Apoptosis or necrosis of neurons in the central nervous system (CNS)

Apoptosis or necrosis of neurons in the central nervous system (CNS) is the hallmark of many neurodegenerative diseases and Traumatic Mind Injury (TBI). for promoting neuronal differentiation and development in CNS with neurodegeneration. applications by culturing many human being and rodent neuronal cells lines including neuroblastoma (SK-N-MC, SY5Y), carcinoma (P-19), and Personal computer-12 (rat pheochromocytoma) cells We display the successful development and proliferation of the cell lines on photoresist produced carbon films which were not really functionalized with normally occurring proteins or synthetically customized biopolymers. Moreover, we proven that Personal computer12 cells, a model program for differentiation research [13], could be induced to differentiate on carbon surface area when put through differentiation-inducing element NGF. One benefit of carbon materials without functionalized with additional biomolecules can be that it could considerably decrease the unwanted immune response, if found in em /em vivo . Other benefits of these carbon products, produced from polymeric precursor, consist of superb biocompatibility, wide electrochemical balance, and availability in high purity, reproducibility, chemical substance inertness, great thermal conductivity, mechanised and dimensional permanence [11C12]. Therefore, our outcomes present evidence for even more substantiation. 2. Methods and Materials 2.1 Fabrication of Carbon substrate Carbon was produced from pyrolysis of photoresist covered on silicon wafer. A 2 size silicon wafer can be cut into several potato chips of approximate size 15.6mm. Silicon chips were washed with 70% ethanol and then air dried. A clean silicon chip was placed in the spin coater. A layer of positive photoresist SPR 220-7.0 was applied order Tedizolid to the silicon chip manually. The spin coater was run at 300 revolutions per minute (rpm) for 3 seconds to spread the photoresist around the wafer then run at 3000 rpm for 30 sec to fully situate the photoresist around the silicon chip. The photoresist coating process was repeated four times to ensure that the desired thickness range had been reached. Following the four coats, order Tedizolid photoresist coated silicon chip was placed on a warm plate set at 95 C for five minutes and was left to cool naturally to room temperature. The chip was placed in a Nitrogen gas atmosphere. The gas atmosphere must be completely free of oxygen. The sample was heated at a rate of 10 C per minute until the desired maximum temperature (700 C to 1100 C) had been reached. Once at the desired maximum temperature, the sample remained under heat at that temperature for one hour. The chip is usually allowed to cool in the nitrogen environment. Once at room temperature, the chip with a layer of carbon was ready to reenter an oxygenated atmosphere [11C12]. 2.2 Cell Culture Carbon chips were placed into wells of 6-well plate, washed with 70% v/v ethanol and sterilized under ultraviolet radiation for 45 minutes. The rat pheochromocytoma PC12 cells were cultured in DMEM high glucose medium supplemented with 5% of FBS and 10% of Horse serum. The cells were induced to differentiate in DMEM high glucose medium (Gibco-11495) with 100 ng/mL of Nerve Growth Factor (Gibco 13257-019). Human neuroblastoma cell lines (SK-N-MC, SY5Y), mouse teratocarcinoma cell line (P-19), were maintained in DMEM high glucose medium (Gibco 11495) supplemented with 10% fetal bovine order Tedizolid serum (Gibco 26140-079) and penicillin-streptomycin (Gibco 15140-122). The cells were incubated at 37 C with 5% CO2 in cell culture dishes. The cells were removed from the cell culture dish with 0.25% trypsin EDTA (Gibco-25200). Removed cells were centrifuged at 1000 rpm for 5 minutes. The pellet was suspended with fresh medium, and counted via a haemocytometer. Approximately 100,000 cells were transferred to the sterilized carbon chip placed in wells of 24-well cell culture plate. 2.3 Scanning Electron Microscopy Differentiated and undifferentiated cells on photoresist derived carbon were fixed by immersion in 2.5% (v/v) glutaraldehyde in 0.5 M Na cacodylate-HCl buffer (pH 7.2) for 1 hr at room temperature. The fixed samples were washed three times in the same buffer. Following the third wash the cells were post fixed for 1 hr in 1% osmium tetroxide (w/v) in the same buffer, cleaned three more times in the same buffer and still left at 4 C in refreshing buffer right away. The next morning hours the samples had Rabbit Polyclonal to FSHR been dehydrated through a graded group of ethanol to 10% and Critical Point Dried out in liquid.