The cell migration transwell assay was performed as described by the manufacturer’s protocol (Corning, NY, USA). SKOV3 cells were seeded onto the Corning Biocoat Matrigel Chambers (Corning #354480) at a density of 25,000 cells/0.5 mL in serum-free RPMI medium, with bottom wells containing RPMI medium with 10% FBS. Cells in the top chambers were incubated with recombinant human IFNγ (0 and 50 ng/mL; R&D Systems, 285-IF-100) in the presence of IL-8 neutralizing monoclonal antibody (2 μg/mL; R&D, MAB208) or control IgG (2 μg/mL; Santa Cruz Biotechnology; sc-2025) for 24 h. After incubation, non-migrating cells were scrubbed from the upper surface of the top chambers using cotton swabs. Migrating cells on the bottom membranes were fixed with 100% methanol, stained with crystal violet, washed, and air dried. Migrating cells were counted in five randomly selected fields under a phase-contrast microscope at 10X magnification, and quantified using ImageJ software as described [37 (link)].
Biocoat matrigel chambers
Manufactured by Corning
BioCoat Matrigel chambers are a cell culture product designed for the in vitro study of cell invasion and migration. The chambers consist of a porous membrane coated with a basement membrane extract, providing a barrier that cells must cross to move from one side of the chamber to the other. This setup allows for the quantitative assessment of the invasive and migratory behavior of various cell types.
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4 protocols using biocoat matrigel chambers
1
Cell Migration Assay with IFNγ and IL-8
2
Quantifying Cell Invasiveness via Matrigel Assay
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Invasiveness was determined using 24-well plate BioCoat Matrigel chambers (Corning). Cells were seeded in the inserts with 1–10% FBS chemotactic gradient. The plates were then incubated for 24/48/72 hours at 37°C and the matrigel layer together with the non-invasive cells was removed with a cotton swab. The cells that have migrated through the Matrigel and porous membrane of the insert were fixed in 4% formaldehyde for 10 minutes before being stained in 0.5% crystal violet. The cells were then visualised at ×40 magnification. Three random fields were counted for each test sample.
3
Wound Healing and Invasion Assay
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Wound healing assay to evaluate cell migration ability was performed as previously described [51 (link)]. Briefly, cells transfected with empty vector or CHD5 construct were allowed to growth until confluent (> 95%). Cell scratches were then created in A498 and HH244 cell lines using 200 μl sterile tips and washed twice with culturing medium. After indicated time points of incubation, cells were imaged under a phase-contrast microscope. The experiments were performed in triplicate. In-vitro invasion assays were carried out in Corning BioCoat Matrigel chambers (Corning, NY) as described previously [51 (link)].
4
Migration and Invasion Assay
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Migration and invasion was evaluated using 6.5 mm 24 well-transwell plates containing 8.0 μm pore polycarbonate membrane inserts and BioCoat Matrigel chambers, respectively (Corning Life Sciences, Corning, NY). Following 24 h of starvation, 2.5 × 104 cells were added to upper chambers with appropriate agent(s), and 700 µL of RPMI 1640 media containing 10% FBS was added to the lower chambers. After 16 h at 37 °C, a cotton-tipped swab was used to remove cells from the top of the membrane and migrating cells attached to the bottom of the membrane were fixed using methanol, stained using 0.2% crystal violet solution, allowed to air dry and imaged and counted using an Echo Revolve microscope.
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