Recombinant human robo 2 fc chimera

Rat embryos were obtained through a cesarean incision from anesthetized Sprague Dawley pregnant dams as described previously for mouse embryos (De Carlos et al., 1996 (link)) and incubated for 24 h at 37°C with an in-shop built rotator device. Three to five embryos were incubated in 5 ml of freshly extracted rat serum or in goat serum (69023, Sigma) containing 1% penicillin-streptomycin mix (15070-063, Gibco) perfused constantly with a gas mix of 95% oxygen/5% carbon dioxide. In control cultures we confirmed that growth in rat or goat serum was comparable. The incubation medium was changed once after 12 h of culture. Embryos were then fixed in 4% PFA in PBS followed by whole brain immunostaining. The following recombinant proteins (all from R&D Systems, Minneapolis, MN), were added to the culture medium as indicated in the results section to a final concentration of 40 ng/ml each: Recombinant rat ROBO I/Fc Chimera (1749-RB-050), Recombinant human ROBO 2/Fc Chimera (3147-RB-050), NCAM-C56 Recombinant human NCAM-I/CD56 (2408-NC-050) or Ig-Fc Recombinant human IgG1 Fc, Human IgG1 (110-Hg-1-100).

Ex vivo cultures of normal lung explants were complemented daily with distinct doses of recombinant rat ROBO1 Fc Chimera (0.04, 0.4, 4 and 40 ng/mL, R&D system; Cat No. 1749-RB-050), recombinant human ROBO2 Fc Chimera (0.04, 0.4, 4 and 40 ng/mL, R&D system; Cat No. 3147-RB-050), or recombinant human IgG1 Fc (used as control at higher dose, R&D; Cat No. 110-HG-100). These doses were selected according to the literature [41 (link), 42 (link)]. Lung explants were obtained in three independent experiments (n ≥ 9 for each dose tested). After 4 days in culture, control, ROBO1 and ROBO2 inhibited lung explants were analyzed for branching morphogenesis in terms of area, external and internal epithelial perimeter, and the number of peripheral airway buds. In addition, SOX2, SOX9, BMP4, total and non-phospho (active) β-Catenin were quantified by Western blot at day 4 in all experimental groups.