Figure 1.
Schema of the experiment procedure and the Hippocampal primary cultures on glass, 15∶1 PDMS, and 35∶1 PDMS coated with poly-l-lysine, fibronectin or laminin over time followed staining with antibody or analyzing with western blot (A).
Hippocampal neurons had short neurites 1 to 3 days after seeding on poly- l-lysine-coated glass coverslips (B, C). Hippocampal neurons had longer neurites 5 and 10 days after being seeded on poly- l-lysine-coated glass coverslips (D, E). The neurites were shorter 1 and 3 days after seeding on 15∶1 PDMS (F, G). Hippocampal neurons had longer neurites 5 and 10 days after seeding on 15∶1 PDMS (H, I). Hippocampal neurites at day 1 and day 3 after seeding on 35∶1 PDMS (J, K). Hippocampal neurons had longer neurites at 5 and 10 days after seeding on 35∶1 PDMS (L, M). Tubulin (green) was used to determine neurite lengths (scale bar = 50 µm, n = 3).
Figure 2.
Hippocampal primary cultures on glass, 15∶1 PDMS, and 35∶1 PDMS coated with fibronectin over time.
Hippocampal neurons had short neurites 1 and 3 days after seeding on fibronectin-coated glass (A, B). Hippocampal neurites were longer 5 and 10 days after seeding on glass coverslips coated with fibronectin (C, D). The neurites were shorter 1 and 3 days after seeding on 15∶1 PDMS (E, F). Hippocampal neurons had longer neurites at 5 and 10 days after seeding on 15∶1 PDMS (G, H). Hippocampal neurites at 1 and 3 days after seeding on 35∶1 PDMS (I, J). Hippocampal neurons had longer neurites at 5 and 10 days after seeding on 35∶1 PDMS (K, L). Tubulin (green) was used to determine neurite lengths (scale bar = 50 µm, n = 3).
Figure 3.
Hippocampal primary cultures on glass, 15∶1 PDMS, and 35∶1 PDMS coated with laminin over time.
Hippocampal neurons had short neurites 1 and 3 days after seeding on laminin-coated glass (A, B). Hippocampal neurites were longer 5 and 10 days after seeding on glass coverslips coated with laminin (C, D). The neurites were shorter 1 and 3 days after seeding on 15∶1 PDMS (E, F). Hippocampal neurons had longer neurites at 5 and 10 days after seeding on 15∶1 PDMS (G, H). Hippocampal neurites at 1 and 3 days after seeding on 35∶1 PDMS (I, J). Hippocampal neurons had longer neurites at 5 and 10 days after seeding on 35∶1 PDMS (K, L). Tubulin (green) was used to determine neurite lengths (scale bar = 50 µm, n = 3).
Figure 4.
Quantification of the percentage of neurite outgrowth and neurite length on substrates with differing elasticity over time.
(A) Hippocampal neurons with neurite were determined for cultures on glass substrates, 15∶1 PDMS and 35∶1 PDMS substrates coated with poly- l-lysine. (B) Percentage of neurite outgrowth over time on substrates with varying elasticity, coated with fibronectin. (C) Percentage of neurite outgrowth over time on substrates with varying elasticity, coated with laminin. (D) Hippocampal neurons appear to have relatively short neurites when grown on 15∶1 PDMS substrates coated with poly- l-lysine. (E) The neurite lengths of hippocampal neurons were determined while culturing hippocampal neurons on glass substrates, 15∶1 and 35∶1 PDMS coated with fibronectin. Hippocampal neurite length increased when seeding on glass substrate, 15∶1and 35∶1 PDMS coated with fibronectin. (F) Hippocampal neurons appear to have relatively short neurites when grown on 15∶1 PDMS substrates coated with laminin. (*p<0.05 compared with glass substrate, **p<0.01 compared with a glass substrate, #p<0.05 compared with 35∶1 PDMS, ##p<0.01 compared with 35∶1 PDMS, n = 50 per groups). The error bars represent s.e.m.
Figure 5.
pPKCα, pFAKy397, pFAKy925, and pERK1/2 were quantified 5 days after plating on substrates coated with poly-l-lysine.
After culturing for 5 days, hippocampal neuron lysates were reacted with (A) pPKCα, (B) pFAKy397, (C) pFAKy925, (D) pERK1/2 antibodies, and (E) β-actin. Three groups shown: glass, 15∶1 PDMS, and 35∶1 PDMS. (n = 3)
Figure 6.
pPKCα, pFAKy397, pFAKy925, and pERK1/2 were quantified 5 days after plating on substrates coated with fibronectin.
After culturing for 5 days, hippocampal neurons lysates were reacted with (A) pPKCα, (B) pFAKy397, (C) pFAKy925, (D) pERK1/2 antibodies, and (E) β-actin. Three groups shown: glass, 15∶1 PDMS, and 35∶1 PDMS. (n = 3)
Figure 7.
Quantification of Western blot results (A) pPKCα, (B) pFAKy397, (C) pFAKy925, (D) pERK1/2 antibodies, Six groups shown: glass, 15∶1 PDMS, and 35∶1 coated with poly-L-lysine or fibronectin.
(*p<.05 compared to poly-g groups, # p< .05 compared to FN-g groups, @ p<.05 compared to FN-15 groups)