Obtaining a Mixed Glial Cell Culture from a Neonatal Mouse Brain

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Growing of Mixed Glial Cultures

1. Decapitate 1- 2-day-old pups quickly with 5.5 inch operating scissors and place the heads immediately in a 50 mL tube on ice. Note that this decapitation is the mode of euthanasia.
2. In a laminar airflow hood, make a small incision in the skull and meninges using 4.5 inch straight micro-dissecting scissors. Begin cutting from the caudal end to the rostral end (nose). Get underneath the skin by using the opening formed by the decapitation.
   1. After the incision, peel one of the hemispheres to the side. Then use a pair of curved or hooked tweezers to remove the entire brain.
3. Immerse the brain(s) in a new 50 mL tube containing 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) for 15 min in a 37 °C water bath. Use 2 mL of trypsin-EDTA per brain.
4. Wash the brain(s) with fresh growth media (10% fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium/Nutrient mixture F12 (DMEM/F12), 1% penicillin/streptomycin, 1% L-glutamine, 1% sodium pyruvate, and 1% non-essential amino acids) by adding and removing the media. Repeat this 4x.
5. For each brain, plate two T-75 flasks containing growth media. Therefore, add 2 mL of growth media per brain to the tube. Thus, it will be an equivalent of 1 mL of homogenized brain with 8-9 mL of growth media per T-75 flask.
6. Homogenize by triturating the brain(s) with pipettes of differing aperture sizes, in order from largest to smallest. When it is visible that the brain tissue is not getting smaller, transition to the next pipette. At the end of the trituration, the suspension should be clear, with no visible chunks.
   1. Use a 25 mL pipette, 5 mL pipette, and then a 10 mL pipette sequentially.
7. Pass each homogenous brain suspension through a 70 µm cell strainer to make it into a single cell culture.
8. For each homogenized brain, plate two T-75 flasks containing growth media, as described in step 1.5 (1 mL of homogenized brain with 8-9 mL of growth media per T-75 flask).
9. Change growth media after 6 d and grow until isolation on the 16^th day.