I am planning to co-culture primary astrocytes with immortalized neurons GT1-7 (both from mice). What is the best way to do this?
Homogenize the brains and plate them in a T75 flask with DMEM 10% FBS. This will highly enrich for astrocytes in the culture. Then seed them accordingly in a 6-well or 24-well accordingly to your preferences. Usually I seed about 35K per well for a 24-well plate. Following 7 days in culture, plate the neurons on top of the astrocytes in the well. Change the media to neuronal basal media with B27 and glutamine.
I'd establish your astrocyte cultures first in their usual growth medium (typically DMEM + 10% FBS) and allow the cells to reach confluence to provide a good monolayer, timing will depend on seeding density in your wells or plates. When you're ready, add your neurons in their preferred culture medium, can supplement with serum to keep the astrocytes happy.
I've co-cultured plenty of combinations of neurons and glia (all primaries), and sometimes you need to tweak the serum concentrations to keep the cells growing well and to find something that works with your assays. Astrocytes probably won't be too upset with being in a serum-free growth medium but there's no harm in having it if you can - the main reason neuron medium is serum free is to minimise glial growth.
Thank you Saravanan Gunaseelan and Katherine A Southam , I'll consider your suggestions!