Cortical, Striatal and Dopaminergic Neurons Cultures Protocol

Abstract

This protocol describes how to obtain primary neuronal cultures from postnatal mice (P0-P1). It can be used to obtain pure cortical neurons cultures or stratal-cortical neurons and dopaminergic-cortical neurons co-cultures.

Before start

Prepare the plates :

Coat plates with poly-L-Lysine 0h 10m 0s at room temperature or at least 2h 0m 0s at 37°C.
Wash thoroughly with distilled water three times. Let it dry under the hood in the dark.

Prepare the following solutions :

- Dissection medium : 10mM HEPES pH7.3 , 0.1% D-glucose, 0.11mg/ml Sodium pyruvate in HBSS.

- DNase solution : one vial of DNAse in 500µL of BME.

- Papain solution : one vial of Papain in 5mL of BME plus 250µL of DNase solution.

- Trypsin inhibitor solution: 15.5mg/ml in BME. Filter the solution.

- Stop solution: 250µL of DNase solution, 600µL of Trypsin inhibitor solution in 5.4mL of BME.

10/10 solution: Trypsin inhibitor 10ug/ml and BSA 10ug/ml in BME. Filter the solution.
Plating medium : 1mL B27 supplement, 125µL L-Glutamine, 500µL Pen/Strep, 500µL N2 supplement in 50mL Neurobasal.

Steps

Dissection

Dissect the cortices, the striatum and the substantia nigra (SN) in cold dissection medium, carefully removing the meninges

Cells Dissociation

Snip the tissues into smaller pieces and place them in Papain solution. Use around 500µL of Papain solution for 5 SN, around 1mL of Papain solution for 5 striata and around 5mL of Papain solution for 5-6 cortices.

Triturate 10 times with a 5ml-pipette

Incubate at 37°C for 0h 40m 0s and mix by inversion every 0h 10m 0s

Triturate 10 times again with a 5ml-pipette

Spin in swing bucket rotor at 1000rpm for 0h 5m 0s

Remove and discard the supernatant

Tap the tubes to move the pellet and add STOP solution. Add 1mL for 5 SN, 1mL for 5 striata and around 3mL for 3 cortices.

Triturate 3 times with a 5ml-pipette

10.

Incubate at room temperature for 0h 10m 0s

11.

Take the supernatant and add to 10/10 solution drop-by-drop (use a volume of 10/10 solution that is equal to the supernatant volume)

12.

Spin at 800rpm for 0h 10m 0s

Neuron Seeding

13.

Discard the supernatant and resuspend in Neurobasal complete medium with AraC. Use 1mL of medium to resuspend 5 striata and 3 cortices.

14.

Count cortical and striatal cells with trypan blue

15.

Plate 500000 cells/well in 24wells-plate.

For striatal-cortical co-culture plate a ratio of 1:2 striatum:cortex.

For SN-cortical co-culture plate one well for each SN and around 500000 cortical neurons/well and also add GDNF to the medium.

16.

Replace half medium about every 7 days by collecting conditioned medium from cells and mixing with fresh complete medium in a ratio of 1:1.