1. From the tube with the brain tissue, remove 1 ml medium and save for step 3, being careful not to remove the tissue. 2. With 1 ml pipettor with sterile blue plastic tip, or silanized 9 inch pasteur pipet with tip barely fire polished (preferable), suck the tissue with medium into the pipet and immediately dispense the contents back into the same container. Take care not to create bubbles. Repeat this trituration step about 10 times or until most all pieces of tissue are dispersed. Higher survival can be obtained by incubating the tissue for 30 min. at 30oC in 2 mg/ml papain (Worthington) in Hibernate, without B27, followed by trituration in Hibernate/B27. 3. Add back 1 ml medium that you removed in step 1 and mix. 4. Let undispersed pieces settle by gravity for 1 min. 5. Transfer supernatant to a new sterile 15 ml tube. 6. Spin 1100 rpm (200 x G), 1 min. Discard supernatant. 7. Flick the tube to disperse the pellet of cells. Resuspend pellet in 1 ml Nuerobasal /10% serum /3 mM glutamine (12 mL provided; more available from www.Invitrogen.com, 1-800- 955-6288). 8. Aliquot 20 ul and mix with 20 ul 0.4% trypan blue. 9. Count in hemacytometer: = cells/ml. 10. Dilute cells with Neurobasal/ serum/glutamine to plate 15 x 103 cells/2 cm2 of substrate in 0.4ml/2 cm2 substrate (or whatever density you desire. Plating at higher densities will result in a mixture of neurons and astrocytes) 11. Incubate 37oC, 5% CO2, 9% oxygen (20% oxygen is O.K.) 12. After 4-6 days astrocytes will be nearly confluent and ready to harvest or pass. If desired, you can change 50% of the medium to Neurobasal / 2% B27/0.5mM glutamine in preparation to harvest the next day for transfer to neuron cultures. If expansion is desired, harvest cells with 0.05% trypsin in Hibernate minus calcium, 37o, 5 minutes. Pellet cells as in step 6 and continue with steps 7-11,