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The following procedures have been found effective with 18-day gestation rat hippocampi and with neuroblastoma cell lines.
Note: Use 0.15 mL/cm2 surface area of poly-D-lysine. Poly-D-lysine solutions are stored at -20°C in polycarbonate tubes. Poly-D-lysine should be prescreened for toxicity.
Note: Vessels can now be used or stored for up to two (2) weeks at 4 to 10°C in sterile, distilled water. If vessels are to be stored, remove water about one (1) hour prior to use.
Note: The addition of fibronectin or vitronectin is usually not necessary when supplementing with B27.
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- Coat culture vessels with a 0.05 mg/mL solution of poly-D-lysine (MW 30,000 - 70,000) and incubate for one (1) hour or overnight:
Note: Use 0.15 mL/cm2 surface area of poly-D-lysine. Poly-D-lysine solutions are stored at -20°C in polycarbonate tubes. Poly-D-lysine should be prescreened for toxicity.
- Wash vessels with sterile, cell culture grade water.
Note: Vessels can now be used or stored for up to two (2) weeks at 4 to 10°C in sterile, distilled water. If vessels are to be stored, remove water about one (1) hour prior to use.
- To NEUROBASAL medium, add 0.5 mM L-glutamine, 25 µM glutamate, and either 2% B27 or 1% N2 supplement.
- If using N2 supplement, prior to adding cells, add human plasma fibronectin (Cat.No. 33016) at a final concentration of 5-10 µg/mL directly to the medium or substitute bovine vitronectin (Cat. No. 12165) for fibronectin at 0.5 to 1.0 mg/mL. Tilt vessel immediately to ensure even distribution.
Note: The addition of fibronectin or vitronectin is usually not necessary when supplementing with B27.
- To prepare human fibronectin - reconstitute 5 mg in 6.75 mL of sterile, distilled H20 for a 740 µg/mL stock. Add 50 µL of stock per 5 mL of culture medium for a final concentration of 7.4 µg/mL.
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For primary hippocampal neurons (i.e. from Sprague -- Dawley rats at 18 days gestation) and other fetal neurons.
- Embryos are recovered by C-section under nembutal anaesthetic and desired region dissected.
- Individual cells are isolated by trituration 10 times in 1 mL Hanks' Balanced Salt Solution w/o Ca++ and Mg++ (Cat. No. 14175) and supplemented with 1.0 mM sodium pyruvate (Cat. No. 11360) and 10 mM HEPES (Cat. No. 15630), pH 7.4 using a 9 inch siliconized pasteur pipet with the tip barely fire polished.
- Divalent cations are restored by dilution with 2 volumes HBSS (Cat. No. 14025) supplemented as above.
