Pore size polycarbonate membrane

For the migration assay, Renca cells were plated in six-well plates at a density of 3 × 10⁵ and incubated with medium alone or serial dilutions of lycorine hydrochloride (0, 1, 2.5, 5, and 10 μM) for 24 h. Renca cells were resuspended in serum-free medium and seeded into trans-well chambers with 8-μm diameter pore size polycarbonate membranes (Corning, NY, USA). Medium containing 30% fetal bovine serum was used in the lower chamber as an attractant. For the invasion assay, trans-well chambers were coated with Corning Matrigel Matrix (Corning, NY, USA). After 12 h, cells on the upper chamber surface were removed using cotton swabs. The migrated or invaded cells in the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The total numbers of cells were captured and analyzed from 10 different fields with the Olympus IX2 microscope.

To detect the chemotactic ability of Ang II-treated Hif1a^fl/fl or Hif1a^ΔSMC VSMCs on macrophages, chemotaxis assay was performed in 24-well Boyden chambers with 5 µm pore size polycarbonate membranes (Corning, NY, USA). The supernatants from 1 μM Ang II-treated Hif1a^fl/fl or Hif1a^ΔSMC VSMCs were added into the bottom chamber as chemoattractant, and 1 × 10⁵ bone marrow-derived macrophages were seeded onto the upper chamber. For the CCL7-neutralizing experiment, 8 μg/mL normal goat IgG control or CCL7-neutralizing antibody was added into the bottom chamber together with the VSMC supernatants. For all chemotaxis assays, after incubation for 6 h at 37 °C, the residual cells on the upper side of the membrane were removed by a cotton swab. The cells that migrated to the lower side of the membrane were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet solution for 5 min, and the cell numbers were counted. Each well represented an independent experiment but not experimental replicate, because the VSMCs from each mouse were cultured separately but not pooled.

For the migration assay, cells were plated in six-well plates at a density of 3 × 10⁵ and incubated in DMEM-0% FBS (serum-free medium) in the presence of BsAb for 8 h and JNJ for 2 h, then were treated with HGF (100 ng/ml). Cells were resuspended in serum-free medium and seeded into transwell chambers with 8-μm diameter pore size polycarbonate membranes (Corning, NY, U.S.A.). Medium containing 30% FBS was used in the lower chamber as an attractant. After 12 h, cells on the upper chamber surface were removed using cotton swabs. The migrated or invaded cells in the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% Crystal Violet. The total numbers of cells were captured and analyzed from ten different fields with the Olympus IX2 microscope.