Trans-well assay was used to detect cell migration and invasion, as per the prescribed protocol [22 (link)].
For migration analysis, U2OS cells transfected with ZFAS1 siRNA or control siRNA were seeded in a 24-well transwell unit (5 × 104 cells/well) with 8-μm polycarbonate nucleopore filters (Corning Costar, Corning, NY, USA). The upper compartment contained a serum-free medium, whereas the lower compartment contained a medium containing 10% FBS. Cells were then incubated for 24 h in a humid incubator under 37°C and 5% CO2 conditions. Crystal violet staining was used to fix and count cells adhering to the lower surface of the filter.
For the invasion assay, the membrane of the trans-well unit was coated with 40 μL Matrigel (BD Biosciences, USA) and incubated at 37°C for 4 h to form a reconstructed basement membrane. The cells were examined using the same techniques as in the migration assay.
For migration analysis, U2OS cells transfected with ZFAS1 siRNA or control siRNA were seeded in a 24-well transwell unit (5 × 104 cells/well) with 8-μm polycarbonate nucleopore filters (Corning Costar, Corning, NY, USA). The upper compartment contained a serum-free medium, whereas the lower compartment contained a medium containing 10% FBS. Cells were then incubated for 24 h in a humid incubator under 37°C and 5% CO2 conditions. Crystal violet staining was used to fix and count cells adhering to the lower surface of the filter.
For the invasion assay, the membrane of the trans-well unit was coated with 40 μL Matrigel (BD Biosciences, USA) and incubated at 37°C for 4 h to form a reconstructed basement membrane. The cells were examined using the same techniques as in the migration assay.
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