title: An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody.
abstract: Astrocytes are a major cell type in the mammalian CNS. Astrocytes are now known to play a number of essential roles in processes including synapse formation and function, as well as blood-brain barrier formation and control of cerebral blood flow. However, our understanding of the molecular mechanisms underlying astrocyte development and function is still rudimentary. This lack of knowledge is at least partly due to the lack of tools currently available for astrocyte biology. ACSA-2 is a commercially available antibody originally developed for the isolation of astrocytes from young postnatal mouse brain, using magnetic cell-sorting methods, but its utility in isolating cells from adult tissue has not yet been published. Using a modified protocol, we now show that this tool can also be used to isolate ultrapure astrocytes from the adult brain. Furthermore, using a variety of techniques (including single-cell sequencing, overexpression and knockdown assays, immunoblotting, and immunohistochemistry), we identify the ACSA-2 epitope for the first time as ATP1B2 and characterize its distribution in the CNS. Finally, we show that ATP1B2 is stably expressed in multiple models of CNS injury and disease. Hence, we show that the ACSA-2 antibody possesses the potential to be an extremely valuable tool for astrocyte research, allowing the purification and characterization of astrocytes (potentially including injury and disease models) without the need for any specialized and expensive equipment. In fact, our results suggest that ACSA-2 should be a first-choice method for astrocyte isolation and characterization.
authors.affiliation: From the Laboratory of Glia Biology, VIB-KU Leuven Center for Brain and Disease Research.|the Laboratory of Glia Biology, KU Leuven Department of Neuroscience, and.
authors.forename: Mykhailo Y
authors.initials: MY
authors.lastname: Batiuk
authors.affiliation: From the Laboratory of Glia Biology, VIB-KU Leuven Center for Brain and Disease Research.|the Laboratory of Glia Biology, KU Leuven Department of Neuroscience, and.
authors.forename: Filip
authors.initials: F
authors.lastname: de Vin
authors.affiliation: From the Laboratory of Glia Biology, VIB-KU Leuven Center for Brain and Disease Research.|the Laboratory of Glia Biology, KU Leuven Department of Neuroscience, and.
authors.forename: Sandra I
authors.initials: SI
authors.lastname: Duqué
authors.affiliation: From the Laboratory of Glia Biology, VIB-KU Leuven Center for Brain and Disease Research.|the Laboratory of Glia Biology, KU Leuven Department of Neuroscience, and.
authors.forename: Chen
authors.initials: C
authors.lastname: Li
authors.affiliation: the Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0106, Japan, and.
authors.forename: Takashi
authors.initials: T
authors.lastname: Saito
authors.affiliation: the Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0106, Japan, and.
authors.forename: Takaomi
authors.initials: T
authors.lastname: Saido
authors.affiliation: the VIB-KU Leuven Center for Brain and Disease Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium.
authors.forename: Mark
authors.initials: M
authors.lastname: Fiers
authors.affiliation: the Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy, and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, United Kingdom.
authors.forename: T Grant
authors.initials: TG
authors.lastname: Belgard
authors.affiliation: From the Laboratory of Glia Biology, VIB-KU Leuven Center for Brain and Disease Research, Matthew.Holt@kuleuven.vib.be.|the Laboratory of Glia Biology, KU Leuven Department of Neuroscience, and.
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Holt
mesh_terms: D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D001253:Astrocytes / Q000378:metabolism* / Q000473:pathology; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D001930:Brain Injuries / Q000378:metabolism* / Q000473:pathology; D027682:Cation Transport Proteins / Q000096:biosynthesis / Q000737:chemistry; D015816:Cell Adhesion Molecules, Neuronal / Q000096:biosynthesis / Q000737:chemistry; D004195:Disease Models, Animal; D000939:Epitopes / Q000096:biosynthesis / Q000737:chemistry; D005260:Female; D005786:Gene Expression Regulation; D008297:Male; D051379:Mice
chemical_list: D000906:Antibodies; C504191:Atp1b2 protein, mouse; D027682:Cation Transport Proteins; D015816:Cell Adhesion Molecules, Neuronal; D000939:Epitopes; D000251:Adenosine Triphosphatases
keywords: astrocyte; cell surface protein; flow cytometry; immunoblotting; immunohistochemistry; immunoisolation; membrane protein; neuroscience; plasma membrane; single-cell RNA-seq