Analysis of the effect of poly-L-lysine coating on contact angles.

<p>(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). *, <i>p<</i>0.001 <i>vs.</i> plastic culture dish.</p

PC12 cell adhesion to an Si₃N₄ surface.

<p>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).</p

Growth of extensions in PC12 cells after NGF stimulation.

<p>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. (*, <i>p<</i>0.001 <i>vs.</i> plastic culture dish).</p

Non-NGF stimulated PC12 cell proliferation on different substrates.

<p>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). *, <i>p<</i>0.001 <i>vs.</i> plastic culture dish.</p

Surface structure analysis of an Si₃N₄ surface under different coating conditions.

<p>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.</p

PC12 cell differentiation on a poly-L-lysine coated Si₃N₄ surface after NGF stimulation.

<p>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.</p

Effect of FBS and NGF on PC12 cell proliferation on a poly-L-lysine coated Si₃N₄ surface.

<p>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 (*, <i>p<</i>0.001 <i>vs.</i> FBS (−) NGF (+)); NGF significantly decreased the cell proliferation rate (*, <i>p<</i>0.001 <i>vs.</i> FBS (+) NGF (+)).</p

Two-Dimensional Microchemical Observation of Mast Cell Biogenic Amine Release as Monitored by a 128 × 128 Array-Type Charge-Coupled Device Ion Image Sensor

Available array-type, chemical-sensing image sensors generally only provide on/off responses to the sensed chemical and produce qualitative information. Therefore, there is a need for an array sensor design that can detect chemical concentration changes to produce quantitative, event-sensitive information. In this study, a 128 × 128 array-type image sensor was modified and applied to imaging of biogenic amines released from stimulated rat mast cells, providing recordable responses of the time course of their release and diffusion. The imaging tool was manufactured by an integrated circuit process, including complementary metal oxide semiconductor and charge-coupled device technology. It was fitted with an amine-sensitive membrane prepared from plasticized poly­(vinyl chloride) including a hydrophobic anion, which allowed the sensor to detect amines, such as histamine and serotonin, in Tyrode’s solution. As mast cells were larger in diameter than the pixel hollows, some pixels monitored amines released from single cells. The image from the array responses yielded sequential snapshots at a practical frame speed that followed amine concentration changes over time, after mast cell amine release was synchronized by chemical stimulation. This sensor was shown to be sensitive to amine release at very low stimulus concentrations and was able to detect localized spots of high amine release. The entire time course of the amine release was recorded, including maximum concentration at 4–6 s and signal disappearance at 30 s after stimulation. With further development, this sensor will increase opportunities to study a variety of biological systems, including neuronal chemical processes