Isolation and characterization of living primary astroglial cells using the new GLAST‐specific monoclonal antibody ACSA‐1

Astrocytesshow large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocytesubpopulations has been hampered by the lack of a suitable antibody for their direct detection and isolation. Here, we describe a new monoclonal antibody, ACSA-1, which was generated by immunization of GLAST1 knockout mice. The antibody specifically detectsan extracellular epitope of the astrocyte-specific L-glutamate/L-aspartate transporter GLAST (EAAT1, Slc1a3). As shown by immunohistochemistry, immunocytochemistry, and flow cytometry, ACSA-1 was cross-reactive for mouse, human, and rat. It labeled virtually all astrocytespositive for GFAP, GS, BLBP, RC2, and Nestin, including protoplastic, fibrous, and reactive astrocytesas well as Bergmann glia, M€ glia, and radial glia. Oligodendrocytes, microglia, neurons, and neuronal progenitors were negative for ACSA-1. Using an immunomagnetic approach, we established a method for the isolation of GLAST-positive cells with high purity. Binding of the antibody to GLAST and subsequent sorting of GLAST-positive cells neither interfered with cellular glutamate transport nor compromised astrocyteviability in vitro. The ACSA-1 antibody is not only a valuable tool to identify and track astrocytesby immunostaining, but also provides the possibility of separation and further analysis of pure astrocytes. V C 2012 Wiley Periodicals, Inc.