8 μm pore transwells

About harvested cells (1 × 10⁵) in 100 μl of serum-free DMEM were added into the upper compartment of 8-μm-pore Transwells (Costar, Corning, Cambridge, MA, USA) and 200 μl of 10% FBS in free medium (Gibco, Invitrogen, Carlsbad, CA, USA) was placed in the lower compartment. The cells were allowed to migrate for 24 h of incubation at 37 °C. For quantification, the cells in the lower compartment were stained with haematoxylin and counted in five randomly selected visual fields (x200 magnification) under a microscope. The experiment was repeated 3 times.

About 1 × 10⁵ cells mixed in 200 μl serum-free media were placed in the upper compartment of 8-μm-pore transwells (Costar, Corning, Cambridge, MA, USA) and 400 μl of 10% FBS in free medium (Gibco, Invitrogen, Carlsbad, CA, USA) was added to the lower compartment. And we have checked the migration every 12 h. The cells were allowed to migrate within 72 h. For quantification, the cells in the lower compartment were stained with crystal violet and counted in five randomly chosen fields (× 200) under a light microscope. The experiment was conducted with three replicates.

The cells were first transfected with lentiviral GFP and then selected by FACS to obtain a high yield of GFP⁺ cells. A total of 5 × 10⁴ GFP⁺ DFSCs and Wnt5a⁺/GFP⁺ DFSCs in 100 μl DMEM were loaded into 8 μm pore Transwells (Corning, Corning, NY, USA) in 24-well plates as per our prior methods [15 (link)]. Following 12-hour incubation, migrated cells were trypsinized and counted, as per our prior methods [11 (link)].

Microglia (10⁵ cells per well) were seeded onto 8-μm-pore Transwells (Corning, Inc., Lowell, MA, USA), and the Transwells were placed on a chamber containing 100 μM ADP. Cells were allowed to migrate for 4–12 h. Non-migrated cells were removed from the top surface of Transwells with a cotton swab. Cells that had migrated to the bottom surface of the Transwells were fixed with 4% paraformaldehyde for 20 min and visualized by staining with 1% Crystal violet (Sigma). The number of migrated cells in nine randomly chosen fields was counted. The FAK inhibitors, FAK 14 and PF573228 (Santa Cruz), were treated for 30 min before adding cells on top surface.

Motility assays were performed in 8 μm-pore transwells (Corning Inc. Life Science, Lowell, MA). Cells were cultured to 80% confluence and then starved for 24 h in DMEM containing 1% FBS. 5x10⁴ cells (or HGF siRNA transfected cells) were plated in 200μl of DMEM plus 1% FBS in the upper wells. Bottom wells contained 500μl of DMEM plus 1% FBS with or without HGF (100 ng/mL) (R&D, Minneapolis, MN). In some cases, both upper and bottom wells received 50 μg/mL HS20 or human IgG (Sigma St. Louis, MO). Cells were incubated at 37°C in humidified 5% CO2 for 16 h. Cells on the transwells were rinsed in PBS, fixed with 1% glutaraldehyde (Sigma, St. Louis, MO) in PBS at room temperature for 15 min, and stained with 0.1% crystal violet (Sigma St. Louis, MO) in water for 30 min. After de-staining in water, non-migrating cells on top of the filter were removed with a cotton swab. Each well was examined and photographed with an AMG EVOS XL microscope. Images were acquired through a phase contrast microscope at 10x magnification. Migrating cells on the bottom of the filter were solubilized in 500 μl of 0.2% Triton X-100 (Invitrogen, Camarillo, CA) at 4°C overnight. The absorbance was measured at 590 nm.

Invasion assay was performed with 5 × 10⁴ PCa cells on 8-μm-pore Transwells (Corning) coated with 50 μg/cm² of reconstituted Matrigel for 16 h, as described in [9 (link)]. Chemotaxis was evaluated by counting the cells migrated to the lower surface of the filters (six randomly chosen fields).