Posted 09 September 2010 - 11:40 PM
[Procedure for mouse postnatal hippocampal neurons
Procedure:
Pups 0-1 days old.
1. Cut heads of the pups, remove brains and dissect out hippocampi. Place them in a cold solution 1.
2. Chop with sterile scalpel blade first one way and thereafter again after turning the petri dish 90o.
3. Take 1 ml of solution 2 37oC (trypsin) add 600 µl to a new 10 ml tube and the rest to the cerebellum. Pipette the cells to the tube. Keep at room temp. Postnatal pups 4 days for 8 min and pups at 7-8 days for 14 min (10-11).
4. Add 3 ml solution 4 (mild trypsin Inhibitor).
5. Cfg. 1500 rpm 2 min.
6. Discard S/N. Fill up with 1 ml solution 3 (trypsin inhibitor) resuspend with the 1 ml blue tip (20 times) to make resuspension, carefully.
7. Leave the tube for 5 min in an angle position to allow remaining tissue blocks to settle. Transfer S/N with pipette to a new tube.
8. Add 3 ml of solution 5 and cfg. 5 min at 700 rpm.
9. Resuspend the cells in a suitable volume of medium neurobasal medium. Count cells.
10. Add 5microM cytosine arabinofuranoside after ca 48 hous to block mitosis of astrocytes by replacing ca half of the medium on neurons. Replace one third of the medium every ca third day and check that moisture level in the incubator is good enough to avoid evaporation of the medium.
Such neurons survived for 9 days-then I fixed them (may be they would survive longer).
Good luck!
MgSO4 stock solution, 150 mM:
3,7g MgSO4- 7(H20) in 100 ml sterile water. Sterile filter 0.2 µm
CaCl2 stock solution, 0,11 M:
0.162g CaCl-2(H20) in 10 ml sterile water. Sterile filter 0.2 µm.
Solutions: Sol 1: (ca. 20 ml/tube)
100 ml HBSS,
0.3 g bovine serum albumin (Sigma A4503),
0.8 ml 3.8% MgSO4-stock solution,
2 ml 1 M HEPES solution (Gibco 15630-056) to 20 mM.
Sol 2: (ca. 1.5 ml/tube)
12.5 ml sol 1
2.45 mg trypsin (Sigma T-0303, bovine pancreas, 15200 BAEE-U/mg prot).
Sterile filter 0.2 µm
Sol 3: (ca. 1.5 ml/tube)
10 ml sol 1
0.8 mg DNAse I (Sigma D5025, 1.5 Kunitz-U/mg solid)
5.2 mg Soybean Trypsin Inhibitor (Sigma T9128 1 mg inhibits 1.7 mg trypsin (10000BAEE-U/mg prot). Sterile filter 0.2 µm
100 µl l 3.8% MgSO4-stock sol.
Sol 4: (ca. 3.5 ml/tube)
10.5 ml sol 1
2 ml sol 3.
Sol 5: (ca. 3.5 ml/tube)
12.5 ml sol 1
15 µl from 1.2% CaCl2-stock sol (stock: 0.11 M CaCl2)
100 µl 3.8% MgSO4-stock sol (3.7 g MgSO4-7(H2O) in 100 ml water - 150 mM),Sterilefilter (Gelmann, 0.2 µm)
All solutions into 10 ml tubes, at –20oc
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(Pictures attached) Somebody can help me? 
Mattek_1_day_6_with_12ul_ara.JPG 20.37K
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(Pictures attached) Somebody can help me? 
