Figure 1.
PC12 cell adhesion to an Si3N4 surface.
PC12 cells were seeded at the same concentration on Si3N4 surfaces coated with different cell adhesion molecules and images of representative areas were captured using an inverted microscope 24 hours later. Non-coated Si3N4 surfaces were used as a control. (A) Non-coated Si3N4 surface, (B) concanavalin A and (C) 0.01% poly-L-lysine coated Si3N4 surface. (Scale bar: 50 µm).
Figure 2.
Surface structure analysis of an Si3N4 surface under different coating conditions.
Three-dimensional atomic force microscope height images. (A) 0.01% poly-L-lysine coated plastic dish, (B) non-coated Si3N4 surface, (C) 0.01% poly-L-lysine coated Si3N4 surface at 1 day after coating and (D) at 5 days after coating, (E) 0.05% poly-L-lysine coated Si3N4 surface at 1 day after coating and (F) at 5 days after coating.
Figure 3.
Analysis of the effect of poly-L-lysine coating on contact angles.
(A) Substrates were coated with poly-L-lysine solution for 2 hours and then rinsed with distilled water. After overnight drying (∼16 hours) at room temperature, images were captured using a contact angle analyzer. The angle was calculated using the instrument software and the values shown represent the averages of the angles from both sides of the droplet. Non-coated surfaces were also tested for comparative purposes. Each picture is representative for each sample group. The light square observed in the center of the droplet of the pictures taken to the plastic dish samples corresponds to the area where light passes perpendicularly through the water so that refraction does not happen, a similar effect than the one produced by a spherical lens when illuminated. It is important to mention that the light source was located at the opposite side of the water droplet in relation to the camera lens. (B) Plastic culture dishes and Si3N4 surfaces were left untreated or coated with 0.01% or 0.05% poly-L-lysine. A contact angle analyzer was used to measure sessile drop contact angles. Values are the mean ± S.E. for twenty samples (Two trials of ten samples each). *, p<0.001 vs. plastic culture dish.
Figure 4.
Non-NGF stimulated PC12 cell proliferation on different substrates.
PC12 cells were seeded on 35 mm2 poly-L-lysine coated Si3N4 surfaces (○) or plastic culture dishes (▵) and counted every 2 days. The cells were cultured in DMEM +10% FBS +10% horse serum. The medium was replaced every 3 days. Values are the mean ± S.E. for twenty samples (Two trials of ten samples each). *, p<0.001 vs. plastic culture dish.
Figure 5.
PC12 cell proliferation on a poly-L-lysine coated Si3N4 surface.
PC12 cells expressing DsRed2 protein were seeded at a concentration of 7×104 cells/ml on poly-L-lysine coated Si3N4 surfaces and images were captured every 2 days using a fluorescence microscope. (A) 3 days, (B) 5 days, (C) 7 days and (D) 9 days after cell seeding. Scale bar: 100 µm.
Figure 6.
Effect of FBS and NGF on PC12 cell proliferation on a poly-L-lysine coated Si3N4 surface.
PC12 cells were seeded onto poly-L-lysine coated Si3N4 surfaces in serum-supplemented growth medium and after 24 hours, cells were stimulated with NGF (50 ng/ml) in the presence/absence of FBS+horse serum. Cells were counted every 2 days. Three treatment groups were established: FBS (+) NGF (−) (○); FBS (+) NGF (+) (□); and FBS (−) NGF (+) (⋄). The values shown are the mean ± S.E. for twenty samples (Two trials of ten samples each). The presence of FBS significantly increased the cell proliferation rate (*, p<0.001 vs. FBS (−) NGF (+)); NGF significantly decreased the cell proliferation rate (*, p<0.001 vs. FBS (+) NGF (+)).
Figure 7.
PC12 cell differentiation on a poly-L-lysine coated Si3N4 surface after NGF stimulation.
PC12 cells expressing DsRed2 protein were seeded onto poly-L-lysine coated Si3N4 surfaces and 24 hours later (considered time for cell attachment), NGF (50 ng/ml) was added to the medium in the absence of FBS and horse serum. Images were captured using a fluorescence microscope at (A) 1 day, (B) 3 days, (C) 5 days and (D) 7 days after NGF stimulation. Scale bar: 100 µm.
Figure 8.
Growth of extensions in PC12 cells after NGF stimulation.
PC12 cells seeded on different substrates were stimulated with NGF (50 ng/ml) in the absence of FBS and horse serum and images were captured every 2 days beginning at 3 days after NGF stimulation. Using AquaCosmos analysis software, the areas of differentiated cell bodies were compared to the areas of the outgrowth extensions. Representative data are shown in the graph. Cells cultured on an Si3N4 surface (○ ) or on a plastic culture dish (▵). The values shown are the mean ± S.E. of 30 samples. (*, p<0.001 vs. plastic culture dish).
Figure 9.
Assessment of PC12 cell proliferation using a BrdU assay.
(A) Cells were seeded on different surfaces and 24 hours after seeding, they were stimulated with NGF (50 ng/ml). After 2 hours incubation in growth medium containing BrdU solution, cells were fixed, permeabilized and stained using an Alexa Fluor 488 conjugated anti-BrdU antibody. Finally, BrdU positive cells were counted in 20 samples and the proportion (%) of labeled cells per counted field was calculated. Standard error is shown using error bars. The following media were tested: FBS (+) NGF (−) (○); FBS (+) NGF (+) (□); and FBS (−) NGF (+) (⋄). (*, p<0.05; * *, p<0.01; * * *, p<0.001 vs. plastic culture dish). (B) Cells were seeded on different surfaces and cultured in the absence of serum and with NGF treatment. They were fixed using a paraformaldehyde-based solution and immunocytochemical detection of the BrdU antigen was performed to identify proliferative (S phase) cells. Images were captured at different time points. Representative images of each condition are shown. Scale bar: 50 µm.