The absorbance at 650 nm was subtracted from the absorbance at 405 nm to determine the relative absorbance of each well, and the amount of NGF was calculated from a standard curve. To determine the biological activity of C6S and NGF, cortical neurons selleck chemical were cultured on gels with and without C6S and NGF. Gels (20 ��l) were prepared according to the previously described protocol39 in silicone inserts (Sigma-Aldrich) placed in chambered glass slides (Nalgene) with two gels per chamber. Silicone inserts were sterilized by sonication with 90% ethanol (VWR Scientific, EM-EX0276) for 20 min. All materials and solutions were filtered (0.2 ��m, Millipore) for cell culture. Cortex tissue (embryonic rat day 18) was purchased from BrainBits. Primary cortical neurons were isolated from E18 cortex tissue following a protocol from BrainBits.
40 The cortical tissue was digested in a Hibernate E media solution (BrainBits, HE) containing 2 mg/ml papain (Worthington Biochemical Corporation, LS003126) at 37��C for 30 min. The tissue was then transferred into a 2% (v/v) solution of B27 supplement (Invitrogen, 17504044) in Hibernate media and triturated. The cell suspension was filtered through a 40-��m nylon cell strainer (BD Falcon) and collected. The filtered suspension was centrifuged at 1100 rpm for 1 min. The supernatant was removed and the cell pellet resuspended in 3 ml B27/Neurobasal media (Invitrogen, 21103049) with 0.5 mM glutamine (Invitrogen, 25030149). The viability and density of the cell suspension was determined by mixing 20 ��l of Trypan Blue (0.
4%, Sigma-Aldrich, T6146) with 20 ��l of the cell suspension. Cell density was counted using a hemocytometer. The cell suspension was diluted to a final concentration of 2 x 105 cells/ml. Supplemented neurobasal media (100 ��l) was added onto each gel, then 6.375 ��l of the cell suspension was placed on each gel. The cells were incubated for 1 h at 37��C and 5% CO2 before an additional 1 ml of media was added to each chamber (2 gels). Cells were incubated for 48 h before fixation. After 2 d of culture, the cells were fixed with warm 4% (v/v) paraformaldehyde (Electron Microscopy Sciences, 19200) in 1xPBS for 1 h at room temperature. The cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787) solution in PBS for 2 h.
After washing 3x with PBS (20 min incubations), Image-iT? FX signal enhancer (Invitrogen, “type”:”entrez-nucleotide”,”attrs”:”text”:”I36933″,”term_id”:”2084893″,”term_text”:”I36933″I36933) was added to the cells and incubated at room temperature for 2 h. The cells were again washed 3x and blocked overnight with 1% bovine serum albumin (BSA, Sigma-Aldrich, A7906) and 10% goat serum GSK-3 (Invitrogen, 50062Z). After blocking, the cells were washed 6x with 0.1% BSA in PBS then incubated in 5 ��g/ml mouse anti-��III-tubulin (R&D Systems, MAB1195) at room temperature for 2 h and overnight at 4��C. The cells were again washed 3x with 0.