Transwell migration chamber

To assay the migration ability of ccRCC cells, a cell suspension containing 1 × 10⁵ cells in serum-free medium was added to the upper inserts of the Transwell migration chambers (Becton Dickinson, USA). For TGF-β1 stimulation, TGF-β1 was added to the medium of the upper inserts to make a final concentration of 10 ng/mL. The lower chambers were filled with 500 μL of culture medium supplemented with 10% FBS. Cell mobility was detected after the indicated incubation period, and cells were fixed and stained with 0.1% crystal violet. The cells that penetrated the membrane were counted in five random fields.

Transwell migration chambers (8 µm pore size; BD Biosciences, USA) were purchased for observing the chemotactic motility of cells. Firstly, each top chamber was filled with 200 μL serum-free medium containing cells, while the bottom chamber was filled with 600 μL RMPI-1640 medium containing 10% FBS. The indicated compounds were then added to both chambers at the same concentration. After a 24 h incubation in a humidified atmosphere, nonmigrated cells were erased by cotton swabs. The migrated cells were fixed with 4% paraformaldehyde and stained with crystal violet solution. Images were photographed using an inverted microscope (Nikon; Japan) and the cells in at least 3 random microscopic fields were counted.

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).

Transwell Insert Assays using Transwell migration chambers (BD Biosciences) with 6.5-mm-diameter polycarbonate filters ( 8 μm pore size) were utilized to measure migration as previously described [45 (link)]. In brief, cells were pretreated with CUR for 1–3 hours as indicated. Thereafter, the bottom chambers were filled with 600 μL of DMEM media containing all supplements. Cell lines (3 × 10⁴ per well) were seeded in top chambers in 100 μL DMEM media without serum. HUVECs were seeded into six-well plates to reach a confluence, which was wounded with a yellow pipette tip. The plates were incubated as above and photographed at 24 h. The migrated cells were quantified by manual counting and five randomly chosen fields were analyzed for each well.

Migration assays were performed using trans-well migration chambers (BD Biosciences). MDA-MB-231 cells were seeded into upper chambers at a density of 0.5 × 10⁵ cells in serum-free medium. Medium containing 5% fetal bovine serum was aliquoted to lower wells and cells were incubated for 22 hours. When measuring migration in the presence of Silvestrol, compound was included in the medium in both upper and lower chambers at the concentrations indicated. To assess cell migration after 22 hours, upper chambers were transferred to wells containing 1× PBS +8 μM Calcenein AM (Life Technologies) and incubated for 40 minutes. Fluorescence was evaluated by excitation at 485 nM wavelength followed by measuring emission at 520 nM.