Boyden chamber polycarbonate membranes

Conditioned media from PCs pretreated with ALK5 siRNA or scrambled siRNA was collected at 72 hours post-transfection and applied to human brain microvascular ECs for 48 hours. ECs were trypsinized and immediately added to the top of Boyden chamber polycarbonate membranes (Corning Costar, 5 μm pores) precoated with 0.1% gelatin. The lower compartment of the Boyden chamber contained serum-free M199 with VEGF-A (100 ng/ml). ECs were allowed to migrate for 10 hours from the upper chamber towards the VEGF-A gradient (lower compartment). The membrane was immersed in 4% paraformaldehyde for 20 minutes, and then its upper surface was scraped with a cotton swab to remove non-migrated cells. Cells on the bottom surface (i.e., migrated cells) were imaged and counted after staining with 0.1% Crystal Violet. For PC migration, following treatment with ALK5 siRNA or scrambled siRNA, human brain PCs were trypsinized, resuspended in LG DMEM with TGFβ1 (5 ng/ml) and immediately added to the top of Boyden chamber polycarbonate membranes (Corning Costar, 8 μm pores) precoated with 0.1% gelatin. The lower compartment of the Boyden chamber contained serum-free LG DMEM with TGFβ1 (5 ng/ml) and recombinant PDGF-BB (R&D, 10 ng/ml). PCs were allowed to migrate for 6 hours. Membranes with migrated PCs were fixed, stained, imaged and quantified as described above.