Abstract
Astrocytes, the most abundant cells in the central nervous system (CNS), are essential for neuronal development, network formation, and overall CNS homeostasis. Primary astrocyte culture has been successfully used as a tool to study astrocyte biology in vitro. In the present protocol, a modified immunopanning method was utilized to obtain and purify primary astrocytes from mouse cortex and spinal cord in a relatively quick and inexpensive way. Purified primary astrocytes were then immortalized through infection of lentivirus expressing the SV40 large T antigens. In addition, we provide protocols to determine the expression levels of astrocyte-specific markers and to perform functional studies measuring the ATP-induced calcium flux in the immortalized astrocytes. Following the described protocols assures that the immortalized astrocytes that one prepares mimic the cell biology of primary astrocytes in culture. Thus, the purification and immortalization protocols for primary astrocytes presented in here provide two models for the studies of astrocyte biology and may be useful for the immortalization of other types of primary cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Primary astrocyte purification by a modified immunopanning method Support Protocol: Serum-free primary astrocyte culture Basic Protocol 2: Primary astrocyte immortalization Basic Protocol 3: Calcium transient detection in astrocytes.