Amygdala/Hippocampal Preparation
1. Prepare HIB and keep it in ice.
! Adjust the pH 7.4 and filter sterile (Millex GP filter unit 22µm - SLGP033RP, Merk Millipore)
the ice cold HIB.
SALT
NaCl
KCl
HEPES
α-D-Glucose
Product Code
S/3160/60
(Sigma-Aldrich)
P5405
(Sigma-Aldrich)
H3375
(Sigma-Aldrich)
158968 (Fisher)
Concentration
(mM)
Molecular
Weight
Grams per litre
120
58.44
7.00
5
74.6
0.37
25
238.3
5.96
9
180.16
1.60
2. Prepare maintenance medium (Neurobasal A 1X) and store it in the fridge.
Neurobasal™-A Medium (1X)
B-27 Supplement (50X)
GlutaMAX™ Supplement
Pen/Strep (10000 units/ml)
Product Code
11540366 (Fisher)
11530536 (Fisher)
11574466 (Fisher)
11548876 (Fisher)
Volume per 500ml
500
10
5
5
3. Prepare plating medium (Neurobasal A 1X + 5% FBS).
Add 2.5ml of Fetal Bovine Serum (F0804, Sigma-Aldrich) into 50ml falcon tube of
maintenance medium. Filter sterilize it using Millex GP filter unit 22µm (SLGP033RP, Merk
Millipore) and warm up it in water bath (37°C).
4. Flame sterilize coverslips (Ø30mm, 631-0174 VWR) and put in six well plate. Add 0.1ml polyD-Lysine (0.1% Solution, P8920 Sigma-Aldrich) in the center of each coverslip and leave for
30 min.
Then, remove poly-D-lysine from coverslips and leave dish open to “air dry” in hood.
5. Prepare enzymes for digestion.
Weight 5mg Protease A from Streptomyces Griseus (P5147, Sigma-Aldrich) and 5mg
Thermolysin from Bacillus thermoproteolyticus rokko (P1512, Sigma-Aldrich) into different
eppendorf and put them in -20°C.
6. Dissect amygdala/hippocampus from pups (p0) under the hood and put into sterile HIB.
Remove excess of HIB and quickly trim and chop into 2mm chunks using scalpel blade.
7. Transfer pieces of tissue with fresh HIB into sterile container and put on ice.
8. Prepare enzymes digestion solution.
Dissolve 5mg Protease A and 5mg Thermolysin using 1ml cold HIB each and transfer in 15ml
falcon tube. Add 8ml of cold fresh HIB (Vf = 10ml) and filter sterilise using Millex GP filter
unit 22µm (SLGP033RP, Merk Millipore).
9. Remove the excess of HIB from the sterile container.
! Be careful do not remove pieces of tissue.
Add 3-5ml of enzymes digestion solution directly onto the amygdala/hippocampal pieces.
Leave for 30-45min at room temperature under the hood.
10. Prepare DNAse (DN25, Sigma-Aldrich).
The DNAse stock is 4000U/ml (=8mg/ml of sterile water). Filter sterilise using Millex GP filter
unit 22µm and store the aliquots (30µl) in -20°C.
When the incubation is over, remove digestion solution and add 3ml of warm plating
medium + 30µl DNAse (! Use as 1:100 dilution to give DNAse 40U/ml final).
11. Triturate with a fire-polished pipette and centrifuge at 250g for 3min.
12. Remove the supernatant and suspend in 3ml of warm plating medium. Triturate with a firepolished pipette and centrifuge at 250g for 3min.
13. Suspend in a small volume of warm plating medium and add 10-40 µl droplets of cells to the
center of each coverslip and put six well plate into incubator.
14. Leave for 15min in the incubator and add 2ml of warm plating medium for each well.
15. Add the selective inhibitor of DNA synthesis.
After 24h, replace medium with 2ml of fresh warm plating medium + 10µM Citosyne β-DArabinofuranoside (Ara-C) each well (C6645, Sigma-Aldrich) in order to block the division of
mitotic cells.
16. After 24-48h, replace medium with 2ml of maintenance medium per each well.
17. ! Feed cells every 3-4 days by replacing 1ml of medium with fresh maintenance medium.
18. ! Transfect neurons using Lipofectamine 2000 Reagent (10696343, Fisher) after 7days.