Modified boyden chamber

Migration was assayed using a modified Boyden chamber (Corning Costar, Fisher Scientific, Pittsburgh, PA) containing a polycarbonate membrane filter (6.5 mm diameter, 8 μm pore size). The upper chamber contained cells in RPMI without FBS, and the lower chamber contained RPMI with 10% FBS (chemoattractant) or without FBS (control). Cells were incubated for 16 hours at 37°C in a 5% CO₂ incubator. Non-migrated cells were scraped off from the upper surface of the membrane with a cotton swab. Migrated cells remaining on the bottom surface were counted after staining with Coomassie Blue.

Cell movement was measured in culture inserts that had microporous membranes (8.0 µm, Corning, NY, USA). A total of 2 × 10⁴ cells/well suspended in culture media were plated in the top chamber of a modified Boyden chamber (Corning Costar, Corning, NY, USA), and PNS was added to the bottom chamber to encourage HUVEC migration. After 48 hours at 37°C under 5% CO₂, the microporous membranes were washed with PBS, fixed for 30 minutes in 4% paraformaldehyde, and stained with 0.5% crystal violet solution. Nonmigrating cells were washed using cotton-tipped swabs on the top side, and the number of migrated cells in each insert was manually counted in five random microscopic fields (100×) [37 (link)].

Cell migration was measured in culture inserts containing microporous membranes (8.0 μm, Corning, NY, USA). Briefly, 5× 10³ cells/well suspended in EBM were seeded in the upper chamber of a modified Boyden chamber (Corning Costar, Corning, NY, USA), and PNS was added to the lower chamber to induce EPC migration. After 72 h incubation at 37 °C and 5% CO₂, the microporous membranes were washed with PBS, fixed in 4% paraformaldehyde for 15 min, and stained with 0.5% crystal violet solution. Non-migrating cells on the top side were removed with cotton-tipped swabs and the number of migrated cells was counted manually in five random microscopic fields (100 ×) in each insert.

Cell migration was assessed in a modified Boyden chamber (Costar), in which two chambers were separated by a polycarbonate membrane (pore diameter, 8.0 μm). MG-63 cells were grown to subconfluence in tissue culture plates and then detached; thereafter, they were centrifuged and rendered into single cell suspensions in serum-free culture medium supplemented with 5 μg/mL BSA. The suspensions containing 5 × 10⁴ cells were added to wells with a membrane placed in the bottom. Medium containing indicated Wnt5a was added to the upper and lower compartment of the Boyden chamber. The cells were allowed to migrate for the indicated periods of time at 37°C in this assay. Thereafter, the medium was discarded, stationary cells were removed with a cotton-tipped applicator and the membranes were cut out of the chamber and stained with 0.5% crystal violet. The response was evaluated in a light microscope by counting the number of cells that had migrated into the membrane.

Assays were performed using a modified Boyden chamber (Corning Costar, Rochester, NY, USA) containing a gelatin-coated polycarbonate membrane filter (8-μm pore size). The upper surface of the filter was coated with 20 μL Matrigel (0.3 mg/ml; BD Biosciences, Bedford, MA, USA). Transfected cells were harvested with a cell dissociation solution (Invitrogen, Carlsbad, CA, USA) and suspended in medium with 1% bovine serum albumin.
Cells (1 × 10⁴) were added to the upper compartment and allowed to migrate for 6 h at 37°C. After 6 h, cells on the upper side of the membrane were removed with a cotton swab. Cells on the bottom surface of the membrane were fixed in 3.7% paraformaldehyde, stained with hematoxylin for quantification. Data were expressed as the average number of cells per insert.