were seeded into a 6-well plate at a density of 1 × 105 cells per well and cultured overnight. A 200 µL micropipette tip was used to generate a 2 mm-wide scratch line in the cell monolayer. Cells were allowed to migrate for 24 h. Images were captured under an inverted light microscope (Olympus, Japan).
For the invasion assay, BEAS-2B cells transfected with LV-FAM13A, shFAM13A, or corresponding negative control were loaded into a Matrigel-coated upper chamber (Corning, Corning, NY, USA) filled with serum-free DMEM. The lower chamber was filled with 500 μL DMEM containing 20% FBS. After incubation at 37 °C for 48 h, nonmigrating or non-invading cells remaining in the upper chamber were removed with a cotton swab. The migrating or invading cells adhering to the lower surface were fixed and stained with crystal violet (0.1%). The stained cells were counted in 3–5 randomly selected fields under an inverted light microscope (Olympus) at × 200 magnification. Images were acquired using an Olympus camera (Olympus, Japan).
For the invasion assay, BEAS-2B cells transfected with LV-FAM13A, shFAM13A, or corresponding negative control were loaded into a Matrigel-coated upper chamber (Corning, Corning, NY, USA) filled with serum-free DMEM. The lower chamber was filled with 500 μL DMEM containing 20% FBS. After incubation at 37 °C for 48 h, nonmigrating or non-invading cells remaining in the upper chamber were removed with a cotton swab. The migrating or invading cells adhering to the lower surface were fixed and stained with crystal violet (0.1%). The stained cells were counted in 3–5 randomly selected fields under an inverted light microscope (Olympus) at × 200 magnification. Images were acquired using an Olympus camera (Olympus, Japan).
7
Quantifying Cell Invasion Dynamics
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Twenty-four hours post-transfection, the cells were reseeded into a Matrigel-coated upper chamber (8 μm pore size) of a Tran-swell assay system (Corning, NY, USA). After the cells were cultured for 24 h, we used a cotton swab to scrape off the noninvading cells on the upper surface of the membrane and stained the invading cells with 0.1% crystal violet. Twenty minutes after staining, cells were counted and imaged with a microscope.
8
Thyroid Cancer Cell Invasion Assay
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1 × 104 thyroid cancer cells with indicated treatment were suspended in 100 μL serum‐free medium and added to each matrigel‐coated upper chamber (Corning, USA), while the lower chambers were filled with 500 μL medium containing 20% foetal bovine serum. After incubation at 37°C for 24 hours, the cells that did not invade through the pores were removed with a cotton swab. The chambers were washed, fixed, stained with 0.1% crystal violet and counted in three random view fields.
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