Boyden chamber

The induced mobility or chemotaxis of neutrophils, macrophages and lymphocytes was evaluated according to the method previously described [36 (link),70 (link)]. Cell suspensions were deposited in the upper compartment of a Boyden chamber separated by a filter of polycarbonate (3 μm in diameter; MercK, Madrid, Spain). The number of cells (neutrophils and lymphocytes) that migrated toward the chemoattractant agent, formyl-Met-Leu-Phe (fMLP, 10⁻⁸ M, Sigma-Aldrich, Madrid, Spain), deposited in the lower compartment of the chamber, was counted in the lower face of the filter that separates the two compartments of the chamber. This number is expressed as Chemotaxis Index (C.I.).

Cells (1 × 10⁵), suspended in DMEM containing 0.1% BSA were added to the top of the Boyden chamber (EMD Millipore, Inc., USA) at 37°C for 2h. The lower chamber contained 10% serum-supplemented medium. After incubation for 24 h at 37°C, a Transwell chamber (EMD Millipore, Inc., USA) was used to determine cell migration ability. A total of 1 × 10⁵ siRNA-p62 transfected A549 cells were suspended in DMEM containing 10% FBS at a density of 5000 cells/well. Cells were subsequently placed onto the top of each chamber. Medium containing 10% FBS was added to the bottom of the chamber. The cells were incubated for 24 h, and then cells on the upside of the membrane were wiped off to remove the non-migrated cells. Cells that had migrated to the underside of the membrane were stained with crystal violet at room temperature for 20 min and visualized under Nikon Eclipse TE2000-U microscope. A total of 4 random fields (magnification, ×100) were scanned and analyzed using the aforementioned ImageJ software.

The migration assay was carried out by using the Boyden chamber (Chemicon, Rosemont, IL) as we previously reported with slight modification [44 (link)]. In brief, ECs (2 × 10⁴ cells) were seeded into the upper compartment of the Boyden chamber. After 24 hrs, the ECs which migrated across the membrane were counted under an inverted light microscope. Ten random microscopic fields were assessed in each well. The average number of cells per field was determined.
The tube formation assay was conducted by using in vitro angiogenesis assay kit (Chemicon). First, the ECMatrix solution were thawed and mixed with the ECMatrix diluent. Then, the ECMatrix mixture were placed in a 96-well tissue culture plate at 37°C for 1 hr to allow the matrix solution to solidify. ECs (1 × 10⁴ cells/well) were seeded onto the solidified matrix and incubated at regular cell culture conditions (5% CO2, 37°C). After 24 hr post-seeding, 2 μg/ml calcein (Fisher scientific) was directly added to the culture well and incubated for 20 mins prior to imaging under an inverted fluorescence microscope. Tubes were defined as a tube structure exhibiting a length 4 times of its width [45 (link)]. Five random microscopic fields were assessed in each well. The average number of tubes per field was determined.