TITLE: Embryonic/Postnatal Mouse Neuron Culture Protocol
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This protocol details the embryonic/postnatal neuron culture procedures.

[GUIDELINES]
*Simplified protocol for neuron culture (hippocampal or cortical)
All procedures on live animals should be performed in accordance with your Institutional Animal Care and Use Committee. 

[STEPS]
SECTION: Coverslip and plate preparation
1. Drop 12 mm coverslips into a 200 ml beaker containing 95% Ethanol (or methanol). (Leave at least 1 min).
SECTION: Coverslip and plate preparation
2. Plate coverslips on a 24-well plate and allow to completely dry (>30 min).
SECTION: Coverslip and plate preparation
3. Dilute PDL to 100 1527 with borate buffer (2.5 mL PDL solution + 47.5 mL borate buffer) and coat coverslips with 0.5 mL PDL for 120 min in the incubator or 120 min in the TC hood. Plastic plates can be coated similarly (100 µL for 96-well).
SECTION: Coverslip and plate preparation
4. Wash coverslips or plates 3-5x in tissue culture water. Remove all culture water from coverslips or plates, then allow to dry for 60 min + in TC hood.

SECTION: Coverslip and plate preparation
5. Add neuron medium to plates and incubate at 37 °C in incubator > 60 min to equilibrate (50 µL for 96-well, 0.5 mL 24-well, 1 mL 12-well, 2 mL 6-well). If using a 96-well plate, add 200 µL of PBS in periphery wells and 150 µL of PBS between wells.  
SECTION: Coverslip and plate preparation
6. Equilibrate a T25 or T75 flask of neuronal medium in the incubator. Can also warm Neurobasal to Room temperature for wash steps.

SECTION: Papain Preparation
7. Make the papain dissociation solution (see Media section). Each T25 flask can hold tissue from 1 to maximum 20 mouse preps.  Sterile filter (syringe filter or Steriflip, depending on volume) and keep in T25 flask in the incubator. 

 

SECTION: Embryonic culture only:
8. Apply CO2 to dam as indicated in IACUC protocol.
SECTION: Embryonic culture only:
9. Dislocate spine.
SECTION: Embryonic culture only:
10. Pin dam, abdomen up, to Styrofoam covered with lab mat.
SECTION: Embryonic culture only:
11. Spray dam with 70% ethanol thoroughly.
SECTION: Embryonic culture only:
12. With dissection scissors, open the abdominal cavity, avoiding internal organs. 

 

SECTION: Embryonic culture only:
13. Grasp the uterus with forceps. Lift and cut the uterus out. Transfer to a 10 cm culture dish.
SECTION: Embryonic culture only:
14. Cut the uterus and remove the embryonic sacs to release embryos into a fresh dish on ice.

SECTION: Pup dissection (embryonic and postnatal):
15. Decapitate pup, allowing head to fall into a 10 cm dish filled with HBSS on ice. 

SECTION: Pup dissection (embryonic and postnatal):
16. Using forceps, hold head by the eyes. Using the other hand and a curved or angled forceps, pinch the scalp just behind the eyes and pull back, tearing it off. 
SECTION: Pup dissection (embryonic and postnatal):
17. Next, use a micro-scissor to cut down the mid-sagittal skull, and gently peel the skull off with forceps, being careful in case any tissue still connects it to the rest of the skull. Gently remove brain (using a spatula or curved forceps) and place into Petri dish filled with ice cold sterile Hibernate Medium.
SECTION: Pup dissection (embryonic and postnatal):
18. Gently separate hemispheres, removing the thalamus, striatum, and brainstem.
SECTION: Pup dissection (embryonic and postnatal):
19. Grasp the olfactory bulb and pull caudal, slowly removing the meninges from the cortex. While still grasping the meninges, flip the hemisphere and remove meninges from the hippocampus.
SECTION: Pup dissection (embryonic and postnatal):
20. Using micro-scissors, cut the hippocampus away from the cortex. 
 

SECTION: Pup dissection (embryonic and postnatal):
21. Hippocampi can be left whole. If cortical tissue is to be used, it should be cut into 1 mm³ segments.
SECTION: Pup dissection (embryonic and postnatal):
22. Place all segments into a conical with Hibernate medium.
SECTION: Pup dissection (embryonic and postnatal):
23. Bring to biosafety cabinet. Everything from here on should be done in a sterile biosafety cabinet. Pipette out Hibernate medium and rinse twice with fresh, Room temperature HBSS. 
SECTION: Trituration
26. Add 50 µL [170 U] DNase/10 mL papain solution and gently rotate until pieces of tissue are separated. 
 

SECTION: Trituration
28. Rinse chunks 2-3 times using 5 ml Neurobasal (warmed to 37 °C). 
 

SECTION: Trituration
29. Remove as much media as possible and resuspend cells in 2 mL of neuron medium.
SECTION: Trituration
30. Mechanically dissociate cells (do not over triturate!). First, pipet up and down with 1000 µL tips ~10-15 times (when done properly the medium becomes slightly opalescent with dissociated cells). Next, use a 200 µL tip ~20 times. The chunks should now be completely dissociated into individual cells.
SECTION: Trituration
31. Bring total volume to 6 mL Neurobasal and strain through a 40 μm cell strainer into a 50 mL conical, rinsing strainer with 2 mL Neurobasal before and after. Transfer cells to 15 mL conical.
SECTION: Trituration
32. Centrifuge cells at 1000 x g, 5 min. Resuspend the pellet in 2 mL of neuron media, mix and count.
SECTION: Trituration
33. Dilute cell suspension to 1,000,000 cells/mL. For 96-well, cells should be diluted to 170,000 cell/mL. 
SECTION: Trituration
34. Add the appropriate volume to the well of the neuron media-containing dishes so that each well contains:

17,000 cells (96-well) 
 

100,000 cells (24-well)
250,000 cells (12-well)
1,000,000 cells (6-well)
SECTION: Trituration
35. Gently agitate plates back and forth in each direction to spread cells. Place in incubator.
SECTION: Trituration
36. Cells can be checked the next day for adherence and even distribution. Neurons will start to sprout neurites within the first few days.
SECTION: Trituration
37. Add additional media to each well once a week:

20 μL (96-well)
115 μL (24-well)
330 μL(12-well)
1 mL (6-well)

SECTION: Trituration
27. Use 10 mL serological pipet to move tissue from T25 flask into 15 mL conical.
SECTION: Papain Digestion
24. Use serological pipet to transfer chunks to papain and move to 37 °C incubator, laid flat.
SECTION: Papain Digestion
25. Digest for approximately 5 min-20 min or until chunks have fallen apart and look something like cotton candy.