Collagen from bovine skin

PC‐12 cells stably expressing the EKAR2G1 construct, described earlier in Ryu et al (2015), and PC‐12 Neuroscreen‐1 (NS‐1, gift from Tobias Meyer) were cultured using low‐glucose DMEM (Sigma) supplemented with 10% horse serum (HS; Sigma), 5% fetal bovine serum (FBS; Sigma), and 1% penicillin/streptomycin. Cells were cultured on plastic tissue culture dishes (TPP) coated with 50 μg/ml collagen from bovine skin (Sigma). Cells were passaged at 70% confluence by detaching cells using a cell scraper (Fisher).

Cells were seeded at a specific density in a 24 well plate. To form the collagen discs as described previously [23 (link)], 150,000 cells were mixed with a neutral solution consisting of collagen from bovine skin (Sigma-Aldrich, U.S.A), 10 x PBS and water. The cell-collagen solution was then loaded to a 24-well plate. Wells with no treatments and no cells were used as a control. The plates were incubated for approximately 2 h at 37°C to allow polymerisation. By using a fine pipette tip (1–10μl) and scoring around the edge carefully, the collagen discs were released from the sides of the wells. Cells were treated with rhSP-A or rhSP-D (10 μg/ml). Using a BioRad machine, images were captured post-treatment at zero min (at time of treatment), 3 and 24 h. The software ImageJ was used to measure the surface area changes.