TITLE: Primary Culture of Mouse Mesencephalic Neurons
AUTHORS: louis-eric.trudeau

[DESCRIPTION]
This protocol details the procedure required for the dissection and collection of primary mouse culture mesencephalic neurons.

[BEFORE_START]
Preparation of solutions for dissociation: all solution must be prepared fresh 

Papain solution: (5 ml of solution for a maximum of 5 brains and/or region / dissection) 
2.2 mg of Cysteine HCl (Sigma C-6852; 2.5 mM; Room temperature) 
dissolve in 5 mL of dissociation solution (stored at 4 °C) 
adjust the pH to ~7.4 with NaOH 1 N 
add 100 Papain Units1  (Worthington LS003126; stored at 4 °C) (volume " x " µl of the stock according to its concentration in Units/mgP) 
incubate at 37 °C for 15 min without shaking to facilitate activation and solubilization 
filter sterilize using a 0.2 µm/28 mm syringe filter (Sartorius 14555306, SFCA membrane, blue). Use it immediately: the activity decreases with time – if not used within 30 min, trash

Trituration solution: (20 ml of trituration solution for 10 brains)
20 mL of Neurcell+ (37 °C) 
20 mg of trypsin inhibitor2  (Sigma T-9253; 4 °C) 
20 mg of BSA (Sigma A-7030; 4 °C) 
47.66 mg of HEPES3  (Sigma H-3375; 10 mM; 37 Room temperature) 
adjust the pH to 7.4 with NaOH 1 N
filter sterilize and store at 37 °C

Centrifugation solution: (5ml of solution for a maximum of 5 brains and/or region / dissection)
5 mL of Neurcell+ (37 °C) 
50 mg of BSA (Sigma A-7030; 4 °C) 
11.9 mg of HEPES (Sigma H-3375; 10 mM; 37 Room temperature) 
adjust the pH to 7.4 with NaOH 1 N. 
filter sterilize and store at 37 °C

*** Prepare a tube with the dissociation solution to rinse the tissues after incubation in papain – this tube also serves as a reference tube for adjusting the pH of the other solutions (the phenol red is pH sensitive, so it allows you to compare the pH). ***

Remember to sterilize enough 35mm petri dishes and Whatman filters in the morning of dissection and leave them in the hood for transfer of coverslips for seeding of neurons.

1Papain is a cysteine protease that catalyzes the cleavage of peptide bonds 
2Protease inhibitor blocks the activity of proteolytic enzymes 
3Used as a buffer solution and preferred over bicarbonate buffer for cell culture due to better stability at physiological pH






















[STEPS]
SECTION: Dissection (preferably in pairs)
1. Prepare two containers of crushed ice.
SECTION: Dissection (preferably in pairs)
2. Clean the dissection surface with 70% alcohol. Place the dissection tools in a beakers filled with 70% alcohol and a Kimwipe (to protect the tips of the tools).
SECTION: Dissection (preferably in pairs)
3. Prepare 1 petri dish with 2 mL for 2 animals (n times). Keep on ice.

SECTION: Dissection (preferably in pairs)
4. Prepare 1 petri with 2 mL for 5 animals (n times) to collect the tissue blocks. Identify the petri and keep on ice.

SECTION: Dissection (preferably in pairs)
5. Prepare one 10 ml syringe per series of 5 animals (n times) with dissociation solution. Keep on ice.

SECTION: Dissection (preferably in pairs)
6. Place the animals on ice. Wait 2 min to 3 min (until they are anesthetized). Put a maximum of 5 pups on ice at a time. Once the dissection begins, add the subsequent series of pups.

