Primary Culture of Cortical Neurons

Materials and Reagents

1. Poly-L-lysine (Sigma-Aldrich, catalog number: P2636 )
   
2. Dulbecco's Modified Eagle Medium, DMEM (Life Technologies, Invitrogen^TM, catalog number: 12800-017 )
   
3. Fetal bovine serum (Life Technologies, Gibco^®, catalog number: 10437 )
   
4. 2.5% Trypsin solution (Life Technologies, Gibco^®, catalog number: 15090-046 )
   
5. DNase (Sigma-Aldrich, catalog number: DN25 )
   
6. Penicillin/streptomycin (Life Technologies, Gibco^®, catalog number: 15140 )
7. NaH₂PO₄^.2H₂O
   
8. Na₂HPO₄^.12H₂O
   
9. NaCl
   
10. DMEM
    
11. FBS
    
12. Serum-containg culture medium (see Recipes)
    
13. PBS (see Recipes)
    

Equipment

1. Cell culture incubator
   
2. Centrifuge
   
3. Stereoscopic microscope
   
4. Scissors, forceps, and knives
   
5. Sterile filter (0.45 μm)
   
6. Cell strainer 70 μm nylon (BD Biosciences, catalog number: 352350 )
7. 24 well culture plate
   

Procedure

1. Coating and washing culture plates.
   1. Dilute poly-L-lysine stock solution with sterile PBS to 100 μg/ml. Add optimal volume of solution to each well of culture plates. In case of 24 well culture plate, 0.5 ml solution is enough. Coating allows neurons to adhere to the culture plates.
      
   2. Leave the plates in incubator for at least 1 h. After that, wash the wells twice with 1 ml/well of sterile PBS. Completely remove the PBS before use. It is not necessary to dry the plate after PBS washing.
      
2. Choose appropriate anesthesia according to the recommendations of your Institutional Animal Care and Use Committee. After anesthetization, cut the scalp and remove the skull. We use postnatal day 1 mice to measure the length of corticospinal neuron. Pick up whole brain from pups and place it in cold PBS on ice (Chen et al., 2007).
   
3. Under a microscope, dissect out the cerebral cortex of brains. Remove meninges and blood vessels. The tissues are cut into as small piece (1 mm cubes) as can with sterile knives.
   
4. Transfer tissues to a 50 ml tube. The tissues are trypsinized in 10 ml of the trypsin solution (0.25% trypsin, 100 μg/ml DNase in PBS), and are incubated at 37 °C for 15 min.
   
5. Add an equal amount of 10% FBS-DMEM, and pipettes up and down a few times to help with tissues dissociation.
   
6. Centrifuge at 300 x g for 3 min at room temperature, discard the supernatant, and resuspend the pellet in fresh DMEM (not 10% FBS-DMEM).
   
7. The cells are filtered through a 70 μm nylon cell strainer to obtain single cell suspension. Centrifuge the resulting cell suspension at 300 x g for 3 min at room temperature. Resuspend the cell pellet in fresh DMEM (not 10% FBS-DMEM).
   
8. Count the number of cells and adjust the concentration using 10% FBS-DMEM. To measure the neurite length, plate the cells on a 4-well chamber slide at a density of 5 x 10⁴ cells/well. Transfer the cell suspension to culture plates and incubate for 24 h at 37 °C with 5% CO₂. To investigate the effect of pharmacological agents, change to the medium containing each agent 2 h after cell plating. Representative image shows the neuronal class III β-Tubulin (Tuj-1) labeled cortical neuron obtained 1 day culture.
   
   
   Figure 1. Image of cultured cortical neuron. Representative image shows the neuronal class III β-Tubulin (Tuj-1) labeled cortical neuron obtained 1 day culture.
   

Recipes

1. Poly-L-lysine: Dissolve of sterile distilled water to make stock solution (10 mg/ml). Keep aliquots at -20 °C. Dilute with sterile PBS right before use.
   
2. Serum-containg culture medium
   

   Reagents	Volume
   DMEM	445 ml
   FBS	50 ml
   Penicillin/streptomycin	5 ml

3. PBS
   

   Reagents	Volume
   NaH2PO4·2H2O	0.312 g
   Na2HPO4·12H2O	2.8652 g
   NaCl	8.5 g
   DW	1 L

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientists (A) (25710006) from the Japan Society for the Promotion of Sciences to RM, the Osaka University Program for the Support of Networking among Present and Future Researchers to RM, and a Grant-in-Aid for Scientific Research (S) from JSPS (25221309) to TY.

References

1. Chen, Y., Balasubramaniyan, V., Peng, J., Hurlock, E. C., Tallquist, M., Li, J. and Lu, Q. R. (2007). Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nat Protocols 2(5): 1044-1051. 
   
2. Muramatsu, R., Takahashi, C., Miyake, S., Fujimura, H., Mochizuki, H. and Yamashita, T. (2012). Angiogenesis induced by CNS inflammation promotes neuronal remodeling through vessel-derived prostacyclin. Nat Med 18(11): 1658-1664.