5. Add 1 mL of HBSS 1Â to each culture insert and keep inside
the incubator until use.
On the culture day
6. Before starting the procedure ensure adequate sterilization of
instruments and supplies, and disinfect the whole working
space.
7. Add ice-cold HBSS 1Â to the 150 mm soda lime silica glass
petri dish and place it on ice.
8. Add ice-cold HBSS 1Â to four p60 plastic dishes and place
them on ice.
9. Prewarm the Neuronal Culture Media-Plating, one aliquot of
10% FBS and 10 mL of HBSS 1Â at 37
C.
10. Let the DNase I and trypsin equilibrate to room temperature.
11. Place the fire-polished glass pipettes inside the cell culture
hood and coat them by pipetting pure FBS up and down.
12. Euthanize female mouse by CO 2 inhalation followed by cervical dislocation.
13. Perform cesarean-section on female and transfer the embryos
with the placenta to the 150 mm glass petri dish with ice-cold
HBSS 1Â.
14. Remove 1 embryo from the placenta and euthanize by
decapitation.
15. Transfer the head to a p60 dish with ice-cold HBSS 1Â.
16. Dissect the brain out of the skull and transfer to a p60 dish with
ice-cold HBSS 1Â.
17. Repeat for the reminder embryos until all brains are
dissected out.
18. Take one brain and transfer to a new p60 dish, cut the brain
along the midline, and remove the cerebellum and hindbrain
(Fig. 1).
19. Remove the meninges and all noncortical brain tissue, exposing the hippocampus.
20. Dissect out the hippocampus from each hemisphere, transfer
to a new collection p60 dish, and repeat with all the remaining
brains.
21. Take the hippocampi to the cell culture hood.
22. Mince the hippocampi gently with a sterile scalpel.
23. Collect all the tissue pieces and transfer them to a 15 mL tube
using a p1000 pipette tip.
24. Let the tissue settle to the bottom of the tube and carefully
remove the HBSS.
25. Add 4.5 mL of trypsin (0.25%) and incubate at 37
C for
15 min with gentle agitation every 5 min.
148
Diego Alzate-Correa et al.
the incubator until use.
On the culture day
6. Before starting the procedure ensure adequate sterilization of
instruments and supplies, and disinfect the whole working
space.
7. Add ice-cold HBSS 1Â to the 150 mm soda lime silica glass
petri dish and place it on ice.
8. Add ice-cold HBSS 1Â to four p60 plastic dishes and place
them on ice.
9. Prewarm the Neuronal Culture Media-Plating, one aliquot of
10% FBS and 10 mL of HBSS 1Â at 37
C.
10. Let the DNase I and trypsin equilibrate to room temperature.
11. Place the fire-polished glass pipettes inside the cell culture
hood and coat them by pipetting pure FBS up and down.
12. Euthanize female mouse by CO 2 inhalation followed by cervical dislocation.
13. Perform cesarean-section on female and transfer the embryos
with the placenta to the 150 mm glass petri dish with ice-cold
HBSS 1Â.
14. Remove 1 embryo from the placenta and euthanize by
decapitation.
15. Transfer the head to a p60 dish with ice-cold HBSS 1Â.
16. Dissect the brain out of the skull and transfer to a p60 dish with
ice-cold HBSS 1Â.
17. Repeat for the reminder embryos until all brains are
dissected out.
18. Take one brain and transfer to a new p60 dish, cut the brain
along the midline, and remove the cerebellum and hindbrain
(Fig. 1).
19. Remove the meninges and all noncortical brain tissue, exposing the hippocampus.
20. Dissect out the hippocampus from each hemisphere, transfer
to a new collection p60 dish, and repeat with all the remaining
brains.
21. Take the hippocampi to the cell culture hood.
22. Mince the hippocampi gently with a sterile scalpel.
23. Collect all the tissue pieces and transfer them to a 15 mL tube
using a p1000 pipette tip.
24. Let the tissue settle to the bottom of the tube and carefully
remove the HBSS.
25. Add 4.5 mL of trypsin (0.25%) and incubate at 37
C for
15 min with gentle agitation every 5 min.
148
Diego Alzate-Correa et al.