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All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Dissection, Dissociation, and Culture of Hippocampal Neurons from Pregnant Mouse/Rat
- Dissection and dissociation of hippocampal neurons for culture
- Thaw protease enzyme solution (3 mg/mL protease 23 in Slice dissection solution, SLDS, Table 1) and pre-warm plating media (Table 2) at 37 °C.
- Dissect the hippocampus and rinse with SLDS.
NOTE: Four hippocampi provide enough cells for one 24-well or one 6-well plate.
- Remove SLDS, add 2 mL of protease enzyme solution, and incubate the sample at 37 °C, 5% CO2, for 15 min.
- Wash the hippocampus explants with 5-10 mL of plating medium twice. Add 5 mL of plating medium. Dissociate hippocampal explants into single cells by generating a small vortex using a pipette with a 1-mL plastic tip. Pipette up and down about 40 times, avoiding the formation of oxygen bubbles, until the solution becomes cloudy and no large pieces of explant remain.
NOTE: Remember to leave some media during washes, the hippocampal explants should remain submerged in the medium during the whole process.
- Centrifuge the cells at 1,125 x g for 3 min. Remove the supernatant with the cells submerged in the media. Resuspend the cells in 145 mL of plating media. Add 1 mL/well of the cell suspension to a 24-well plate (3 mL/well to a 6-well plate).
NOTE: 145 mL of plating media is used to produce a cell suspension that has a low cell density. This volume will allow the plating of cells into six 24-well plates, eight 6-well plates, or various combinations of 24- or 6-well plates, depending on the experiments to be conducted. Each well of the 24-well plate will have about 3 x 104 cells.
- Incubate the cells in the plating media at 37 °C, 5% CO2 for 2-4 h, then remove all plating media and replace it with fresh maintenance media.
- After 2 days (2 days in vitro, 2DIV), replace half of the media with 2 µM Ara-C (arabinosylcytosine) dissolved in maintenance media, which inhibits growth of fibroblasts, endothelial, and glial cells. After exposing cells to Ara-C for two days, replace the media with fresh maintenance media.
Table 1: Coverslip coating solution. Provides the recipe for preparing the coverslip coating solution used to adhere cultured cells to the coverslips.
| Coverslip coating solution |
| 780 µL | Acetic acid17 mM stock: 50 µL acetic acid in 50 mL of H2O |
| 200 µL | Poly-D-lysine (0.5 mg/mL stock) |
| 20 µL | Rat tail collagen (3 mg/mL stock) |
| The total volume is determined based on the number of coverslips to be coated. |
Table 2: Cell culture media recipes. Provides the recipes for preparing media utilized in the culturing of primary hippocampal neuron cells.
| 1x Slice dissection solution (SLDS), filtered, pH= 7.4, store at 4 °C |
| 1 L | ddH2O |
| 82 mM | Na2SO4 |
| 30 mM | K2SO4 |
| 10 mM | HEPES (free acid) |
| 10 mM | D-glucose |
| 5 mM | MgCl2 |
| 0.00% | Phenol red (optional) |
| Plating Media (PM, filter, store at 4 °C) |
| 439 mL | MEM Earle's Salts |
| 50 mL | FBS |
| 11.25 mL | 20% D-glucose |
| 5 mL | Sodium pyruvate (100 mM,) |
| 62.5 µL | L-glutamine (200 mM) |
| 5 mL | Penicillin/Streptomycin (P/S, 100x) |
| Maintenance Media (MM, filter, store at 4 °C) |
| 484 mL | Neurobasal |
| 10 mL | B27 50x supplement |
| 1.25 mL | L-glutamine (200 mM) |
| 5 mL | 100x P/S |
| All solutions are sterilized using a filter with 0.2 mm pore size. |