Obtaining a Mixed Glial Cell Culture from a Neonatal Mouse Brain
Transcription
Transfer the brain to a 50-milliliter tube containing 2 milliliters of 0.25% trypsin-EDTA, and incubate for 15 minutes in a 37 degrees Celsius water bath. Wash the brain with fresh growth medium, by adding and removing the medium. After completion of the washes, add 2 milliliters of growth medium per brain, and homogenize by triturating the brain.
When the brain tissue does not get smaller, transition to a pipette with a smaller aperture. At the end of the titration when the suspension is clear with no visible chunks, pass the suspension through a 70-micron cell strainer to create a single-cell culture.
Then, pipette 8 to 9 millimeters of growth medium into T75 flasks, two flasks per brain, and add 1 milliliter of brain homogenate to each flask. Grow the cells until isolation on the 16th day.
