Migration and invasion assays were run in triplicate porous (8-μm pore size) Transwell migration chambers (BD Biosciences, Bedford, MA, USA) as described previously (Chia et al., 2007 (link); Kusuma et al., 2012 (link); Sloan et al., 2006 (link)). Transwells were coated with ECM proteins overnight at 4°C (Chia et al., 2007 (link)). Recombinant human laminin-511 (alpha5beta1gamma1) was isolated as previously described (Doi et al., 2002 (link)), and vitronectin was obtained from Sigma. For migration assays, cells (2×105/200 μl) in serum-free medium (SFM) were seeded into the top chamber of the Transwell. For invasion assays, a cell suspension of 1×105 cells in 50 μl of SFM was mixed with 50 μl Matrigel (BD Biosciences). 80 μl of the mixture was placed in the Transwell and allowed to set for 30 minutes, followed by addition of 100 μl of SFM. Cells were allowed to migrate for 4–5 hours or invade for 18 hours. The data represent the mean number of migrated or invaded cells ± s.d. of a representative experiment (n=3).
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