Materials

Sku Name Size Price Qty
33016015 Fibronectin Human Protein, Plasma 5 mg USD 356.00
PHE0011 Vitronectin Human Protein, Natural 100 µg USD 230.00
15230162 Distilled Water 500 mL USD 12.75
10888022 1X Neurobasal®-A Medium 500 mL USD 58.00
25030081 200 mM L-Glutamine 100 mL USD 23.40
17504044 B-27® Supplement (50X), serum free 10 mL USD 85.00
17502048 N-2 Supplement (100X) 5 mL USD 70.00
14175095 HBSS, no calcium, no magnesium, no phenol red 500 mL USD 18.35
11360070 100 mM Sodium Pyruvate 100 mL USD 9.30
15630080 HEPES (1 M) 100 mL USD 53.25
14025092 HBSS, calcium, magnesium, no phenol red 500 mL USD 18.35

Protocol for Isolation and Digestion of Prenatal Primary Hippocampal and Cortical Neurons

The following procedures have been found effective with 18-day gestation rat hippocampi and with neuroblastoma cell lines.

  1. 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.

  1. 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.

  1. 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.

  1. 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.
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