BCPAP (7.5 × 105), SW1736 (1 × 106), Cal62 (1 × 106), and C643 cells (1 × 106) were starved in RPMI with 0.1% FBS and treated with DMSO, dasatinib, PF-562,271, the combination, or the neutralizing antibody anti-IL1β, at the indicated concentrations. For conditioned media experiments, cell media was changed to 1% FBS. After 24 hours, cells were harvested and seeded in the upper chambers of Matrigel-coated transwells (24-well, 8 μM pore size; BD Biosciences, San Jose, CA) in 0.1% FBS RPMI with the appropriate drug/combination. RPMI with 10% FBS and the appropriate drug/combination was added to the lower chamber. For conditioned media experiments, conditioned media with or without drug, was added to either the upper or lower chamber, with 1% FBS with or without drug in the opposite chamber, as indicated. For MMP inhibitor treatments, SB-3CT (6 μM) was added to the upper and lower chamber. Cells were allowed to invade for 24 hours and assessed as previously described (36 ). Migration assays were performed similarly, except cells were plated in uncoated transwells (24-well, 8.0 μm pore size; Falcon) and allowed to migrate towards 10% FBS.
Uncoated transwells
Manufactured by BD
Sourced in United States
Uncoated transwells are a type of laboratory equipment used in cell culture experiments. They consist of a porous membrane that separates two compartments, allowing for the study of cell migration, permeability, and other biological processes across a defined barrier.
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Lab products found in correlation
2 protocols using uncoated transwells
1
Invasion and Migration Assays for Thyroid Cancer
2
Cancer Cell Migration and Invasion Assay
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Migration and invasion assays to measure the mobility of cancer cells were performed using Transwell chambers as previously described [11 (link)]. Cells were cultured 24 h after transfection, and then starved for 12 h in serum‐free medium. The cells were digested and adjusted to 1.25 × 105 cells·mL−1 (migration assay) or 2.5 × 105 cells·mL−1 (invasion assay), and 400 μL of the cell suspension was added to the upper chamber. Uncoated Transwells (353097, FALCON, Los Angeles, CA, USA) were used for the migration assay, and Matrigel‐coated Transwells (356234, CORNING, Corning, NY, USA) were used for the invasion assay. Afterwards, 500 μL of 10% serum medium was added to the lower chamber. Cells were incubated for 24–36 h (migration assay) or 36–48 h (invasion assay). At the end of the culture, non‐migrated cells in the upper chamber were carefully removed with a cotton swab. The migrated cells that passed through the membrane and adhered to the lower surface of the membrane were fixed with 4% paraformaldehyde for 15 min and then stained with crystal violet for 5 min. The number of migrated or invaded cells was counted in 10 random fields photographed using a 200× microscope (IX73, OLYMPUS, Tokyo, Japan).
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