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

Abstract

Astrocytes show large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocyte subpopulations 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 detects an 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 astrocytes positive for GFAP, GS, BLBP, RC2, and Nestin, including protoplastic, fibrous, and reactive astrocytes as well as Bergmann glia, Müller 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 astrocyte viability in vitro. The ACSA-1 antibody is not only a valuable tool to identify and track astrocytes by immunostaining, but also provides the possibility of separation and further analysis of pure astrocytes.

Neuronal vs glial glutamate uptake: Resolving the conundrum

“…This needed an explanation (see below). (Storck et al, 1992;Tanaka, 1993b;Arriza et al, 1994) is the only glutamate transporter that appears to be selectively expressed in astroglial cells in the central nervous system (Danbolt, 1994;Lehre et al, 1993;Lehre et al, 1995;Ginsberg et al, 1995;Rothstein et al, 1995;Schmitt et al, 1997;Ullensvang et al, 1997;Berger and Hediger, 1998;Banner et al, 2002;Kugler and Beyer, 2003;Regan et al, 2007;Berger and Hediger, 2000;Jungblut et al, 2012) including retina (Rauen et al, 1996;Lehre et al, 1997;Rauen et al, 1998;Harada et al, 1998;Pow and Barnett, 1999;Rauen, 2000;Bringmann et al, 2013). It is also expressed in supporting cells in the inner ear (Furness and Lehre, 1997;Takumi et al, 1997) as well as in several peripheral organs including, the heart, fat cells and taste buds (Lawton et al, 2000;Berger and Hediger, 2006;Adachi et al, 2007;Martinov et al, 2014).…”

Isolating astrocytes and neurons sequentially from postnatal murine brains with a magnetic cell separation technique

“…In this protocol, instead of isolating neurons and astrocytes separately from different brains, we describe a novel application of magnetic cell sorting approach to purify them sequentially from the same mouse brain sample. For astrocytes, we used the ACSA-2 antibody, which specifically recognizes an epitope that is comparably expressed as the astrocyte-specific L-glutamate/L-aspartate transporter GLAST (EAAT1, Slc1a3) [8,9]we describe a new monoclonal antibody, ACSA-1, which was generated by immunization of GLAST1 knockout mice. The antibody specifically detects an extracellular epitope of the astrocyte-specific L-glutamate/L-aspartate transporter GLAST (EAAT1, Slc1a3.…”

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

“…In addition to embryonic sources, neural stem or progenitor cells can be extracted from the neurogenic regions of adult brain 27 . Moreover, flow cytometry and FACS can be exploited to quantify, analyze and isolate various cell populations including mature neurons 54 , astroglia 33 , microglial 55 , oligodendrocytes 56 , or endothelial cells 57 from postnatal brain tissue.…”

“…Flow cytometry can hence play a crucial role in resolving cellular heterogeneity and, thereby, facilitate biomedical applications (in vitro assays, cell therapy) and optimize quantitative readout by focusing on the most relevant subset , a combination of the CD15/CD24/CD29 surface antigens for the isolation of NSC, differentiated neuron and neural crest cells 28 or CD15/CD24/CD44/CD184/CD271 to isolate neural and glial subsets 25 , among other signatures 29,30 . Beyond neurons, glial markers include A2B5 31 , CD44 25 , NG2 32 and GLAST 33 . A recent publication has exploited the midbrain floorplate precursor marker CORIN 34,35 to enrich for dopaminergic precursors in Parkinson cell transplantation paradigms 36 .…”