Culturing and aging of primary Astrocyte
The 1-5-days old C57BL/6J mouse pups were used to culture the primary astrocytes. CO2 inhalation was used to euthanize, and the brains were collected in a sterile dish containing cold media. The meninges and blood vessels were removed from the brain, and brain tissue was transferred to a culture dish containing cold DMEM media. The brain tissue was cut into smaller pieces using sterile scissors, followed by 2-3 PBS washes. After that, brain tissue pieces were incubated for 20-25 minutes in a 0.25% trypsin-EDTA solution at 37 °C. The trypsin-EDTA solution was neutralized with 10% FBS-DMEM, and the cell suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in complete DMEM followed by culture in humidified incubator at 37 °C with 5% CO2. After 48 h, the media containing non-adherent cells were replaced with fresh complete DMEM (Sigma-D5523). The media were changed every fourth day until the astrocytes were confluent and ready for subculture. The astrocytes were aged by serial passaging until the fourth passage.