Materials

Sku Name Size Price Qty
A1247501 Hibernate®-A Medium 500 mL USD 93.00
25030081 L-Glutamine (200 mM) 100 mL USD 23.40
17504044 B-27® Supplement (50X), serum free 10 mL USD 85.00

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

Isolation of tissue

  1. Hippocampi are rapidly dissected from the brain in 2 mL HIBERNATE™ A supplemented with B27 (Cat. No. 17504) and 0.5 mM L-glutamine (Cat. No. 25030) at 4°C in a 35 mm diameter dish.
  2. Meninges and excess white matter are removed in the same medium to a second dish at 4°C.
  3. Hippocampi are transferred to sterile paper prewet with the same medium on the cooled stage of a MacIlwain tissue chopper.
  4. 0.5 mm thickness slices are made perpendicular to the long axes of the hippocampi and transferred to a tube at 4°C of the same medium.
  5. After shaking for 8 minutes at 30°C, slices are transferred with a wide bore pipette to another tube at 30°C containing papain.

Digestion of tissue

  1. Papain (Worthington 3119, 15-23 U/mg protein and not activated by cysteine) is prepared by dissolving 12 mg in 6 mL of HIBERNATE-A, warming for 5 minutes at 37°C and then filter sterilizing. The papain solution should be used within 3 hours.
  2. Slices are incubated for 30 minutes in a 30°C water bath with a platform rotating at a speed sufficient to suspend the slices (e.g.. 170 rpm).
  3. Slices are transferred to a 15 mL tube containing 2 mL of HIBERNATE-A/B27 at 30°C and allowed to sit for 5 minutes at room temperature.
  4. Slices are triturated 10 times (in about 30 seconds) with a siliconized 9 inch Pasteur pipette with the tip fire polished to an opening of 0.7 to 0.9 mm diameter.
  5. The pieces are allowed to settle for 2 minutes and the supernatant transferred to another tube.
  6. The sediment from the first tube is resuspended in 2 mL HIBERNATE-A/B27