SECTION: Dissection (preferably in pairs)
7. The first person removes the brains from the skulls once the animals are anesthetized (nonresponsive to manipulation and paw pressing). Wipe the skull and neck area with 70% alcohol and dry with a Kimwipe.
SECTION: Dissection (preferably in pairs)
8. Hold the animal by the skin under the throat. Cut the skin around top of the skull (starting behind the ear). Take care not to damage the brain, keep the tips of the scissors facing upwards.
SECTION: Dissection (preferably in pairs)
9. Remove the cut part of the skin and the skull with the curved forceps (pointing upwards).
SECTION: Dissection (preferably in pairs)
10. Rinse the brain thoroughly with cold dissociation medium (2 ml per animal).
SECTION: Dissection (preferably in pairs)
11. Using the curved forceps gently remove the brain and place it in a petri dish (start at the olfactory bulbs and finish by cutting the base of the brain stem).
SECTION: Dissection (preferably in pairs)
12. The second person, using a binocular magnifying glass, dissects the isolated brains, one by one, preparing the slices and tissue blocks: Place the brain with the ventral region up and hold it gently with the thin forceps at the frontal lobe.
SECTION: Dissection (preferably in pairs)
13. Using the scalpel blade, cut a thin coronal slice of the brain at the "midbrain flexure"; using the Willis circle as a reference (slice 1 mm thick).
SECTION: Dissection (preferably in pairs)
14. Isolate the VTA and substantia nigra with a scalpel.
SECTION: Dissection (preferably in pairs)
15. In the sterile hood, using a 10 ml sterile pipette, transfer the blocks of tissue obtained from a maximum of 5 brains into the 15 ml tube containing papain solution #1, taking care to transfer as little dissociation solution as possible with the tissue blocks, to reduce papain dilution. Incubate with stirring for 20 min at 37 °C. Quickly continue dissecting the brains of the other mice, transferring the resulting tissue blocks into the tubes containing papain solution #2, #3 and #4 (as needed) and incubate each with agitation 20 min at 37 °C.
 





SECTION: Dissociation of Cells
16. Prior to starting, flame polish the tip of a 5 ml glass pipette to a diameter of 1.5 mm and a second to a smaller diameter (~0.5 mm, see annex 1).
SECTION: Dissociation of Cells
17. After 20 min of agitation, replace the papain solution in tube #1 with 2 mL. Repeat this rinse a second time.

SECTION: Dissociation of Cells
18. Rinse with 2 mL, remove and add 1 mL.

SECTION: Dissociation of Cells
19. Gently triturate 20 times with the 1.5 mm pipette and 40 times with the 0.5 mm pipette (use a rubber bulb). 
 

SECTION: Dissociation of Cells
20. Repeat steps 17 to 19 with papain tube #2, #3 and #4 (as needed).
SECTION: Dissociation of Cells
21. Gently transfer the dissociated cells, from the different tubes, on top of the centrifugation solution (no more than 2 ml of suspended cells per 5 ml centrifugation tube).
SECTION: Dissociation of Cells
22. Centrifuge for 2 min at position 3 (1150 rpm), then 3 min at position 4 (1400 rpm) on a clinical IEC centrifuge (the centrifuge tubes must be balanced, calibrate the centrifuge with a 15 ml tube filled with water if needed).

SECTION: Dissociation of Cells
23. Remove the supernatant and resuspend the cells in a 500 µL. Mix 10 µL with 10 µL, take 10 µL and count the cell density using a hemacytometer. Add to the cell suspension the required volume of the trituration medium (supplemented with Neurocell+ if necessary) to achieve the desired concentration. For the volume to add according to the number of cells and the desired concentration, see the calculation table. Combine the tubes of the same cell type in a single tube.

 

SECTION: Dissociation of Cells
24. Apply 65 µL to the coverslips covered with astrocyte monolayer. Remove 5 petri dishes at a time. Dry the bottom of the coverslips by placing them carefully one by one on a sterile Whatman filter paper. Place the coverslips in pre-sectioned (see annex 2) sterile dry petri dishes, and add the cell suspension at the desired concentrations as quickly as possible to avoid over drying the astrocyte monolayer. Do this step in pairs, the first person removes the coverslips from the old petri dishes, drying them on the Whatman filter paper, the second one recovers them and places them in a new petri, adding immediately 65 µL. Place the petri in the incubator.
 



SECTION: Dissociation of Cells
25. After 180 min, add 2.5 mL (Mix Neurocell+/EMEM+ for dopamine neurons, Neurocell+ only if striatal cells) to each petri dish and put back all the petris in the incubator.

SECTION: Dissociation of Cells
26. After 1440 min, add 10 micromolar (µM) (from 2 mM stock; 12.5 µl/2.5 ml) to inhibit glial proliferation.

SECTION: Dissociation of Cells
27. If necessary, following 7 days of culture, add 0.5 millimolar (mM) (from 125 mM stock, 10 µl/2.5 ml) to prevent the toxicity of glutamate release.

SECTION: Dissociation of Cells
28. If necessary, add 500 µL (Mix Neurocell+/EMEM+) every 5 days to all the petri dishes to compensate evaporation and to feed the cells.